structure-based-simulation-sampling-transcription-factor-protein

Structure-Based Simulation and Sampling of Transcription Factor Protein Movements along DNA from Atomic-Scale Stepping to Coarse-Grained Diffusion

One-dimensional (1-D) sliding of transcription factor (TF) protein along DNA is essential for facilitated diffusion of the TF to locate target DNA site ...

Shanghai Jiao Tong University

Taste receptor T1R1-T1R3 can be activated by binding to several natural ligands, e.g., l-glutamate and 5'-ribonucleotides etc., thereby stimulating the umami taste. The molecular mechanism of umami recognition at atomic details, however, remains elusive. Here, using homology modeling, molecular docking and molecular dynamics (MD) simulations, we investigate the effects of five natural umami ligands on the structural dynamics of T1R1-T1R3. Our work identifies the key residues that are directly involved in recognizing the binding ligands. In addition, two adjacent binding sites in T1R1 are determined for substrate binding, and depending on the molecular size and chemical properties of the incoming ligand, one or both binding sites can be occupied. More interestingly, the ligand binding can modulate the pocket size, which is likely correlated with the closing and opening motions of T1R1. We then classify these five ligands into two groups according to their different binding effects on T1R1, which likely associate with the distinct umami signals stimulated by various ligands. This work warrants new experimental assays to further validate the theoretical model and provides guidance to design more effective umami ligands.

Understanding the molecular mechanism of umami recognition by T1R1-T1R3 using molecular dynamics simulations.

Ellagitannin-derived ellagic acid (EA) and colonic metabolite urolithins are functional dietary ingredients for cancer prevention, but the underlying mechanism need elucidation. Mucin-type O-glycosylation, initiated by polypeptide N-acetyl-α-galactosaminyltransferases (ppGalNAc-Ts), fine-tunes multiple biological processes and is closely associated with cancer progression. Herein, we aim to explore how specific tannin-based polyphenols affect tumor behavior of colorectal cancer cells (CRC) by modulating O-glycosylation. Utilizing HPLC-based enzyme assay, we find urolithin D (UroD), EA and gallic acid (GA) potently inhibit ppGalNAc-Ts. In particular, UroD inhibits ppGalNAc-T2 through a peptide/protein-competitive manner with nanomolar affinity. Computational simulations combined with site-directed mutagenesis further support the inhibitors' mode of action. Moreover, lectin analysis and metabolic labelling reveal that UroD can reduce cell O-glycans but not N-glycans. Transwell experiments prove that UroD inhibits migration and invasion of CRC cells. Our work proves that specific tannin-based polyphenols can potently inhibit ppGalNAc-Ts activity to reduce cell O-glycosylation and lead to lowering the migration and invasion of CRC cells, suggesting that disturbance of mucin-type O-glycosylation is an important mechanism for the function of dietary polyphenols.

Inhibition of polypeptide N-acetyl-α-galactosaminyltransferases is an underlying mechanism of dietary polyphenols preventing colorectal tumorigenesis.

The known benzonaphthyridine alkaloid, albogrisin A (), and six new compounds, including two pyrazinone stereoisomers, albogrisin B ()/B' (), together with four 4-pyrroloquinolinones, two diastereoisomers, albogrisin C ()/C' (), and their methyl esters, albogrisin D ()/D' (), were isolated from mangrove-derived  MGR072.  and  are converted into  in acidic aqueous solution but into / and / in 0.05% trifluoroacetic acid acetonitrile.  and  are new indoleamine 2,3-dioxygenase 1 inhibitors.

Characterization and Nonenzymatic Transformation of Three Types of Alkaloids from  MGR072 and Discovery of Inhibitors of Indoleamine 2,3-Dioxygenase.

