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Find video protocols related to scientific articles indexed in Pubmed.
Charge reduction stabilizes intact membrane protein complexes for mass spectrometry.
J. Am. Chem. Soc.
PUBLISHED: 11-18-2014
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The study of intact soluble protein assemblies by means of mass spectrometry is providing invaluable contributions to structural biology and biochemistry. A recent breakthrough has enabled similar study of membrane protein complexes, following their release from detergent micelles in the gas phase. Careful optimization of mass spectrometry conditions, particularly with respect to energy regimes, is essential for maintaining compact folded states as detergent is removed. However many of the saccharide detergents widely employed in structural biology can cause unfolding of membrane proteins in the gas phase. Here, we investigate the potential of charge reduction by introducing three membrane protein complexes from saccharide detergents and show how reducing their overall charge enables generation of compact states, as evidenced by ion mobility mass spectrometry. We find that charge reduction stabilizes the oligomeric state and enhances the stability of lipid-bound complexes. This finding is significant since maintain-ing native-like membrane proteins enables ligand binding to be assessed from a range of detergents that retain solubility while protecting the overall fold.
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A comparative cross-linking strategy to probe conformational changes in protein complexes.
Nat Protoc
PUBLISHED: 08-21-2014
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Chemical cross-linking, together with mass spectrometry (MS), is a powerful combination for probing subunit interactions within static protein assemblies. To probe conformational changes in response to stimuli, we have developed a comparative cross-linking strategy, using lysine-specific deuterated and nondeuterated bis(sulfosuccinimidyl)suberate cross-linking reagents (BS3). Here we describe the experimental procedures as well as the data analysis, validation and interpretation. The protocol involves first assigning cross-linked peptides in the complex without ligand binding, or with post-translational modifications (PTMs) at natural abundance, using a standard procedure with labeled cross-linkers, proteolysis and assignment of cross-linked peptides after liquid chromatography-tandem MS (LC-MS/MS) and database searching. An aliquot of the protein complex is then exposed to a stimulus: either ligand binding or incubation with a phosphatase or kinase to bring about changes in PTMs. Two solutions--one containing the apo/untreated complex and the other containing the enzymatically modified/ligand-bound complex--are then cross-linked independently. Typically, nondeuterated BS3-d0 is used for the untreated complex and deuterated BS3-d4 is used for the experiment. The two aliquots are then incubated at equal concentrations, digested and processed as before. The ratios of labeled and unlabeled cross-linked peptides provide a direct readout of the effect of the stimulus. We exemplify our method by quantifying changes in subunit interactions induced by dephosphorylation of an ATP synthase. The protocol can also be used to determine the conformational changes in protein complexes induced by various stimuli including ligand/drug binding, oligomerization and other PTMs. Application of the established protocol takes ~9 d, including protein complex purification.
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SpyAvidin hubs enable precise and ultrastable orthogonal nanoassembly.
J. Am. Chem. Soc.
PUBLISHED: 08-21-2014
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The capture of biotin by streptavidin is an inspiration for supramolecular chemistry and a central tool for biological chemistry and nanotechnology, because of the rapid and exceptionally stable interaction. However, there is no robust orthogonal interaction to this hub, limiting the size and complexity of molecular assemblies that can be created. Here we combined traptavidin (a streptavidin variant maximizing biotin binding strength) with an orthogonal irreversible interaction. SpyTag is a peptide engineered to form a spontaneous isopeptide bond to its protein partner SpyCatcher. SpyTag or SpyCatcher was successfully fused to the C-terminus of Dead streptavidin subunits. We were able to generate chimeric tetramers with n (0 ? n ? 4) biotin binding sites and 4-n SpyTag or SpyCatcher binding sites. Chimeric SpyAvidin tetramers bound precise numbers of ligands fused to biotin or SpyTag/SpyCatcher. Mixing chimeric tetramers enabled assembly of SpyAvidin octamers (8 subunits) or eicosamers (20 subunits). We validated assemblies using electrophoresis and native mass spectrometry. Eicosameric SpyAvidin was used to cluster trimeric major histocompatibility complex (MHC) class I:?2-microglobulin:peptide complexes, generating an assembly with up to 56 components. MHC eicosamers surpassed the conventional MHC tetramers in acting as a powerful stimulus to T cell signaling. Combining ultrastable noncovalent with irreversible covalent interaction, SpyAvidins enable a simple route to create robust nanoarchitectures.
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Mass-selective soft-landing of protein assemblies with controlled landing energies.
Anal. Chem.
PUBLISHED: 07-31-2014
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Selection and soft-landing of bionanoparticles in vacuum is potentially a preparative approach to separate heterogeneous mixtures for high-resolution structural study or to deposit homogeneous materials for nanotechnological applications. Soft-landing of intact protein assemblies however remains challenging, due to the difficulties of manipulating these heavy species in mass-selective devices and retaining their structure during the experiment. We have developed a tandem mass spectrometer with the capability for controlled ion soft-landing and ex situ visualization of the soft-landed particles by means of transmission electron microscopy. The deposition conditions can be controlled by adjusting the kinetic energies of the ions by applying accelerating or decelerating voltages to a set of ion-steering optics. To validate this approach, we have examined two cage-like protein complexes, GroEL and ferritin, and studied the effect of soft-landing conditions on the method's throughput and the preservation of protein structure. Separation, based on mass-to-charge ratio, of holo- and apo-ferritin complexes after electrospray ionization enabled us to soft-land independently the separated complexes on a grid suitable for downstream transmission electron microscopy analysis. Following negative staining, images of the soft-landed complexes reveal that their structural integrity is largely conserved, with the characteristic central cavity of apoferritin, and iron core of holoferritin, surviving the phase transition from liquid to gas, soft-landing, and dehydration in vacuum.
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A hydrodynamic comparison of solution and gas phase proteins and their complexes.
J Phys Chem B
PUBLISHED: 07-03-2014
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The extent to which protein structures are preserved on transfer from solution to gas phase is a central question for native mass spectrometry. Here we compare the collision cross sections (?) of a wide range of different proteins and protein complexes (15-500 kDa) with their corresponding Stokes radii (RS). Using these methods, we find that ? and RS are well correlated, implying overall preservation of protein structure in the gas phase. Accounting for protein hydration, a scaling term is required to bring ? and RS into parity. Interestingly, the magnitude of this scaling term agrees almost entirely with the drag factor proposed by Millikan. RS were then compared with various different predicted values of ? taken from their atomic coordinates. We find that many of the approaches used to obtained ? from atomic coordinates miscalculate the physical sizes of the proteins in solution by as much as 20%. Rescaling of ? estimated from atomic coordinates may therefore seem appropriate as a general method to bring theoretical values in line with those observed in solution.
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Structural basis for Pan3 binding to Pan2 and its function in mRNA recruitment and deadenylation.
EMBO J.
PUBLISHED: 05-28-2014
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The conserved eukaryotic Pan2-Pan3 deadenylation complex shortens cytoplasmic mRNA 3' polyA tails to regulate mRNA stability. Although the exonuclease activity resides in Pan2, efficient deadenylation requires Pan3. The mechanistic role of Pan3 is unclear. Here, we show that Pan3 binds RNA directly both through its pseudokinase/C-terminal domain and via an N-terminal zinc finger that binds polyA RNA specifically. In contrast, isolated Pan2 is unable to bind RNA. Pan3 binds to the region of Pan2 that links its N-terminal WD40 domain to the C-terminal part that contains the exonuclease, with a 2:1 stoichiometry. The crystal structure of the Pan2 linker region bound to a Pan3 homodimer shows how the unusual structural asymmetry of the Pan3 dimer is used to form an extensive high-affinity interaction. This binding allows Pan3 to supply Pan2 with substrate polyA RNA, facilitating efficient mRNA deadenylation by the intact Pan2-Pan3 complex.
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Flexible membrane proteins: functional dynamics captured by mass spectrometry.
Curr. Opin. Struct. Biol.
PUBLISHED: 05-22-2014
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Membrane proteins are flexible molecular machines, responsible for the exchange of molecules in and out of the cell, which have evolved to perform specific and complex tasks with great efficiency. Obtaining accurate descriptions of their dynamics in the context of their function represents a major challenge for structural biology. Here we chart recent developments in mass spectrometry designed to characterize changes in the dynamics of membrane proteins in response to ligand binding or post-translational modifications. We focus on cooperative movements and structural changes across a range of timescales, from milliseconds to minutes, and highlight the contributions of mass spectrometry to our understanding of molecular mechanisms of diverse transmembrane processes.
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Membrane proteins bind lipids selectively to modulate their structure and function.
Nature
PUBLISHED: 04-28-2014
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Previous studies have established that the folding, structure and function of membrane proteins are influenced by their lipid environments and that lipids can bind to specific sites, for example, in potassium channels. Fundamental questions remain however regarding the extent of membrane protein selectivity towards lipids. Here we report a mass spectrometry approach designed to determine the selectivity of lipid binding to membrane protein complexes. We investigate the mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis and aquaporin Z (AqpZ) and the ammonia channel (AmtB) from Escherichia coli, using ion mobility mass spectrometry (IM-MS), which reports gas-phase collision cross-sections. We demonstrate that folded conformations of membrane protein complexes can exist in the gas phase. By resolving lipid-bound states, we then rank bound lipids on the basis of their ability to resist gas phase unfolding and thereby stabilize membrane protein structure. Lipids bind non-selectively and with high avidity to MscL, all imparting comparable stability; however, the highest-ranking lipid is phosphatidylinositol phosphate, in line with its proposed functional role in mechanosensation. AqpZ is also stabilized by many lipids, with cardiolipin imparting the most significant resistance to unfolding. Subsequently, through functional assays we show that cardiolipin modulates AqpZ function. Similar experiments identify AmtB as being highly selective for phosphatidylglycerol, prompting us to obtain an X-ray structure in this lipid membrane-like environment. The 2.3?Å resolution structure, when compared with others obtained without lipid bound, reveals distinct conformational changes that re-position AmtB residues to interact with the lipid bilayer. Our results demonstrate that resistance to unfolding correlates with specific lipid-binding events, enabling a distinction to be made between lipids that merely bind from those that modulate membrane protein structure and/or function. We anticipate that these findings will be important not only for defining the selectivity of membrane proteins towards lipids, but also for understanding the role of lipids in modulating protein function or drug binding.
