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In JoVE (1)
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Articles by Tommi A. White in JoVE
JoVE Immunology and Infection
Fastställande av molekylära strukturer av HIV Envelope Glycoproteins med Cryo-elektrontomografi och automatisk Sub-tomogram Snittar
1Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 2The Medical Research Council Mitochondrial Biology Unit, University of Cambridge, 3National Library of Medicine, National Institutes of Health, 4Massachusetts Institute of Technology, 5William Fremd High School, 6University of Virginia, 7Duke University, 8Yale University, 9University of Notre Dame, 10Washington University in St. Louis, 11Bioinformatics and Computational Biosciences Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12Thomas Jefferson High School for Science and Technology
Protokollet beskriver en hög genomströmning metod för att fastställa strukturer av membranproteiner med Cryo-elektron tomografi och 3D bildbehandling. Den omfattar uppgifter om extraktion, datainsamling, databearbetning och tolkning, och avslutar med produktionen av en representant mål för strategin, HIV-1 kuvert glykoprotein. Dessa computational förfaranden är utformade på ett sätt som gör att forskare och studenter att arbeta på distans och bidra till databehandling och strukturell analys.
Other articles by Tommi A. White on PubMed
Acute and Chronic Treatment of Ob/ob and Db/db Mice with AICAR Decreases Blood Glucose Concentrations
The enzyme 5'AMP-activated protein kinase (AMPK) is activated by increases in intracellular AMP concentration through a complex interaction of phosphorylation and allosteric regulation. Actions of AMPK elucidated thus far suggest that AMPK may be a viable target for pharmacologic intervention in type II diabetes. Activation of AMPK is believed to mediate both the acute increase in skeletal muscle glucose uptake during exercise, as well as the adaptive responses to chronic exercise such as regulation of expression of components of the muscle glucose uptake system. In addition, AMPK is known to inhibit key enzymes involved in lipid and cholesterol synthesis, suggesting that activation of this kinase may also ameliorate dyslipidemia. To investigate the effects of AMPK activation in animal models of type II diabetes, db/db and ob/ob mice were administered 5-aminoimidazole-4-carboxamide 1-beta-ribofuranoside (AICAR) subcutaneously either acutely (single injection) or twice per day for 8 days (chronic treatment). Blood glucose was lowered transiently in both db/db and ob/ob mice by acute AICAR treatment, returning to basal levels approximately 3 h after AICAR administration. In response to chronic treatment, blood glucose (measured 18 h post-AICAR administration) was significantly decreased in both mouse models when compared to vehicle control groups, with morning blood glucose values on Day 8 being decreased approximately 30-35% in both mouse models. Chronic AICAR administration also resulted in an elevation of total Glut4 concentration in skeletal muscle from ob/ob mice, but not db/db mice. In contrast to the beneficial effects on glucose metabolism, AICAR treatment of db/db and ob/ob mice led to approximately a 2.5-3-fold increase in serum triglyceride levels compared to vehicle-treated controls. These data suggest that pharmacological activation of AMPK may enhance glucose uptake in individuals with type II diabetes, however, this benefit may be offset by the concomitant elevation in triglycerides.
Structures of the Escherichia Coli PutA Proline Dehydrogenase Domain in Complex with Competitive Inhibitors
Proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the flavin-dependent oxidation of proline to Delta(1)-pyrroline-5-carboxylate. Here we present a structure-based study of the PRODH active site of the multifunctional Escherichia coli proline utilization A (PutA) protein using X-ray crystallography, enzyme kinetic measurements, and site-directed mutagenesis. Structures of the PutA PRODH domain complexed with competitive inhibitors acetate (K(i) = 30 mM), L-lactate (K(i) = 1 mM), and L-tetrahydro-2-furoic acid (L-THFA, K(i) = 0.2 mM) have been determined to high-resolution limits of 2.1-2.0 A. The discovery of acetate as a competitive inhibitor suggests that the carboxyl is the minimum functional group recognized by the active site, and the structures show how the enzyme exploits hydrogen-bonding and nonpolar interactions to optimize affinity for the substrate. The PRODH/L-THFA complex is the first structure of PRODH with a five-membered ring proline analogue bound in the active site and thus provides new insights into substrate recognition and the catalytic mechanism. The ring of L-THFA is nearly parallel to the middle ring of the FAD isoalloxazine, with the inhibitor C5 atom 3.3 A from the FAD N5. This geometry suggests direct hydride transfer as a plausible mechanism. Mutation of conserved active site residue Leu432 to Pro caused a 5-fold decrease in k(cat) and a severe loss in thermostability. These changes are consistent with the location of Leu432 in the hydrophobic core near residues that directly contact FAD. Our results suggest that the molecular basis for increased plasma proline levels in schizophrenic subjects carrying the missense mutation L441P is due to decreased stability of human PRODH2.