HIV-1 entry is mediated firstly by the molecular recognition between the viral glycoprotein gp120 and its receptor CD4 on host T-cells. As a key antigen that can be targeted by neutralizing antibodies, gp120 has been a focus for extensive studies with efforts to understand its structural properties and conformational dynamics upon receptor binding. An atomistic-level revelation of gp120 opening dynamics activated by CD4, however, is still unknown. Here, by constructing a Markov State Model (MSM) based on hundreds of Molecular Dynamics (MD) simulations with an aggregated simulation time of ∼20 microseconds (μs), we identify the key metastable states of gp120 during its opening dynamics upon CD4 binding. The MSM provides a clear dynamic model whereby the identified metastable states coexist and can reach an equilibrium. More importantly, a hydrophobic core flanked by variable loops (V1V2 and V3) and the β20/21 region plays an essential role in triggering the gp120 opening. Any destabilizing effects introduced into the hydrophobic core, therefore, can be expected to promote transition of gp120 to an open state. Moreover, the variable loops demonstrate high flexibilities in fully open gp120. In particular, the V3 region is capable of exploring both closed and open conformations, even with the V1/V2 loops largely adopting an open form. In addition, the bridging sheet formation in gp120 is likely induced by the incoming co-receptor/antibody recognitions, since the V1/V2 structure is highly heterogeneous so that the bridging-sheet formed conformation is not the most populated state. Our studies provide deep insights into the dynamic features of gp120 and its molecular recognitions to the broadly neutralizing antibodies, which guides future attempts to design more effective gp120 immunogens.

Opening dynamics of HIV-1 gp120 upon receptor binding is dictated by a key hydrophobic core.

XimA is a unique amide synthetase that belongs to the ANL superfamily of adenylating enzymes, but with a special structural fold. In order to improve the enzyme promiscuity, we engineered XimA by site-directed mutagenesis at a specific position based on our theoretical model of XimA. Thus, we were able to produce diverse benzopyran derivatives with up to 15 different l-form and d-form amino acid substitutions, catalyzed by several XimA variants. Molecular docking and molecular dynamics simulations conducted for various XimA systems provide further structural insights into the substitution effects of the phenylalanine-201 as an active site residue on protein dynamics and enzyme catalysis.

Rational engineering of amide synthetase enables bioconversion to diverse xiamenmycin derivatives.

The HIV-1 infection is triggered by the binding of the viral envelope glycoprotein (Env) gp120-gp41 trimer to host-cell receptor CD4 and co-receptor CCR5/CXCR4, which leads to substantial conformational changes of Env, that is, structural transition of gp120 from a closed to an open state followed by gp41 refolding from pre-fusion to post-fusion states. The latter finally promotes membrane fusion, likely via visiting a critical pre-hairpin state of gp41. The complete conformational dynamics of the pre-hairpin formation at atomic resolution, however, is still unknown. Here, by constructing a Markov state model based on the all-atom molecular dynamics (MD) with an aggregated simulation time of ∼24 μs, we reveal the gp41 refolding dynamics from pre-fusion to pre-hairpin state and the key metastable states involved. Moreover, we further explored the drug resistance mechanism of two C-terminal heptad repeat-derived gp41 inhibitors, T20 and sifuvirtide, based on the constructed inhibitor-bound gp41 pre-hairpin complexes. The results indicate that these two inhibitors have distinct binding sites on gp41 but share a common drug resistance region that usually exhibits a helical structure in the pre-hairpin state yet adopts various secondary structures in other metastable states. Moreover, we conducted several mutant MD simulations to further investigate the mechanisms of how some drug-resistant mutations might affect the pre-hairpin formation, which in turn prevent the inhibitor recognition. Our findings provide deep structural insights into the molecular mechanisms of the pre-hairpin formation for gp41, which helps to guide future anti-HIV drug design.

Refolding Dynamics of gp41 from Pre-fusion to Pre-hairpin States during HIV-1 Entry.

Mangrove-derived actinobacteria strains are well-known for producing novel secondary metabolites. The polycyclic tetramate macrolactam (PTM), ikarugamycin (IKA) isolated from Streptomyces xiamenensis 318, exhibits antiproliferative activities against pancreatic ductal adenocarcinoma (PDAC) in vitro. However, the protein target for bioactive IKA is unclear. In this study, whole transcriptome-based profiling revealed that the glycolysis pathway is significantly affected by IKA. Metabolomic studies demonstrated that IKA treatment induces a significant drop in glucose-6-phosphate and a slight increase in intracellular glucose level. Analysis of glucose consumption, lactate production, and the extracellular acidification rate confirmed the inhibitory role of IKA on the glycolytic flux in PDAC cells. Surface plasmon resonance (SPR) experiments and docking studies identified the key enzyme of glycolysis, hexokinase 2 (HK2), as a molecular target of IKA. Moreover, IKA reduced tumor size without overt cytotoxicity in mice with PDAC xenografts and increased chemotherapy response to gemcitabine in PDAC cells in vitro. Taken together, IKA can block glycolysis in pancreatic cancer by targeting HK2, which may be a potential drug candidate for PDAC treatment.