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The structured core domain of ?B-crystallin can prevent amyloid fibrillation and associated toxicity.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 04-07-2014
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Mammalian small heat-shock proteins (sHSPs) are molecular chaperones that form polydisperse and dynamic complexes with target proteins, serving as a first line of defense in preventing their aggregation into either amorphous deposits or amyloid fibrils. Their apparently broad target specificity makes sHSPs attractive for investigating ways to tackle disorders of protein aggregation. The two most abundant sHSPs in human tissue are ?B-crystallin (ABC) and HSP27; here we present high-resolution structures of their core domains (cABC, cHSP27), each in complex with a segment of their respective C-terminal regions. We find that both truncated proteins dimerize, and although this interface is labile in the case of cABC, in cHSP27 the dimer can be cross-linked by an intermonomer disulfide linkage. Using cHSP27 as a template, we have designed an equivalently locked cABC to enable us to investigate the functional role played by oligomerization, disordered N and C termini, subunit exchange, and variable dimer interfaces in ABC. We have assayed the ability of the different forms of ABC to prevent protein aggregation in vitro. Remarkably, we find that cABC has chaperone activity comparable to that of the full-length protein, even when monomer dissociation is restricted through disulfide linkage. Furthermore, cABC is a potent inhibitor of amyloid fibril formation and, by slowing the rate of its aggregation, effectively reduces the toxicity of amyloid-? peptide to cells. Overall we present a small chaperone unit together with its atomic coordinates that potentially enables the rational design of more effective chaperones and amyloid inhibitors.
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Mass Spectrometry Quantifies Protein Interactions-From Molecular Chaperones to Membrane Porins.
Angew. Chem. Int. Ed. Engl.
PUBLISHED: 03-26-2014
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Proteins possess an intimate relationship between their structure and function, with folded protein structures generating recognition motifs for the binding of ligands and other proteins. Mass spectrometry (MS) can provide information on a number of levels of protein structure, from the primary amino acid sequence to its three-dimensional fold and quaternary interactions. Given that MS is a gas-phase technique, with its foundations in analytical chemistry, it is perhaps counter-intuitive to use it to study the structure and non-covalent interactions of proteins that form in solution. Herein we show, however, that MS can go beyond simply preserving protein interactions in the gas phase by providing new insight into dynamic interaction networks, dissociation mechanisms, and the cooperativity of ligand binding. We consider potential pitfalls in data interpretation and place particular emphasis on recent studies that revealed quantitative information about dynamic protein interactions, in both soluble and membrane-embedded assemblies.
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The structures of cytosolic and plastid-located glutamine synthetases from Medicago truncatula reveal a common and dynamic architecture.
Acta Crystallogr. D Biol. Crystallogr.
PUBLISHED: 03-19-2014
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The first step of nitrogen assimilation in higher plants, the energy-driven incorporation of ammonia into glutamate, is catalyzed by glutamine synthetase. This central process yields the readily metabolizable glutamine, which in turn is at the basis of all subsequent biosynthesis of nitrogenous compounds. The essential role performed by glutamine synthetase makes it a prime target for herbicidal compounds, but also a suitable intervention point for the improvement of crop yields. Although the majority of crop plants are dicotyledonous, little is known about the structural organization of glutamine synthetase in these organisms and about the functional differences between the different isoforms. Here, the structural characterization of two glutamine synthetase isoforms from the model legume Medicago truncatula is reported: the crystallographic structure of cytoplasmic GSII-1a and an electron cryomicroscopy reconstruction of plastid-located GSII-2a. Together, these structural models unveil a decameric organization of dicotyledonous glutamine synthetase, with two pentameric rings weakly connected by inter-ring loops. Moreover, rearrangement of these dynamic loops changes the relative orientation of the rings, suggesting a zipper-like mechanism for their assembly into a decameric enzyme. Finally, the atomic structure of M. truncatula GSII-1a provides important insights into the structural determinants of herbicide resistance in this family of enzymes, opening new avenues for the development of herbicide-resistant plants.
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Structural basis for hijacking siderophore receptors by antimicrobial lasso peptides.
Nat. Chem. Biol.
PUBLISHED: 03-13-2014
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The lasso peptide microcin J25 is known to hijack the siderophore receptor FhuA for initiating internalization. Here, we provide what is to our knowledge the first structural evidence on the recognition mechanism, and our biochemical data show that another closely related lasso peptide cannot interact with FhuA. Our work provides an explanation on the narrow activity spectrum of lasso peptides and opens the path to the development of new antibacterials.
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The p97-FAF1 protein complex reveals a common mode of p97 adaptor binding.
J. Biol. Chem.
PUBLISHED: 03-11-2014
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p97, also known as valosin-containing protein, is a versatile participant in the ubiquitin-proteasome system. p97 interacts with a large network of adaptor proteins to process ubiquitylated substrates in different cellular pathways, including endoplasmic reticulum-associated degradation and transcription factor activation. p97 and its adaptor Fas-associated factor-1 (FAF1) both have roles in the ubiquitin-proteasome system during NF-?B activation, although the mechanisms are unknown. FAF1 itself also has emerging roles in other cell-cycle pathways and displays altered expression levels in various cancer cell lines. We have performed a detailed study the p97-FAF1 interaction. We show that FAF1 binds p97 stably and in a stoichiometry of 3 to 6. Cryo-EM analysis of p97-FAF1 yielded a 17 ? reconstruction of the complex with FAF1 above the p97 ring. Characteristics of p97-FAF1 uncovered in this study reveal common features in the interactions of p97, providing mechanistic insight into how p97 mediates diverse functionalities.
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eIF2B is a decameric guanine nucleotide exchange factor with a ?2?2 tetrameric core.
Nat Commun
PUBLISHED: 02-19-2014
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eIF2B facilitates and controls protein synthesis in eukaryotes by mediating guanine nucleotide exchange on its partner eIF2. We combined mass spectrometry (MS) with chemical cross-linking, surface accessibility measurements and homology modelling to define subunit stoichiometry and interactions within eIF2B and eIF2. Although it is generally accepted that eIF2B is a pentamer of five non-identical subunits (?-?), here we show that eIF2B is a decamer. MS and cross-linking of eIF2B complexes allows us to propose a model for the subunit arrangements within eIF2B where the subunit assembly occurs through catalytic ?- and ?-subunits, with regulatory subunits arranged in asymmetric trimers associated with the core. Cross-links between eIF2 and eIF2B allow modelling of interactions that contribute to nucleotide exchange and its control by eIF2 phosphorylation. Finally, we identify that GTP binds to eIF2B?, prompting us to propose a multi-step mechanism for nucleotide exchange.
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Analysis of the subunit organization of the eIF2B complex reveals new insights into its structure and regulation.
FASEB J.
PUBLISHED: 02-14-2014
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Eukaryotic initiation factor 2B (eIF2B) is the guanine nucleotide exchange factor for eIF2 and a critical regulator of protein synthesis, (e.g., as part of the integrated stress response). Certain mutations in the EIF2B genes cause leukoencephalopathy with vanishing white matter (VWM), an often serious neurological disorder. Comprising 5 subunits, ?-? (eIF2B? being the catalytic one), eIF2B has always been considered an ????? heteropentamer. We have analyzed the subunit interactions within mammalian eIF2B by using a combination of mass spectrometry and in vivo studies of overexpressed complexes to gain further insight into the subunit arrangement of the complex. Our data reveal that eIF2B is actually decameric, a dimer of eIF2B(????) tetramers stabilized by 2 copies of eIF2B?. We also demonstrate a pivotal role for eIF2B? in the formation of eIF2B(????) tetramers. eIF2B(?????)2 decamers show greater binding to eIF2 than to eIF2B(????) tetramers, which may underlie the increased activity of the former. We examined the levels of eIF2B subunits in a panel of different mouse tissues and identified different levels of eIF2B subunits, particularly eIF2B?, which implies heterogeneity in the cellular proportions of eIF2B(?????) and eIF2B(????) complexes, with important implications for the regulation of translation in individual cell types.
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A mass spectrometry-based hybrid method for structural modeling of protein complexes.
Nat. Methods
PUBLISHED: 02-09-2014
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We describe a method that integrates data derived from different mass spectrometry (MS)-based techniques with a modeling strategy for structural characterization of protein assemblies. We encoded structural data derived from native MS, bottom-up proteomics, ion mobility-MS and chemical cross-linking MS into modeling restraints to compute the most likely structure of a protein assembly. We used the method to generate near-native models for three known structures and characterized an assembly intermediate of the proteasomal base.
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Dynamic protein ligand interactions--insights from MS.
FEBS J.
PUBLISHED: 01-21-2014
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Proteins undergo dynamic interactions with carbohydrates, lipids and nucleotides to form catalytic cores, fine-tuned for different cellular actions. The study of dynamic interactions between proteins and their cognate ligands is therefore fundamental to the understanding of biological systems. During the last two decades MS, and its associated techniques, has become accepted as a method for the study of protein-ligand interactions, not only for covalent complexes, where the use of MS is well established, but also, and significantly for protein-ligand interactions, for noncovalent assemblies. In this review, we employ a broad definition of a ligand to encompass protein subunits, drug molecules, oligonucleotides, carbohydrates, and lipids. Under the appropriate conditions, MS can reveal the composition, heterogeneity and dynamics of these protein-ligand interactions, and in some cases their structural arrangements and binding affinities. Herein, we highlight MS approaches for studying protein-ligand complexes, including those containing integral membrane subunits, and showcase examples from recent literature. Specifically, we tabulate the myriad of methodologies, including hydrogen exchange, proteomics, hydroxyl radical footprinting, intact complexes, and crosslinking, which, when combined with MS, provide insights into conformational changes and subtle modifications in response to ligand-binding interactions.