Exploring Structurally Conserved Solvent Sites in Protein Families
Protein-bound water molecules are important components of protein structure, and therefore, protein function and energetics. Although structural conservation of solvent has been studied in a few protein families, a lack of suitable computational tools has hindered more comprehensive analyses. Herein we present a semiautomated computational approach for identifying solvent sites that are conserved among proteins sharing a common three-dimensional structure. This method is tested on six protein families: (1) monodomain cytochrome c, (2) fatty-acid binding protein, (3) lactate/malate dehydrogenase, (4) parvalbumin, (5) phospholipase A2, and (6) serine protease. For each family, the method successfully identified previously known conserved solvent sites. Moreover, the method discovered 22 novel conserved solvent sites, some of which have higher degrees of conservation than the previously known sites. All six families studied had solvent sites with more than 90% conservation and these sites were invariably located in regions of the protein with very high sequence conservation. These results suggest that highly conserved solvent sites, by virtue of their proximity to conserved residues, should be considered as one of the defining three-dimensional structural characteristics of protein families and folds.
Structure and Kinetics of Monofunctional Proline Dehydrogenase from Thermus Thermophilus
Proline dehydrogenase (PRODH) and Delta(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH) catalyze the two-step oxidation of proline to glutamate. They are distinct monofunctional enzymes in all eukaryotes and some bacteria but are fused into bifunctional enzymes known as proline utilization A (PutA) in other bacteria. Here we report the first structure and biochemical data for a monofunctional PRODH. The 2.0-A resolution structure of Thermus thermophilus PRODH reveals a distorted (betaalpha)(8) barrel catalytic core domain and a hydrophobic alpha-helical domain located above the carboxyl-terminal ends of the strands of the barrel. Although the catalytic core is similar to that of the PutA PRODH domain, the FAD conformation of T. thermophilus PRODH is remarkably different and likely reflects unique requirements for membrane association and communication with P5CDH. Also, the FAD of T. thermophilus PRODH is highly solvent-exposed compared with PutA due to a 4-A shift of helix 8. Structure-based sequence analysis of the PutA/PRODH family led us to identify nine conserved motifs involved in cofactor and substrate recognition. Biochemical studies show that the midpoint potential of the FAD is -75 mV and the kinetic parameters for proline are K(m) = 27 mm and k(cat) = 13 s(-1). 3,4-Dehydro-l-proline was found to be an efficient substrate, and l-tetrahydro-2-furoic acid is a competitive inhibitor (K(I) = 1.0 mm). Finally, we demonstrate that T. thermophilus PRODH reacts with O(2) producing superoxide. This is significant because superoxide production underlies the role of human PRODH in p53-mediated apoptosis, implying commonalities between eukaryotic and bacterial monofunctional PRODHs.
Structural Basis for the Inactivation of Thermus Thermophilus Proline Dehydrogenase by N-propargylglycine
The flavoenzyme proline dehydrogenase catalyzes the first step of proline catabolism, the oxidation of proline to pyrroline-5-carboxylate. Here we report the first crystal structure of an irreversibly inactivated proline dehydrogenase. The 1.9 A resolution structure of Thermus thermophilus proline dehydrogenase inactivated by the mechanism-based inhibitor N-propargylglycine shows that N5 of the flavin cofactor is covalently connected to the -amino group of Lys99 via a three-carbon linkage, consistent with the mass spectral analysis of the inactivated enzyme. The isoalloxazine ring has a butterfly angle of 25 degrees , which suggests that the flavin cofactor is reduced. Two mechanisms can account for these observations. In both, N-propargylglycine is oxidized to N-propargyliminoglycine. In one mechanism, this alpha,beta-unsaturated iminium compound is attacked by the N5 atom of the now reduced flavin to produce a 1,4-addition product. Schiff base formation between Lys99 and the imine of the 1,4-addition product releases glycine and links the enzyme to the modified flavin. In the second mechanism, hydrolysis of N-propargyliminoglycine yields propynal and glycine. A 1,4-addition reaction with propynal coupled with Schiff base formation between Lys99 and the carbonyl group tethers the enzyme to the flavin via a three-carbon chain. The presumed nonenzymatic hydrolysis of N-propargyliminoglycine and the subsequent rebinding of propynal to the enzyme make the latter mechanism less likely.
Crystal Structure of the Bifunctional Proline Utilization A Flavoenzyme from Bradyrhizobium Japonicum
The bifunctional proline catabolic flavoenzyme, proline utilization A (PutA), catalyzes the oxidation of proline to glutamate via the sequential activities of FAD-dependent proline dehydrogenase (PRODH) and NAD(+)-dependent Delta(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains. Although structures for some of the domains of PutA are known, a structure for the full-length protein has not previously been solved. Here we report the 2.1 A resolution crystal structure of PutA from Bradyrhizobium japonicum, along with data from small-angle x-ray scattering, analytical ultracentrifugation, and steady-state and rapid-reaction kinetics. PutA forms a ring-shaped tetramer in solution having a diameter of 150 A. Within each protomer, the PRODH and P5CDH active sites face each other at a distance of 41 A and are connected by a large, irregularly shaped cavity. Kinetics measurements show that glutamate production occurs without a lag phase, suggesting that the intermediate, Delta(1)-pyrroline-5-carboxylate, is preferably transferred to the P5CDH domain rather than released into the bulk medium. The structural and kinetic data imply that the cavity serves both as a microscopic vessel for the hydrolysis of Delta(1)-pyrroline-5-carboxylate to glutamate semialdehyde and a protected conduit for the transport of glutamate semialdehyde to the P5CDH active site.