Ikarugamycin inhibits pancreatic cancer cell glycolysis by targeting hexokinase 2.

Knowledge of how DNA bending facilitates the target-base searching by Thymine DNA glycosylase (TDG) is of major importance for unraveling the recognition mechanism between DNA and TDG in DNA repair process. An atomic-level understanding of the initial encounter between TDG and DNA before base-flipping, however, is still elusive. Here, we employ all-atom molecular dynamics (MD) simulations with an integrated simulation time of ∼3 μs to investigate how TDG responses to different DNA bending conformations. By constructing several TDG-DNA complexes with varied DNA bend angles (ranging from ∼0° to 60°), we pinpoint the key TDG motifs responsible for recognizing certain DNA bending conformations. Particularly, several positively charged residues, i.e., Lys232, Lys240, and Lys246, are critical for the tight binding with DNA backbones. Importantly, the roll-angle patterns, rather than the tilt and twist angles, are found to be strongly correlated with the extent of DNA bending, which in turn, governs the TDG recognition. Further comparisons between the naked and TDG-bound DNA conformations reveal that the TDG binding can impose a substantial DNA deformation, resulting in profound roll-angle alterations. Our studies warrant further experimental validations and provide deep structural insights into the recognition mechanism between TDG and DNA during their initial encounter.

Key structural motifs in Thymine DNA glycosylase responsible for recognizing certain DNA bent conformation revealed by atomic simulations.

The mutations of cancers can encode the seeds of their own destruction, in the form of T-cell recognizable immunogenic peptides, also known as neoantigens. It is computationally challenging, however, to accurately prioritize the potential neoantigen candidates according to their ability of activating the T-cell immunoresponse, especially when the somatic mutations are abundant. Although a few neoantigen prioritization methods have been proposed to address this issue, advanced machine learning model that is specifically designed to tackle this problem is still lacking. Moreover, none of the existing methods considers the original DNA loci of the neoantigens in the perspective of 3D genome which may provide key information for inferring neoantigens' immunogenicity.

DeepAntigen: a novel method for neoantigen prioritization via 3D genome and deep sparse learning.

High-throughput sequencing technology has yielded reliable and ultra-fast sequencing for DNA and RNA. For tumor cells of cancer patients, when combining the results of DNA and RNA sequencing, one can identify potential neoantigens that stimulate the immune response of the T cell. However, when the somatic mutations are abundant, it is computationally challenging to efficiently prioritize the identified neoantigen candidates according to their ability of activating the T cell immuno-response.

A novel neoantigen discovery approach based on chromatin high order conformation.

Thymine DNA glycosylase (TDG), as a repair enzyme, plays essential roles in maintaining the genome integrity by correcting several mismatched/damaged nucleobases. TDG acquires an efficient strategy to search for the lesions among a vast number of cognate base pairs. Currently, atomic-level details of how TDG translocates along DNA as it approaches the lesion site and the molecular mechanisms of the interplay between TDG and DNA are still elusive. Here, by constructing the Markov state model based on hundreds of molecular dynamics simulations with an integrated simulation time of ∼25 μs, we reveal the rotation-coupled sliding dynamics of TDG along a 9 bp DNA segment containing one G·T mispair. We find that TDG translocates along DNA at a relatively faster rate when distant from the lesion site, but slows down as it approaches the target, accompanied by deeply penetrating into the minor-groove, opening up the mismatched base pair and significantly sculpturing the DNA shape. Moreover, the electrostatic interactions between TDG and DNA are found to be critical for mediating the TDG translocation. Notably, several uncharacterized TDG residues are identified to take part in regulating the conformational switches of TDG occurred in the site-transfer process, which warrants further experimental validations.

Atomic resolution of short-range sliding dynamics of thymine DNA glycosylase along DNA minor-groove for lesion recognition.