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Mass spectrometry defines the C-terminal dimerization domain and enables modeling of the structure of full-length OmpA.
Structure
PUBLISHED: 01-20-2014
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The transmembrane domain of the outer membrane protein A (OmpA) from Escherichia coli is an excellent model for structural and folding studies of ?-barrel membrane proteins. However, full-length OmpA resists crystallographic efforts, and the link between its function and tertiary structure remains controversial. Here we use site-directed mutagenesis and mass spectrometry of different constructs of OmpA, released in the gas phase from detergent micelles, to define the minimal region encompassing the C-terminal dimer interface. Combining knowledge of the location of the dimeric interface with molecular modeling and ion mobility data allows us to propose a low-resolution model for the full-length OmpA dimer. Our model of the dimer is in remarkable agreement with experimental ion mobility data, with none of the unfolding or collapse observed for full-length monomeric OmpA, implying that dimer formation stabilizes the overall structure and prevents collapse of the flexible linker that connects the two domains.
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Proteolytic cleavage of Ser52Pro variant transthyretin triggers its amyloid fibrillogenesis.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 01-13-2014
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The Ser52Pro variant of transthyretin (TTR) produces aggressive, highly penetrant, autosomal-dominant systemic amyloidosis in persons heterozygous for the causative mutation. Together with a minor quantity of full-length wild-type and variant TTR, the main component of the ex vivo fibrils was the residue 49-127 fragment of the TTR variant, the portion of the TTR sequence that previously has been reported to be the principal constituent of type A, cardiac amyloid fibrils formed from wild-type TTR and other TTR variants [Bergstrom J, et al. (2005) J Pathol 206(2):224-232]. This specific truncation of Ser52Pro TTR was generated readily in vitro by limited proteolysis. In physiological conditions and under agitation the residue 49-127 proteolytic fragment rapidly and completely self-aggregates into typical amyloid fibrils. The remarkable susceptibility to such cleavage is likely caused by localized destabilization of the ?-turn linking strands C and D caused by loss of the wild-type hydrogen-bonding network between the side chains of residues Ser52, Glu54, Ser50, and a water molecule, as revealed by the high-resolution crystallographic structure of Ser52Pro TTR. We thus provide a structural basis for the recently hypothesized, crucial pathogenic role of proteolytic cleavage in TTR amyloid fibrillogenesis. Binding of the natural ligands thyroxine or retinol-binding protein (RBP) by Ser52Pro variant TTR stabilizes the native tetrameric assembly, but neither protected the variant from proteolysis. However, binding of RBP, but not thyroxine, inhibited subsequent fibrillogenesis.
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Ion mobility-mass spectrometry of a rotary ATPase reveals ATP-induced reduction in conformational flexibility.
Nat Chem
PUBLISHED: 01-08-2014
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Rotary ATPases play fundamental roles in energy conversion as their catalytic rotation is associated with interdomain fluctuations and heterogeneity of conformational states. Using ion mobility mass spectrometry we compared the conformational dynamics of the intact ATPase from Thermus thermophilus with those of its membrane and soluble subcomplexes. Our results define regions with enhanced flexibility assigned to distinct subunits within the overall assembly. To provide a structural context for our experimental data we performed molecular dynamics simulations and observed conformational changes of the peripheral stalks that reflect their intrinsic flexibility. By isolating complexes at different phases of cell growth and manipulating nucleotides, metal ions and pH during isolation, we reveal differences that can be related to conformational changes in the Vo complex triggered by ATP binding. Together these results implicate nucleotides in modulating flexibility of the stator components and uncover mechanistic detail that underlies operation and regulation in the context of the holoenzyme.
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Structure of Cu(I)-Bound DJ-1 Reveals a Biscysteinate Metal Binding Site at the Homodimer Interface: Insights into Mutational Inactivation of DJ-1 in Parkinsonism.
J. Am. Chem. Soc.
PUBLISHED: 10-21-2013
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The Parkinsonism-associated protein DJ-1 has been suggested to activate the Cu-Zn superoxide dismutase (SOD1) by providing its copper cofactor. The structural and chemical means by which DJ-1 could support this function is unknown. In this study, we characterize the molecular interaction of DJ-1 with Cu(I). Mass spectrometric analysis indicates binding of one Cu(I) ion per DJ-1 homodimer. The crystal structure of DJ-1 bound to Cu(I) confirms metal coordination through a docking accessible biscysteinate site formed by juxtaposed cysteine residues at the homodimer interface. Spectroscopy in crystallo validates the identity and oxidation state of the bound metal. The measured subfemtomolar dissociation constant (Kd = 6.41 × 10(-16) M) of DJ-1 for Cu(I) supports the physiological retention of the metal ion. Our results highlight the requirement of a stable homodimer for copper binding by DJ-1. Parkinsonism-linked mutations that weaken homodimer interactions will compromise this capability.
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A nanobody binding to non-amyloidogenic regions of the protein human lysozyme enhances partial unfolding but inhibits amyloid fibril formation.
J Phys Chem B
PUBLISHED: 09-24-2013
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We report the effects of the interaction of two camelid antibody fragments, generally called nanobodies, namely cAb-HuL5 and a stabilized and more aggregation-resistant variant cAb-HuL5G obtained by protein engineering, on the properties of two amyloidogenic variants of human lysozyme, I56T and D67H, whose deposition in vital organs including the liver, kidney, and spleen is associated with a familial non-neuropathic systemic amyloidosis. Both NMR spectroscopy and X-ray crystallographic studies reveal that cAb-HuL5 binds to the ?-domain, one of the two lobes of the native lysozyme structure. The binding of cAb-HuL5/cAb-HuL5G strongly inhibits fibril formation by the amyloidogenic variants; it does not, however, suppress the locally transient cooperative unfolding transitions, characteristic of these variants, in which the ?-domain and the C-helix unfold and which represents key early intermediate species in the formation of amyloid fibrils. Therefore, unlike two other nanobodies previously described, cAb-HuL5/cAb-HuL5G does not inhibit fibril formation via the restoration of the global cooperativity of the native structure of the lysozyme variants to that characteristic of the wild-type protein. Instead, it inhibits a subsequent step in the assembly of the fibrils, involving the unfolding and structural reorganization of the ?-domain. These results show that nanobodies can protect against the formation of pathogenic aggregates at different stages in the structural transition of a protein from the soluble native state into amyloid fibrils, illustrating their value as structural probes to study the molecular mechanisms of amyloid fibril formation. Combined with their amenability to protein engineering techniques to improve their stability and solubility, these findings support the suggestion that nanobodies can potentially be developed as therapeutics to combat protein misfolding diseases.
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Subunit Dynamics and Nucleotide-Dependent Asymmetry of an AAA(+) Transcription Complex.
J. Mol. Biol.
PUBLISHED: 07-29-2013
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Bacterial enhancer binding proteins (bEBPs) are transcription activators that belong to the AAA(+) protein family. They form higher-order self-assemblies to regulate transcription initiation at stress response and pathogenic promoters. The precise mechanism by which these ATPases utilize ATP binding and hydrolysis energy to remodel their substrates remains unclear. Here we employed mass spectrometry of intact complexes to investigate subunit dynamics and nucleotide occupancy of the AAA(+) domain of one well-studied bEBP in complex with its substrate, the ?(54) subunit of RNA polymerase. Our results demonstrate that the free AAA(+) domain undergoes significant changes in oligomeric states and nucleotide occupancy upon ?(54) binding. Such changes likely correlate with one transition state of ATP and are associated with an open spiral ring formation that is vital for asymmetric subunit function and interface communication. We confirmed that the asymmetric subunit functionality persists for open promoter complex formation using single-chain forms of bEBP lacking the full complement of intact ATP hydrolysis sites. Outcomes reconcile low- and high-resolution structures and yield a partial sequential ATP hydrolysis model for bEBPs.
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Genetic testing for Duchenne/Becker muscular dystrophy in Johannesburg, South Africa.
S. Afr. Med. J.
PUBLISHED: 07-16-2013
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Background. Genetic testing for Duchenne/Becker muscular dystrophy (DMD/BMD) mutations initially involved multiplex polymerase chain reaction (mPCR), which targeted two mutation hotspots in the gene and detected deletions in affected males. A newer technology, multiplex ligation-dependent probe amplification (MLPA), was introduced for diagnostic testing in 2007.Objectives. To evaluate MLPA relative to mPCR as a technique for DMD/BMD diagnostic testing and to establish whether the mutation profile in affected individuals differs between different South African ethnic groups. Methods. From January 2000 - May 2007, genetic diagnostic testing for DMD/BMD was undertaken in 128 male patients using mPCR. From May 2007 onwards, MLPA replaced this technique and 261 males were investigated. MLPA is a kit-based technology available from MRC-Holland.Results. Of the 128 and 261 probands tested using mPCR and MLPA, respectively, 31% and 34% were found to carry a deletion mutation. Further, MLPA could detect duplication mutations (11.5%), complex rearrangements (1.5%) and small mutations (1.5%). In black patients, deletion mutations were found to cluster in the 3 region of the gene. No population-specific pathogenic mutations were found. Conclusions. The mutation detection rate for mPCR and MLPA is similar for deletion mutations, but MLPA proved to be a better diagnostic approach as it could detect other types of mutations as well, including duplications, complex rearrangements and small mutations. MLPA could also diagnose mutation status in at-risk female relatives, which is not possible with mPCR. 
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Twenty years of gas phase structural biology.