Molecular Architectures of Trimeric SIV and HIV-1 Envelope Glycoproteins on Intact Viruses: Strain-dependent Variation in Quaternary Structure
The initial step in target cell infection by human, and the closely related simian immunodeficiency viruses (HIV and SIV, respectively) occurs with the binding of trimeric envelope glycoproteins (Env), composed of heterodimers of the viral transmembrane glycoprotein (gp41) and surface glycoprotein (gp120) to target T-cells. Knowledge of the molecular structure of trimeric Env on intact viruses is important both for understanding the molecular mechanisms underlying virus-cell interactions and for the design of effective immunogen-based vaccines to combat HIV/AIDS. Previous analyses of intact HIV-1 BaL virions have already resulted in structures of trimeric Env in unliganded and CD4-liganded states at ~20 Å resolution. Here, we show that the molecular architectures of trimeric Env from SIVmneE11S, SIVmac239 and HIV-1 R3A strains are closely comparable to that previously determined for HIV-1 BaL, with the V1 and V2 variable loops located at the apex of the spike, close to the contact zone between virus and cell. The location of the V1/V2 loops in trimeric Env was definitively confirmed by structural analysis of HIV-1 R3A virions engineered to express Env with deletion of these loops. Strikingly, in SIV CP-MAC, a CD4-independent strain, trimeric Env is in a constitutively "open" conformation with gp120 trimers splayed out in a conformation similar to that seen for HIV-1 BaL Env when it is complexed with sCD4 and the CD4i antibody 17b. Our findings suggest a structural explanation for the molecular mechanism of CD4-independent viral entry and further establish that cryo-electron tomography can be used to discover distinct, functionally relevant quaternary structures of Env displayed on intact viruses.
Three-dimensional Structures of Soluble CD4-bound States of Trimeric Simian Immunodeficiency Virus Envelope Glycoproteins Determined by Using Cryo-electron Tomography
The trimeric envelope glycoprotein (Env) spikes displayed on the surfaces of simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) virions are composed of three heterodimers of the viral glycoproteins gp120 and gp41. Although binding of gp120 to cell surface CD4 and a chemokine receptor is known to elicit conformational changes in gp120 and gp41, changes in quaternary structure of the trimer have only recently been elucidated. For the HIV-1 BaL isolate, CD4 attachment results in a striking rearrangement of the trimer from a "closed" to an "open" conformation. The effect of CD4 on SIV trimers, however, has not been described. Using cryo-electron tomography, we have now determined molecular architectures of the soluble CD4 (sCD4)-bound states of SIV Env trimers for three different strains (SIVmneE11S, SIVmac239, and SIV CP-MAC). In marked contrast to HIV-1 BaL, SIVmneE11S and SIVmac239 Env showed only minor conformational changes following sCD4 binding. In SIV CP-MAC, where trimeric Env displays a constitutively "open" conformation similar to that seen for HIV-1 BaL Env in the sCD4-complexed state, we show that there are no significant further changes in conformation upon the binding of either sCD4 or 7D3 antibody. The density maps also show that 7D3 and 17b antibodies target epitopes on gp120 that are on opposites sides of the coreceptor binding site. These results provide new insights into the structural diversity of SIV Env and show that there are strain-dependent variations in the orientation of sCD4 bound to trimeric SIV Env.
Computational Separation of Conformational Heterogeneity Using Cryo-electron Tomography and 3D Sub-volume Averaging
We have previously used cryo-electron tomography combined with sub-volume averaging and classification to obtain 3D structures of macromolecular assemblies in cases where a single dominant species was present, and applied these methods to the analysis of a variety of trimeric HIV-1 and SIV envelope glycoproteins (Env). Here, we extend these studies by demonstrating automated, iterative, missing wedge-corrected 3D image alignment and classification methods to distinguish multiple conformations that are present simultaneously. We present a method for measuring the spatial distribution of the vector elements representing distinct conformational states of Env. We identify data processing strategies that allow clear separation of the previously characterized closed and open conformations, as well as unliganded and antibody-liganded states of Env when they are present in mixtures. We show that identifying and removing spikes with the lowest signal-to-noise ratios improves the overall accuracy of alignment between individual Env sub-volumes, and that alignment accuracy, in turn, determines the success of image classification in assessing conformational heterogeneity in heterogeneous mixtures. We validate these procedures for computational separation by successfully separating and reconstructing distinct 3D structures for unliganded and antibody-liganded as well as open and closed conformations of Env present simultaneously in mixtures.