Structure
PUBLISHED: 07-10-2013
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Over the past two decades, mass spectrometry (MS) of protein complexes from their native state has made inroads into structural biology. To coincide with the 20(th) anniversary of Structure, and given that it is now approximately 20 years since the first mass spectra of noncovalent protein complexes were reported, it is timely to consider progress of MS as a structural biology tool. Early reports focused on soluble complexes, contributing to ligand binding studies, subunit interaction maps, and topological models. Recent discoveries have enabled delivery of membrane complexes, encapsulated in detergent micelles, prompting new opportunities. By maintaining interactions between membrane and cytoplasmic subunits in the gas phase, it is now possible to investigate the effects of lipids, nucleotides, and drugs on intact membrane assemblies. These investigations reveal allosteric and synergistic effects of small molecule binding and expose the consequences of posttranslational modifications. In this review, we consider recent progress in the study of protein complexes, focusing particularly on complexes extracted from membranes, and outline future prospects for gas phase structural biology.
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Intrinsically disordered protein threads through the bacterial outer-membrane porin OmpF.
Science
PUBLISHED: 07-02-2013
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Porins are ?-barrel outer-membrane proteins through which small solutes and metabolites diffuse that are also exploited during cell death. We have studied how the bacteriocin colicin E9 (ColE9) assembles a cytotoxic translocon at the surface of Escherichia coli that incorporates the trimeric porin OmpF. Formation of the translocon involved ColE9s unstructured N-terminal domain threading in opposite directions through two OmpF subunits, capturing its target TolB on the other side of the membrane in a fixed orientation that triggers colicin import. Thus, an intrinsically disordered protein can tunnel through the narrow pores of an oligomeric porin to deliver an epitope signal to the cell to initiate cell death.
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Evidence that the catenane form of CS2 hydrolase is not an artefact.
Chem. Commun. (Camb.)
PUBLISHED: 06-18-2013
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CS2 hydrolase, a zinc-dependent enzyme that converts carbon disulfide to carbon dioxide and hydrogen sulfide, exists as a mixture of octameric ring and hexadecameric catenane forms in solution. A combination of size exclusion chromatography, multi-angle laser light scattering, and mass spectrometric analyses revealed that the unusual catenane structure is not an artefact, but a naturally occurring structure.
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Structure of the CRISPR interference complex CSM reveals key similarities with cascade.
Mol. Cell
PUBLISHED: 06-04-2013
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The Clustered Regularly Interspaced Palindromic Repeats (CRISPR) system is an adaptive immune system in prokaryotes. Interference complexes encoded by CRISPR-associated (cas) genes utilize small RNAs for homology-directed detection and subsequent degradation of invading genetic elements, and they have been classified into three main types (I-III). Type III complexes share the Cas10 subunit but are subclassifed as type IIIA (CSM) and type IIIB (CMR), depending on their specificity for DNA or RNA targets, respectively. The role of CSM in limiting the spread of conjugative plasmids in Staphylococcus epidermidis was first described in 2008. Here, we report a detailed investigation of the composition and structure of the CSM complex from the archaeon Sulfolobus solfataricus, using a combination of electron microscopy, mass spectrometry, and deep sequencing. This reveals a three-dimensional model for the CSM complex that includes a helical component strikingly reminiscent of the backbone structure of the type I (Cascade) family.
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Detergent-free mass spectrometry of membrane protein complexes.
Nat. Methods
PUBLISHED: 05-22-2013
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We developed a method that allows release of intact membrane protein complexes from amphipols, bicelles and nanodiscs in the gas phase for observation by mass spectrometry (MS). Current methods involve release of membrane protein complexes from detergent micelles, which reveals subunit composition and lipid binding. We demonstrated that oligomeric complexes or proteins requiring defined lipid environments are stabilized to a greater extent in the absence of detergent.
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Mass spectrometry reveals synergistic effects of nucleotides, lipids, and drugs binding to a multidrug resistance efflux pump.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 05-20-2013
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Multidrug resistance is a serious barrier to successful treatment of many human diseases, including cancer, wherein chemotherapeutics are exported from target cells by membrane-embedded pumps. The most prevalent of these pumps, the ATP-Binding Cassette transporter P-glycoprotein (P-gp), consists of two homologous halves each comprising one nucleotide-binding domain and six transmembrane helices. The transmembrane region encapsulates a hydrophobic cavity, accessed by portals in the membrane, that binds cytotoxic compounds as well as lipids and peptides. Here we use mass spectrometry (MS) to probe the intact P-gp small molecule-bound complex in a detergent micelle. Activation in the gas phase leads to formation of ions, largely devoid of detergent, yet retaining drug molecules as well as charged or zwitterionic lipids. Measuring the rates of lipid binding and calculating apparent KD values shows that up to six negatively charged diacylglycerides bind more favorably than zwitterionic lipids. Similar experiments confirm binding of cardiolipins and show that prior binding of the immunosuppressant and antifungal antibiotic cyclosporin A enhances subsequent binding of cardiolipin. Ion mobility MS reveals that P-gp exists in an equilibrium between different states, readily interconverted by ligand binding. Overall these MS results show how concerted small molecule binding leads to synergistic effects on binding affinities and conformations of a multidrug efflux pump.
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Dodecyl maltoside protects membrane proteins in vacuo.
Biophys. J.
PUBLISHED: 05-14-2013
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Molecular dynamics simulations have been used to characterize the effects of transfer from aqueous solution to a vacuum to inform our understanding of mass spectrometry of membrane-protein-detergent complexes. We compared two membrane protein architectures (an ?-helical bundle versus a ?-barrel) and two different detergent types (phosphocholines versus an alkyl sugar) with respect to protein stability and detergent packing. The ?-barrel membrane protein remained stable as a protein-detergent complex in vacuum. Zwitterionic detergents formed conformationally destabilizing interactions with an ?-helical membrane protein after detergent micelle inversion driven by dehydration in vacuum. In contrast, a nonionic alkyl sugar detergent resisted micelle inversion, maintaining the solution-phase conformation of the protein. This helps to explain the relative stability of membrane proteins in the presence of alkyl sugar detergents such as dodecyl maltoside.
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Detergent release prolongs the lifetime of native-like membrane protein conformations in the gas-phase.
J. Am. Chem. Soc.
PUBLISHED: 04-10-2013
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Recent studies have suggested that detergents can protect the structure of membrane proteins during their transition from solution to the gas-phase. Here we provide mechanistic insights into this process by interrogating the structures of membrane protein-detergent assemblies in the gas-phase using ion mobility mass spectrometry. We show a clear correlation between the population of native-like protein conformations and the degree of detergent attachment to the protein in the gas-phase. Interrogation of these protein-detergent assemblies, by tandem mass spectrometry, enables us to define the mechanism by which detergents preserve native-like protein conformations in a solvent free environment. We show that the release of detergent is more central to the survival of these conformations than the physical presence of detergent bound to the protein. We propose that detergent release competes with structural collapse for the internal energy of the ion and permits the observation of transient native-like membrane protein conformations that are otherwise lost to structural rearrangement in the gas-phase.
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The role of salt bridges, charge density, and subunit flexibility in determining disassembly routes of protein complexes.
Structure
PUBLISHED: 03-18-2013
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Mass spectrometry can be used to characterize multiprotein complexes, defining their subunit stoichiometry and composition following solution disruption and collision-induced dissociation (CID). While CID of protein complexes in the gas phase typically results in the dissociation of unfolded subunits, a second atypical route is possible wherein compact subunits or subcomplexes are ejected without unfolding. Because tertiary structure and subunit interactions may be retained, this is the preferred route for structural investigations. How can we influence which pathway is adopted? By studying properties of a series of homomeric and heteromeric protein complexes and varying their overall charge in solution, we found that low subunit flexibility, higher charge densities, fewer salt bridges, and smaller interfaces are likely to be involved in promoting dissociation routes without unfolding. Manipulating the charge on a protein complex therefore enables us to direct dissociation through structurally informative pathways that mimic those followed in solution.
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Steroid-based facial amphiphiles for stabilization and crystallization of membrane proteins.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 03-11-2013
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Amphiphile selection is a critical step for structural studies of membrane proteins (MPs). We have developed a family of steroid-based facial amphiphiles (FAs) that are structurally distinct from conventional detergents and previously developed FAs. The unique FAs stabilize MPs and form relatively small protein-detergent complexes (PDCs), a property considered favorable for MP crystallization. We attempted to crystallize several MPs belonging to different protein families, including the human gap junction channel protein connexin 26, the ATP binding cassette transporter MsbA, the seven-transmembrane G protein-coupled receptor-like bacteriorhodopsin, and cytochrome P450s (peripheral MPs). Using FAs alone or mixed with other detergents or lipids, we obtained 3D crystals of the above proteins suitable for X-ray crystallographic analysis. The fact that FAs enhance MP crystallizability compared with traditional detergents can be attributed to several properties, including increased protein stability, formation of small PDCs, decreased PDC surface flexibility, and potential to mediate crystal lattice contacts.
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Mass spectrometry of intact membrane protein complexes.
Nat Protoc
PUBLISHED: 03-07-2013
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Mass spectrometry (MS) of intact soluble protein complexes has emerged as a powerful technique to study the stoichiometry, structure-function and dynamics of protein assemblies. Recent developments have extended this technique to the study of membrane protein complexes, where it has already revealed subunit stoichiometries and specific phospholipid interactions. Here we describe a protocol for MS of membrane protein complexes. The protocol begins with the preparation of the membrane protein complex, enabling not only the direct assessment of stoichiometry, delipidation and quality of the target complex but also the evaluation of the purification strategy. A detailed list of compatible nonionic detergents is included, along with a protocol for screening detergents to find an optimal one for MS, biochemical and structural studies. This protocol also covers the preparation of lipids for protein-lipid binding studies and includes detailed settings for a quadrupole time-of-flight (Q-TOF) mass spectrometer after the introduction of complexes from gold-coated nanoflow capillaries.
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Protein complexes are under evolutionary selection to assemble via ordered pathways.
Cell
PUBLISHED: 02-05-2013
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Is the order in which proteins assemble into complexes important for biological function? Here, we seek to address this by searching for evidence of evolutionary selection for ordered protein complex assembly. First, we experimentally characterize the assembly pathways of several heteromeric complexes and show that they can be simply predicted from their three-dimensional structures. Then, by mapping gene fusion events identified from fully sequenced genomes onto protein complex assembly pathways, we demonstrate evolutionary selection for conservation of assembly order. Furthermore, using structural and high-throughput interaction data, we show that fusion tends to optimize assembly by simplifying protein complex topologies. Finally, we observe protein structural constraints on the gene order of fusion that impact the potential for fusion to affect assembly. Together, these results reveal the intimate relationships among protein assembly, quaternary structure, and evolution and demonstrate on a genome-wide scale the biological importance of ordered assembly pathways.
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Class I HDACs share a common mechanism of regulation by inositol phosphates.
Mol. Cell
PUBLISHED: 01-29-2013
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Class I histone deacetylases (HDAC1, HDAC2, and HDAC3) are recruited by cognate corepressor proteins into specific transcriptional repression complexes that target HDAC activity to chromatin resulting in chromatin condensation and transcriptional silencing. We previously reported the structure of HDAC3 in complex with the SMRT corepressor. This structure revealed the presence of inositol-tetraphosphate [Ins(1,4,5,6)P4] at the interface of the two proteins. It was previously unclear whether the role of Ins(1,4,5,6)P4 is to act as a structural cofactor or a regulator of HDAC3 activity. Here we report the structure of HDAC1 in complex with MTA1 from the NuRD complex. The ELM2-SANT domains from MTA1 wrap completely around HDAC1 occupying both sides of the active site such that the adjacent BAH domain is ideally positioned to recruit nucleosomes to the active site of the enzyme. Functional assays of both the HDAC1 and HDAC3 complexes reveal that Ins(1,4,5,6)P4 is a bona fide conserved regulator of class I HDAC complexes.
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Comparative cross-linking and mass spectrometry of an intact F-type ATPase suggest a role for phosphorylation.
Nat Commun
PUBLISHED: 01-29-2013
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F-type ATPases are highly conserved enzymes used primarily for the synthesis of ATP. Here we apply mass spectrometry to the F1FO-ATPase, isolated from spinach chloroplasts, and uncover multiple modifications in soluble and membrane subunits. Mass spectra of the intact ATPase define a stable lipid plug in the FO complex and reveal the stoichiometry of nucleotide binding in the F1 head. Comparing complexes formed in solution from an untreated ATPase with one incubated with a phosphatase reveals that the dephosphorylated enzyme has reduced nucleotide occupancy and decreased stability. By contrasting chemical cross-linking of untreated and dephosphorylated forms we show that cross-links are retained between the head and base, but are significantly reduced in the head, stators and stalk. Conformational changes at the catalytic interface, evidenced by changes in cross-linking, provide a rationale for reduced nucleotide occupancy and highlight a role for phosphorylation in regulating nucleotide binding and stability of the chloroplast ATPase.
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Integrative modelling coupled with ion mobility mass spectrometry reveals structural features of the clamp loader in complex with single-stranded DNA binding protein.
J. Mol. Biol.
PUBLISHED: 01-25-2013
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DNA polymerase III, a decameric 420-kDa assembly, simultaneously replicates both strands of the chromosome in Escherichia coli. A subassembly of this holoenzyme, the seven-subunit clamp loader complex, is responsible for loading the sliding clamp (?2) onto DNA. Here, we use structural information derived from ion mobility mass spectrometry (IM-MS) to build three-dimensional models of one form of the full clamp loader complex, ?3???? (254 kDa). By probing the interaction between the clamp loader and a single-stranded DNA (ssDNA) binding protein (SSB4) and by identifying two distinct conformational states, with and without ssDNA, we assemble models of ??-SSB4 (108 kDa) and the clamp loader-SSB4 (340 kDa) consistent with IM data. A significant increase in measured collision cross-section (~10%) of the clamp loader-SSB4 complex upon DNA binding suggests large conformational rearrangements. This DNA bound conformation represents the active state and, along with the presence of ??, stabilises the clamp loader-SSB4 complex. Overall, this study of a large heteromeric complex analysed by IM-MS, coupled with integrative modelling, highlights the potential of such an approach to reveal structural features of previously unknown complexes of high biological importance.
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Structural model of lymphocyte receptor NKR-P1C revealed by mass spectrometry and molecular modeling.
Anal. Chem.
PUBLISHED: 01-14-2013
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NKR-P1C is an activating immune receptor expressed on the surface of mouse natural killer cells. It has been widely used as a marker for NK cell identification in different mice strains. Recently we solved a crystal structure of the C-type lectin-like domain of a homologous protein, NKR-P1A, using X-ray crystallography and also described the strategy for rapid characterization of the protein conformation in solution. This procedure utilized chemical cross-linking, hydrogen/deuterium exchange, and molecular modeling. It was found that the solution structure differs from the crystal structure in the conformation of the loop region. The loop, detached from the protein compact core in the crystal structure, is closely attached to the core of the protein in solution. Here we present and interpret the solution structure of the C-type lectin-like domain of NKR-P1C using chemical cross-linking and molecular modeling. The validation of the model and conformation of the loop region in NKR-P1C were addressed using ion-mobility mass spectrometry.
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Crystal structure of the human short coiled coil protein and insights into SCOC-FEZ1 complex formation.
PLoS ONE
PUBLISHED: 01-01-2013
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The short coiled coil protein (SCOC) forms a complex with fasciculation and elongation protein zeta 1 (FEZ1). This complex is involved in autophagy regulation. We determined the crystal structure of the coiled coil domain of human SCOC at 2.7 Å resolution. SCOC forms a parallel left handed coiled coil dimer. We observed two distinct dimers in the crystal structure, which shows that SCOC is conformationally flexible. This plasticity is due to the high incidence of polar and charged residues at the core a/d-heptad positions. We prepared two double mutants, where these core residues were mutated to either leucines or valines (E93V/K97L and N125L/N132V). These mutations led to a dramatic increase in stability and change of oligomerisation state. The oligomerisation state of the mutants was characterized by multi-angle laser light scattering and native mass spectrometry measurements. The E93V/K97 mutant forms a trimer and the N125L/N132V mutant is a tetramer. We further demonstrate that SCOC forms a stable homogeneous complex with the coiled coil domain of FEZ1. SCOC dimerization and the SCOC surface residue R117 are important for this interaction.
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The N-Terminal Region of the Human Autophagy Protein ATG16L1 Contains a Domain That Folds into a Helical Structure Consistent with Formation of a Coiled-Coil.
PLoS ONE
PUBLISHED: 01-01-2013
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Autophagy is a fundamental cellular process required for organelle degradation and removal of invasive pathogens. Autophagosome formation involves the recruitment of, and interaction between, multiple proteins produced from autophagy-related (ATG) genes. One of the key complexes in autophagosome formation is the ATG12-ATG5-ATG16L1 complex. ATG16L1 functions as a molecular scaffold mediating protein-protein interactions necessary for formation of the autophagosome in response to both classical and pathogen-related autophagy stimuli. The coiled-coil domain of the yeast ortholog, ATG16, exists as a homodimer both in solution and in the crystal form. The yeast and human orthologs show poor sequence identity. Here we have sought to determine the minimal boundaries of the human ATG16L1 coiled-coil domain and ascertain its oligomeric status in solution. Using a range of biochemical and biophysical techniques we show that the secondary structure of the human ATG16L1 coiled-coil has the expected helical composition and that the domain forms a homodimer in solution. We also observe extensive sequence conservation across vertebrates providing strong support for the crucial functional role of the ATG16L1 coiled-coil.
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Hsp70 oligomerization is mediated by an interaction between the interdomain linker and the substrate-binding domain.
PLoS ONE
PUBLISHED: 01-01-2013
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Oligomerization in the heat shock protein (Hsp) 70 family has been extensively documented both in vitro and in vivo, although the mechanism, the identity of the specific protein regions involved and the physiological relevance of this process are still unclear. We have studied the oligomeric properties of a series of human Hsp70 variants by means of nanoelectrospray ionization mass spectrometry, optical spectroscopy and quantitative size exclusion chromatography. Our results show that Hsp70 oligomerization takes place through a specific interaction between the interdomain linker of one molecule and the substrate-binding domain of a different molecule, generating dimers and higher-order oligomers. We have found that substrate binding shifts the oligomerization equilibrium towards the accumulation of functional monomeric protein, probably by sequestering the helical lid sub-domain needed to stabilize the chaperone: substrate complex. Taken together, these findings suggest a possible role of chaperone oligomerization as a mechanism for regulating the availability of the active monomeric form of the chaperone and for the control of substrate binding and release.
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Structural basis for DNA recognition and loading into a viral packaging motor.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 12-29-2011
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Genome packaging into preformed viral procapsids is driven by powerful molecular motors. The small terminase protein is essential for the initial recognition of viral DNA and regulates the motors ATPase and nuclease activities during DNA translocation. The crystal structure of a full-length small terminase protein from the Siphoviridae bacteriophage SF6, comprising the N-terminal DNA binding, the oligomerization core, and the C-terminal ?-barrel domains, reveals a nine-subunit circular assembly in which the DNA-binding domains are arranged around the oligomerization core in a highly flexible manner. Mass spectrometry analysis and four further crystal structures show that, although the full-length protein exclusively forms nine-subunit assemblies, protein constructs missing the C-terminal ?-barrel form both nine-subunit and ten-subunit assemblies, indicating the importance of the C terminus for defining the oligomeric state. The mechanism by which a ring-shaped small terminase oligomer binds viral DNA has not previously been elucidated. Here, we probed binding in vitro by using EPR and surface plasmon resonance experiments, which indicated that interaction with DNA is mediated exclusively by the DNA-binding domains and suggested a nucleosome-like model in which DNA binds around the outside of the protein oligomer.
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Structural characterization of drug-like compounds by ion mobility mass spectrometry: comparison of theoretical and experimentally derived nitrogen collision cross sections.
Anal. Chem.
PUBLISHED: 12-27-2011
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We present the use of drug-like molecules as a traveling wave (T-wave) ion mobility (IM) calibration sample set, covering the m/z range of 122.1-609.3, the nitrogen collision cross-section (?(N(2))) range of 124.5-254.3 Å(2) and the helium collision cross-section (?(He)) range of 63.0-178.8 Å(2). Absolute ?(N(2)) and ?(He) values for the drug-like calibrants and two diastereomers were measured using a drift-tube instrument with radio frequency (RF) ion confinement. T-wave drift-times for the protonated diastereomers betamethasone and dexamethasone are reproducibly different. Calibration of these drift-times yields T-wave ?(N(2)) values of 189.4 and 190.4 Å(2), respectively. These results demonstrate the ability of T-wave IM spectrometry to differentiate diastereomers differing in ?(N(2)) value by only 1 Å(2), even though the resolution of these IM experiments were ?40 (?/??). Demonstrated through density functional theory optimized geometries and ionic electrostatic surface potential analysis, the small but measurable mobility difference between the two diastereomers is mainly due to short-range van der Waals interactions with the neutral buffer gas and not long-range charge-induced dipole interactions. The experimental RF-confining drift-tube and T-wave ?(N(2)) values were also evaluated using a nitrogen based trajectory method, optimized for T-wave operating temperature and pressures, incorporating additional scaling factors to the Lennard-Jones potentials. Experimental ?(He) values were also compared to the original and optimized helium based trajectory methods.
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Joining forces: integrating proteomics and cross-linking with the mass spectrometry of intact complexes.
Mol. Cell Proteomics
PUBLISHED: 12-16-2011
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Protein assemblies are critical for cellular function and understanding their physical organization is the key aim of structural biology. However, applying conventional structural biology approaches is challenging for transient, dynamic, or polydisperse assemblies. There is therefore a growing demand for hybrid technologies that are able to complement classical structural biology methods and thereby broaden our arsenal for the study of these important complexes. Exciting new developments in the field of mass spectrometry and proteomics have added a new dimension to the study of protein-protein interactions and protein complex architecture. In this review, we focus on how complementary mass spectrometry-based techniques can greatly facilitate structural understanding of protein assemblies.
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Mass spectrometry of intact V-type ATPases reveals bound lipids and the effects of nucleotide binding.
Science
PUBLISHED: 10-25-2011
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The ability of electrospray to propel large viruses into a mass spectrometer is established and is rationalized by analogy to the atmospheric transmission of the common cold. Much less clear is the fate of membrane-embedded molecular machines in the gas phase. Here we show that rotary adenosine triphosphatases (ATPases)/synthases from Thermus thermophilus and Enterococcus hirae can be maintained intact with membrane and soluble subunit interactions preserved in vacuum. Mass spectra reveal subunit stoichiometries and the identity of tightly bound lipids within the membrane rotors. Moreover, subcomplexes formed in solution and gas phases reveal the regulatory effects of nucleotide binding on both ATP hydrolysis and proton translocation. Consequently, we can link specific lipid and nucleotide binding with distinct regulatory roles.
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Heterogeneity and dynamics in the assembly of the heat shock protein 90 chaperone complexes.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 10-19-2011
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The Hsp90 cycle depends on the coordinated activity of a range of cochaperones, including Hop, Hsp70 and peptidyl-prolyl isomerases such as FKBP52. Using mass spectrometry, we investigate the order of addition of these cochaperones and their effects on the stoichiometry and composition of the resulting Hsp90-containing complexes. Our results show that monomeric Hop binds specifically to the Hsp90 dimer whereas FKBP52 binds to both monomeric and dimeric forms of Hsp90. By preforming Hsp90 complexes with either Hop, followed by addition of FKBP52, or with FKBP52 and subsequent addition of Hop, we monitor the formation of a predominant asymmetric ternary complex containing both cochaperones. This asymmetric complex is subsequently able to interact with the chaperone Hsp70 to form quaternary complexes containing all four proteins. Monitoring the population of these complexes during their formation and at equilibrium allows us to model the complex formation and to extract 14 different K(D) values. This simultaneous calculation of the K(D)s from a complex system with the same method, from eight deferent datasets under the same buffer conditions delivers a self-consistent set of values. In this case, the K(D) values afford insights into the assembly of ten Hsp90-containing complexes and provide a rationale for the cellular heterogeneity and prevalence of intermediates in the Hsp90 chaperone cycle.
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Non-homologous end-joining partners in a helical dance: structural studies of XLF-XRCC4 interactions.
Biochem. Soc. Trans.
PUBLISHED: 09-23-2011
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XRCC4 (X-ray cross-complementation group 4) and XLF (XRCC4-like factor) are two essential interacting proteins in the human NHEJ (non-homologous end-joining) pathway that repairs DNA DSBs (double-strand breaks). The individual crystal structures show that the dimeric proteins are homologues with protomers containing head domains and helical coiled-coil tails related by approximate two-fold symmetry. Biochemical, mutagenesis, biophysical and structural studies have identified the regions of interaction between the two proteins and suggested models for the XLF-XRCC4 complex. An 8.5 Å (1 Å = 0.1 nm) resolution crystal structure of XLF-XRCC4 solved by molecular replacement, together with gel filtration and nano-ESI (nano-electrospray ionization)-MS results, demonstrates that XLF and XRCC4 dimers interact through their head domains and form an alternating left-handed helical structure with polypeptide coiled coils and pseudo-dyads of individual XLF and XRCC4 dimers at right angles to the helical axis.
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Structure of a Blinkin-BUBR1 complex reveals an interaction crucial for kinetochore-mitotic checkpoint regulation via an unanticipated binding Site.
Structure
PUBLISHED: 07-11-2011
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The maintenance of genomic stability relies on the spindle assembly checkpoint (SAC), which ensures accurate chromosome segregation by delaying the onset of anaphase until all chromosomes are properly bioriented and attached to the mitotic spindle. BUB1 and BUBR1 kinases are central for this process and by interacting with Blinkin, link the SAC with the kinetochore, the macromolecular assembly that connects microtubules with centromeric DNA. Here, we identify the Blinkin motif critical for interaction with BUBR1, define the stoichiometry and affinity of the interaction, and present a 2.2 Å resolution crystal structure of the complex. The structure defines an unanticipated BUBR1 region responsible for the interaction and reveals a novel Blinkin motif that undergoes a disorder-to-order transition upon ligand binding. We also show that substitution of several BUBR1 residues engaged in binding Blinkin leads to defects in the SAC, thus providing the first molecular details of the recognition mechanism underlying kinetochore-SAC signaling.
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Evidence for the assembly of a bacterial tripartite multidrug pump with a stoichiometry of 3:6:3.
J. Biol. Chem.
PUBLISHED: 05-24-2011
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The multiple transferable resistance (mTR) pump from Neisseria gonorrhoeae MtrCDE multidrug pump is assembled from the inner and outer membrane proteins MtrD and MtrE and the periplasmic membrane fusion protein MtrC. Previously we established that while there is a weak interaction of MtrD and MtrE, MtrC binds with relatively high affinity to both MtrD and MtrE. MtrD conferred antibiotic resistance only when it was expressed with MtrE and MtrC, suggesting that these proteins form a functional tripartite complex in which MtrC bridges MtrD and MtrE. Furthermore, we demonstrated that MtrC interacts with an intraprotomer groove on the surface of MtrE, inducing channel opening. However, a second groove is apparent at the interface of the MtrE subunits, which might also be capable of engaging MtrC. We have now established that MtrC can be cross-linked to cysteines placed in this interprotomer groove and that mutation of residues in the groove impair the ability of the pump to confer antibiotic resistance by locking MtrE in the closed channel conformation. Moreover, MtrE K390C forms an intermolecular disulfide bond with MtrC E149C locking MtrE in the open channel conformation, suggesting that a functional salt bridge forms between these residues during the transition from closed to open channel conformations. MtrC forms dimers that assemble into hexamers, and electron microscopy studies of single particles revealed that these hexamers are arranged into ring-like structures with an internal aperture sufficiently large to accommodate the MtrE trimer. Cross-linking of single cysteine mutants of MtrC to stabilize the dimer interface in the presence of MtrE, trapped an MtrC-MtrE complex with a molecular mass consistent with a stoichiometry of 3:6 (MtrE(3)MtrC(6)), suggesting that dimers of MtrC interact with MtrE, presumably by binding to the two grooves. As both MtrE and MtrD are trimeric, our studies suggest that the functional pump is assembled with a stoichiometry of 3:6:3.
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Advances in the mass spectrometry of membrane proteins: from individual proteins to intact complexes.
Annu. Rev. Biochem.
PUBLISHED: 05-10-2011
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Rapid advances in structural genomics and in large-scale proteomic projects have yielded vast amounts of data on soluble proteins and their complexes. Despite these advances, progress in studying membrane proteins using mass spectrometry (MS) has been slow. This is due in part to the inherent solubility and dynamic properties of these proteins, but also to their low abundance and the absence of polar side chains in amino acid residues. Considerable progress in overcoming these challenges is, however, now being made for all levels of structural characterization. This progress includes MS studies of the primary structure of membrane proteins, wherein sophisticated enrichment and trapping procedures are allowing multiple posttranslational modifications to be defined through to the secondary structure level in which proteins and peptides have been probed using hydrogen exchange, covalent, or radiolytic labeling methods. Exciting possibilities now exist to go beyond primary and secondary structure to reveal the tertiary and quaternary interactions of soluble and membrane subunits within intact assemblies of more than 700 kDa.
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Acetylation of lysine 120 of p53 endows DNA-binding specificity at effective physiological salt concentration.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 04-27-2011
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Lys120 in the DNA-binding domain (DBD) of p53 becomes acetylated in response to DNA damage. But, the role and effects of acetylation are obscure. We prepared p53 specifically acetylated at Lys120, AcK120p53, by in vivo incorporation of acetylated lysine to study biophysical and structural consequences of acetylation that may shed light on its biological role. Acetylation had no affect on the overall crystal structure of the DBD at 1.9-? resolution, but significantly altered the effects of salt concentration on specificity of DNA binding. p53 binds DNA randomly in vitro at effective physiological salt concentration and does not bind specifically to DNA or distinguish among its different response elements until higher salt concentrations. But, on acetylation, AcK120p53 exhibited specific DNA binding and discriminated among response elements at effective physiological salt concentration. AcK120p53 and p53 had the highest affinity to the same DNA sequence, although acetylation reduced the importance of the consensus C and G at positions 4 and 7, respectively. Mass spectrometry of p53 and AcK120p53 DBDs bound to DNA showed they preferentially segregated into complexes that were either DNA(p53DBD)(4) or DNA(AcK120DBD)(4), indicating that the different DBDs prefer different quaternary structures. These results are consistent with electron microscopy observations that p53 binds to nonspecific DNA in different, relaxed, quaternary states from those bound to specific sequences. Evidence is accumulating that p53 can be sequestered by random DNA, and target search requires acetylation of Lys120 and/or interaction with other factors to impose specificity of binding via modulating changes in quaternary structure.
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?B-crystallin polydispersity is a consequence of unbiased quaternary dynamics.
J. Mol. Biol.
PUBLISHED: 04-25-2011
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The inherent heterogeneity of many protein assemblies complicates characterization of their structure and dynamics, as most biophysical techniques require homogeneous preparations of isolated components. For this reason, quantitative studies of the molecular chaperone ?B-crystallin, which populates a range of interconverting oligomeric states, have been difficult, and the physicochemical basis for its polydispersity has remained unknown. Here, we perform mass spectrometry experiments to study ?B-crystallin and extract detailed information as to its oligomeric distribution and exchange of subunits under a range of conditions. This allows a determination of the thermodynamic and kinetic parameters that govern the polydisperse ensemble and enables the construction of a simple energy profile for oligomerization. We find that the quaternary structure and dynamics of the protein can be explained using a simple model with just two oligomer-independent interactions (i.e., interactions that are energetically identical in all oligomers from 10mers to 40mers) between constituent monomers. As such, the distribution of oligomers is governed purely by the dynamics of individual monomers. This provides a new means for understanding the polydispersity of ?B-crystallin and a framework for interrogating other heterogeneous protein assemblies.
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Diabetic retinopathy is related to both endothelium-dependent and -independent responses of skin microvascular flow.
Diabetes Care
PUBLISHED: 04-22-2011
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Endothelial dysfunction has been hypothesized as a possible pathogenic factor in the development of diabetic retinopathy (DR). We examined the relationship of DR to endothelium-dependent and endothelium-independent responses in skin microvascular flow.
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Multimeric assembly and biochemical characterization of the Trax-translin endonuclease complex.
Nat. Struct. Mol. Biol.
PUBLISHED: 04-13-2011
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Trax-translin heteromers, also known as C3PO, have been proposed to activate the RNA-induced silencing complex (RISC) by facilitating endonucleolytic cleavage of the siRNA passenger strand. We report on the crystal structure of hexameric Drosophila C3PO formed by truncated translin and Trax, along with electron microscopic and mass spectrometric studies on octameric C3PO formed by full-length translin and Trax. Our studies establish that Trax adopts the translin fold, possesses catalytic centers essential for C3POs endoRNase activity and interacts extensively with translin to form an octameric assembly. The catalytic pockets of Trax subunits are located within the interior chamber of the octameric scaffold. Truncated C3PO, like full-length C3PO, shows endoRNase activity that leaves 3-hydroxyl-cleaved ends. We have measured the catalytic activity of C3PO and shown it to cleave almost stoichiometric amounts of substrate per second.
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A?40 and A?42 amyloid fibrils exhibit distinct molecular recycling properties.
J. Am. Chem. Soc.
PUBLISHED: 04-12-2011
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A critical aspect to understanding the molecular basis of Alzheimers disease (AD) is the characterization of the kinetics of interconversion between the different species present during amyloid-? protein (A?) aggregation. By monitoring hydrogen/deuterium exchange in A? fibrils using electrospray ionization mass spectrometry, we demonstrate that the A? molecules comprising the fibril continuously dissociate and reassociate, resulting in molecular recycling within the fibril population. Investigations on A?40 and A?42 amyloid fibrils reveal that molecules making up A?40 fibrils recycle to a much greater extent than those of A?42. By examining factors that could influence molecular recycling and by running simulations, we show that the rate constant for dissociation of molecules from the fibril (k(off)) is much greater for A?40 than that for A?42. Importantly, the k(off) values obtained for A?40 and A?42 reveal that recycling occurs on biologically relevant time scales. These results have implications for understanding the role of A? fibrils in neurotoxicity and for designing therapeutic strategies against AD.
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Architecture and dynamics of an A-kinase anchoring protein 79 (AKAP79) signaling complex.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 04-04-2011
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A-kinase anchoring protein 79 (AKAP79) is a human anchoring protein that organizes cAMP-dependent protein kinase (PKA), Ca(2+)/calmodulin (CaM)-dependent protein phosphatase (PP2B), and protein kinase C (PKC) for phosphoregulation of synaptic signaling. Quantitative biochemical analyses of selected AKAP79 complexes have determined the quaternary structure of these signaling complexes. We show that AKAP79 dimerizes, and we demonstrate that, upon addition of a lysine-reactive cross-linker, parallel homomeric dimers are stabilized through K328-K328 and K333-K333 cross-links. An assembly of greater complexity comprising AKAP79, PP2B, a type II regulatory subunit fragment (RII 1-45) of PKA, and CaM was reconstituted in vitro. Using native MS, we determined the molecular mass of this complex as 466 kDa. This indicates that dimeric AKAP79 coordinates two RII 1-45 homodimers, four PP2B heterodimers, and two CaM molecules. Binding of Ca(2+)/CaM to AKAP79 stabilizes the complex by generating a second interface for PP2B. This leads to activation of the anchored phosphatases. Our architectural model reveals how dimeric AKAP79 concentrates pockets of second messenger responsive enzyme activities at the plasma membrane.
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Protein-nucleic acid complexes and the role of mass spectrometry in their structure determination.
Crit. Rev. Biochem. Mol. Biol.
PUBLISHED: 03-23-2011
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Mass spectrometry is now established as a powerful tool for the study of the stoichiometry, interactions, dynamics, and subunit architecture of large protein assemblies and their subcomplexes. Recent evidence has suggested that the 3D structure of protein complexes can be maintained intact in the gas phase, highlighting the potential of ion mobility to contribute to structural biology. A key challenge is to integrate the compositional and structural information from ion mobility mass spectrometry with molecular modelling approaches to produce 3D models of intact protein complexes. In this review, we focus on the mass spectrometry of protein-nucleic acid assemblies with particular attention to the application of ion mobility, an emerging technique in structural studies. We also discuss the challenges that lie ahead for the full integration of ion mobility mass spectrometry with structural biology.
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Tandem differential mobility analysis-mass spectrometry reveals partial gas-phase collapse of the GroEL complex.
J Phys Chem B
PUBLISHED: 03-11-2011
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A parallel-plate differential mobility analyzer and a time-of-flight mass spectrometer (DMA-MS) are used in series to measure true mobility in dry atmospheric pressure air for mass-resolved electrosprayed GroEL tetradecamers (14-mers; ~800 kDa). Narrow mobility peaks are found (2.6-2.9% fwhm); hence, precise mobilities can be obtained for these ions without collisional activation, just following their generation by electrospray ionization. In contrast to previous studies, two conformers are found with mobilities (Z) differing by ~5% at charge state z ~ 79. By extrapolating to small z, a common mobility/charge ratio Z(0)/z = 0.0117 cm(2) V(-1) s(-1) is found for both conformers. When interpreted as if the GroEL ion surface were smooth and the gas molecule-protein collisions were perfectly elastic and specular, this mobility yields an experimental collision cross section, ?, 11% smaller than in an earlier measurement, and close to the cross section, A(C,crystal), expected for the crystal structure (determined by a geometric approximation). However, the similarity between ? and A(C,crystal) does not imply a coincidence between the native and gas-phase structures. The nonideal nature of protein-gas molecule collisions introduces a drag enhancement factor, ? = 1.36, with which the true cross section A(C) is related to ? via A(C) = ?/?. Therefore, A(C) for GroEL 14-mer ions determined by DMA measurements is 0.69A(C,crystal). The factor 1.36 used here is based on the experimental Stokes-Millikan equation, as well as on prior and new numerical modeling accounting for multiple scattering events via exact hard-sphere scattering calculations. Therefore, we conclude that the gas-phase structure of the GroEL complex as electrosprayed is substantially more compact than the corresponding X-ray crystal structure.
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Assembly states of the nucleosome assembly protein 1 (NAP-1) revealed by sedimentation velocity and non-denaturing MS.
Biochem. J.
PUBLISHED: 02-26-2011
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Proteins often exist as ensembles of interconverting states in solution which are often difficult to quantify. In the present manuscript we show that the combination of MS under nondenaturing conditions and AUC-SV (analytical ultracentrifugation sedimentation velocity) unambiguously clarifies a distribution of states and hydrodynamic shapes of assembled oligomers for the NAP-1 (nucleosome assembly protein 1). MS established the number of associated units, which was utilized as input for the numerical analysis of AUC-SV profiles. The AUC-SV analysis revealed that less than 1% of NAP-1 monomer exists at the micromolar concentration range and that the basic assembly unit consists of dimers of yeast or human NAP-1. These dimers interact non-covalently to form even-numbered higher-assembly states, such as tetramers, hexamers, octamers and decamers. MS and AUC-SV consistently showed that the formation of the higher oligomers was suppressed with increasing ionic strength, implicating electrostatic interactions in the formation of higher oligomers. The hydrodynamic shapes of the NAP-1 tetramer estimated from AUC-SV agreed with the previously proposed assembly models built using the known three-dimensional structure of yeast NAP-1. Those of the hexamer and octamer could be represented by new models shown in the present study. Additionally, MS was used to measure the stoichiometry of the interaction between the human NAP-1 dimer and the histone H2A-H2B dimer or H3-H4 tetramer. The present study illustrates a rigorous procedure for the analysis of protein assembly and protein-protein interactions in solution.
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Interaction of the p53 DNA-binding domain with its n-terminal extension modulates the stability of the p53 tetramer.
J. Mol. Biol.
PUBLISHED: 02-11-2011
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The tetrameric tumor suppressor p53 plays a pivotal role in the control of the cell cycle and provides a paradigm for an emerging class of oligomeric, multidomain proteins with structured and intrinsically disordered regions. Many of its biophysical and functional properties have been extrapolated from truncated variants, yet the exact structural and functional role of certain segments of the protein is unclear. We found from NMR and X-ray crystallography that the DNA-binding domain (DBD) of human p53, usually defined as residues 94-292, extends beyond these domain boundaries. Trp91, in the hinge region between the disordered proline-rich N-terminal domain and the DBD, folds back onto the latter and has a cation-? interaction with Arg174. These additional interactions increase the melting temperature of the DBD by up to 2 °C and inhibit aggregation of the p53 tetramer. They also modulate the dissociation of the p53 tetramer. The absence of the Trp91/Arg174 packing presumably allows nonnative DBD-DBD interactions that both nucleate aggregation and stabilize the interface. These data have important implications for studies of multidomain proteins in general, highlighting the fact that weak ordered-disordered domain interactions can modulate the properties of proteins of complex structure.
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Structural basis for the subunit assembly of the anaphase-promoting complex.
Nature
PUBLISHED: 02-11-2011
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The anaphase-promoting complex or cyclosome (APC/C) is an unusually large E3 ubiquitin ligase responsible for regulating defined cell cycle transitions. Information on how its 13 constituent proteins are assembled, and how they interact with co-activators, substrates and regulatory proteins is limited. Here, we describe a recombinant expression system that allows the reconstitution of holo APC/C and its sub-complexes that, when combined with electron microscopy, mass spectrometry and docking of crystallographic and homology-derived coordinates, provides a precise definition of the organization and structure of all essential APC/C subunits, resulting in a pseudo-atomic model for 70% of the APC/C. A lattice-like appearance of the APC/C is generated by multiple repeat motifs of most APC/C subunits. Three conserved tetratricopeptide repeat (TPR) subunits (Cdc16, Cdc23 and Cdc27) share related superhelical homo-dimeric architectures that assemble to generate a quasi-symmetrical structure. Our structure explains how this TPR sub-complex, together with additional scaffolding subunits (Apc1, Apc4 and Apc5), coordinate the juxtaposition of the catalytic and substrate recognition module (Apc2, Apc11 and Apc10 (also known as Doc1)), and TPR-phosphorylation sites, relative to co-activator, regulatory proteins and substrates.
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Structures of SAS-6 suggest its organization in centrioles.
Science
PUBLISHED: 01-27-2011
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Centrioles are cylindrical, ninefold symmetrical structures with peripheral triplet microtubules strictly required to template cilia and flagella. The highly conserved protein SAS-6 constitutes the center of the cartwheel assembly that scaffolds centrioles early in their biogenesis. We determined the x-ray structure of the amino-terminal domain of SAS-6 from zebrafish, and we show that recombinant SAS-6 self-associates in vitro into assemblies that resemble cartwheel centers. Point mutations are consistent with the notion that centriole formation in vivo depends on the interactions that define the self-assemblies observed here. Thus, these interactions are probably essential to the structural organization of cartwheel centers.
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The catalytic activity of Ubp6 enhances maturation of the proteasomal regulatory particle.
Mol. Cell
PUBLISHED: 01-25-2011
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The 26S proteasome is a 2.5 MDa macromolecular machine responsible for targeted protein degradation. Recently, four chaperones were identified that promote the assembly of the 19S regulatory particle (RP). Here, we probe the dynamic architecture of the proteasome by applying quantitative proteomics and mass spectrometry (MS) of intact complexes to provide a detailed characterization of how Ubp6 assists this assembly process. Our MS data demonstrate stoichiometric binding of chaperones and Ubp6 to the basal part of the RP. Genetic interactions of Ubp6 with Hsm3, but not with the other chaperones, indicate a functional overlay with Hsm3. Our biochemical data identified Ubp6 as an additional member of the Hsm3 module. Deletions of ubp6 with hsm3 perturb 26S proteasome assembly, which we attribute to an accumulation of ubiquitylated substrates on these assembly precursors. We therefore propose that Ubp6 facilitates proteasomal assembly by clearing ubiquitylated substrates from assembly precursors by its deubiquitylating activity.
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Mass spectrometry reveals stable modules in holo and apo RNA polymerases I and III.
Structure
PUBLISHED: 01-12-2011
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RNA polymerases are essential enzymes which transcribe DNA into RNA. Here, we obtain mass spectra of the cellular forms of apo and holo eukaryotic RNA polymerase I and III, defining their composition under different solution conditions. By recombinant expression of subunits within the initiation heterotrimer of Pol III, we derive an interaction network and couple this data with ion mobility data to define topological restraints. Our data agree with available structural information and homology modeling and are generally consistent with yeast two hybrid data. Unexpectedly, elongation complexes of both Pol I and III destabilize the assemblies compared with their apo counterparts. Increasing the pH and ionic strength of apo and holo forms of Pol I and Pol III leads to formation of at least ten stable subcomplexes for both enzymes. Uniquely for Pol III many subcomplexes contain only one of the two largest catalytic subunits. We speculate that these stable subcomplexes represent putative intermediates in assembly pathways.
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Opening of the outer membrane protein channel in tripartite efflux pumps is induced by interaction with the membrane fusion partner.
J. Biol. Chem.
PUBLISHED: 11-29-2010
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The multiple transferable resistance (MTR) pump, from Neisseria gonorrhoeae, is typical of the specialized machinery used to translocate drugs across the inner and outer membranes of Gram-negative bacteria. It consists of a tripartite complex composed of an inner-membrane transporter, MtrD, a periplasmic membrane fusion protein, MtrC, and an outer-membrane channel, MtrE. We have expressed the components of the pump in Escherichia coli and used the antibiotic vancomycin, which is too large to cross the outer-membrane by passive diffusion, to test for opening of the MtrE channel. Cells expressing MtrCDE are not susceptible to vancomycin, indicating that the channel is closed; but become susceptible to vancomycin in the presence of transported substrates, consistent with drug-induced opening of the MtrE channel. A mutational analysis identified residues Asn-198, Glu-434, and Gln-441, lining an intraprotomer groove on the surface of MtrE, to be important for pump function; mutation of these residues yielded cells that were sensitive to vancomycin. Pull-down assays and micro-calorimetry measurements indicated that this functional impairment is not due to the inability of MtrC to interact with the MtrE mutants; nor was it due to the MtrE mutants adopting an open conformation, because cells expressing these MtrE mutants alone are relatively insensitive to vancomycin. However, cells expressing the MtrE mutants with MtrC are sensitive to vancomycin, indicating that residues lining the intra-protomer groove control opening of the MtrE channel in response to binding of MtrC.
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Trapping of palindromic ligands within native transthyretin prevents amyloid formation.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 11-08-2010
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Transthyretin (TTR) amyloidosis is a fatal disease for which new therapeutic approaches are urgently needed. We have designed two palindromic ligands, 2,2-(4,4-(heptane-1,7-diylbis(oxy))bis(3,5-dichloro-4,1-phenylene)) bis(azanediyl)dibenzoic acid (mds84) and 2,2-(4,4-(undecane-1,11-diylbis(oxy))bis(3,5-dichloro-4,1-phenylene)) bis(azanediyl)dibenzoic acid (4ajm15), that are rapidly bound by native wild-type TTR in whole serum and even more avidly by amyloidogenic TTR variants. One to one stoichiometry, demonstrable in solution and by MS, was confirmed by X-ray crystallographic analysis showing simultaneous occupation of both T4 binding sites in each tetrameric TTR molecule by the pair of ligand head groups. Ligand binding by native TTR was irreversible under physiological conditions, and it stabilized the tetrameric assembly and inhibited amyloidogenic aggregation more potently than other known ligands. These superstabilizers are orally bioavailable and exhibit low inhibitory activity against cyclooxygenase (COX). They offer a promising platform for development of drugs to treat and prevent TTR amyloidosis.
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Ion mobility-mass spectrometry reveals the influence of subunit packing and charge on the dissociation of multiprotein complexes.
Anal. Chem.
PUBLISHED: 11-05-2010
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The composition, stoichiometry, and organization of protein complexes can be determined by collision-induced dissociation (CID) coupled to tandem mass spectrometry (MS/MS). The increased use of this approach in structural biology prompts a better understanding of the dissociation mechanism(s). Here we report a detailed investigation of the CID of two dodecameric, heat-stable and toroidally shaped complexes: heat shock protein 16.9 (HSP16.9) and stable protein 1 (SP-1). While HSP16.9 dissociates by sequential loss of unfolded monomers, SP-1 ejects not only monomers, but also its building blocks (dimers), and multiples thereof (tetramers and hexamers). Unexpectedly, the dissociation of SP-1 is strongly charge-dependent: loss of the building blocks increases with higher charge states of this complex. By combining MS/MS with ion mobility (IM-MS/MS), we have monitored the unfolding and dissociation events for these complexes in the gas phase. For HSP16.9 unfolding occurs at lower energies than the ejection of subunits, whereas for SP-1 unfolding and dissociation take place simultaneously. We consider these results in the light of the structural organization of HSP16.9 and SP-1 and hypothesize that SP-1 is unable to unfold extensively due to its particular quaternary structure and unusually high charge density. This investigation increases our understanding of the factors governing the CID of protein complexes and moves us closer to the goal of obtaining structural information on subunit interactions and packing from gas-phase experiments.
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