Repressor element-1 silencing transcription factor (REST) or neuron-restrictive silencer factor (NRSF) is a zinc-finger (ZF) containing transcriptional repressor that recognizes thousands of neuron-restrictive silencer elements (NRSEs) in mammalian genomes. How REST/NRSF regulates gene expression remains incompletely understood. Here, we investigate the binding pattern and regulation mechanism of REST/NRSF in the clustered protocadherin (PCDH) genes. We find that REST/NRSF directionally forms base-specific interactions with NRSEs via tandem ZFs in an anti-parallel manner but with striking conformational changes. In addition, REST/NRSF recruitment to the HS5-1 enhancer leads to the decrease of long-range enhancer-promoter interactions and downregulation of the clustered PCDHα genes. Thus, REST/NRSF represses PCDHα gene expression through directional binding to a repertoire of NRSEs within the distal enhancer and variable target genes.

Mechanism of REST/NRSF regulation of clustered protocadherin α genes.

The RecQ family of helicases are important for maintenance of genomic integrity. Although functions of constructive subdomains of this family of helicases have been extensively studied, the helical hairpin (HH) in the RecQ-C-terminal domain (RQC) has been underappreciated and remains poorly understood. Here by using single-molecule fluorescence resonance energy transfer, we found that HH in the human BLM transiently intercepts different numbers of nucleotides when it is unwinding a double-stranded DNA. Single-site mutations in HH that disrupt hydrogen bonds and/or salt bridges between DNA and HH change the DNA binding conformations and the unwinding features significantly. Our results, together with recent clinical tests that correlate single-site mutations in HH of human BLM with the phenotype of cancer-predisposing syndrome or Bloom's syndrome, implicate pivotal roles of HH in BLM's DNA unwinding activity. Similar mechanisms might also apply to other RecQ family helicases, calling for more attention to the RQC helical hairpin.

RQC helical hairpin in Bloom's syndrome helicase regulates DNA unwinding by dynamically intercepting nascent nucleotides.

Aβ is believed to have critical role in early aggregation of full length amyloids that are associated with the Alzheimer's disease and can aggregate to form amyloid fibrils. However, the early aggregation mechanism is still unsolved. Here, multiple long-term molecular dynamics simulations combining with Markov state model were used to probe the early oligomerization mechanism of Aβ peptides. The identified dimeric form adopted either globular random-coil or extended β-strand like conformations. The observed dimers of these variants shared many overall conformational characteristics but differed in several aspects at detailed level. In all cases, the most common type of secondary structure was intermolecular antiparallel β-sheets. The inter-state transitions were very frequent ranges from few to hundred nanoseconds. More strikingly, those states which contain fraction of β secondary structure and significant amount of extended coiled structures, therefore exposed to the solvent, were majorly participated in aggregation. The assembly of low-energy dimers, in which the peptides form antiparallel β sheets, occurred by multiple pathways with the formation of an obligatory intermediates. We proposed that these states might facilitate the Aβ aggregation through a significant component of the conformational selection mechanism, because they might increase the aggregates population by promoting the inter-chain hydrophobic and the hydrogen bond contacts. The formation of early stage antiparallel β sheet structures is critical for oligomerization, and at the same time provided a flat geometry to seed the ordered β-strand packing of the fibrils. Our findings hint at reorganization of this part of the molecule as a potentially critical step in Aβ aggregation and will insight into early oligomerization for large β amyloids.

Article	Year
Understanding the molecular mechanism of umami recognition by T1R1-T1R3 using molecular dynamics simulations. Biochemical and biophysical research communications\| PubMed ID: 31092329	2019
Inhibition of polypeptide N-acetyl-α-galactosaminyltransferases is an underlying mechanism of dietary polyphenols preventing colorectal tumorigenesis. Bioorganic & medicinal chemistry\| PubMed ID: 31227364	2019
Characterization and Nonenzymatic Transformation of Three Types of Alkaloids from MGR072 and Discovery of Inhibitors of Indoleamine 2,3-Dioxygenase. Organic letters\| PubMed ID: 31613107	2019
Opening dynamics of HIV-1 gp120 upon receptor binding is dictated by a key hydrophobic core. Physical chemistry chemical physics : PCCP\| PubMed ID: 31764922	2019
Rational engineering of amide synthetase enables bioconversion to diverse xiamenmycin derivatives. Chemical communications (Cambridge, England)\| PubMed ID: 31768510	2019
Refolding Dynamics of gp41 from Pre-fusion to Pre-hairpin States during HIV-1 Entry. Journal of chemical information and modeling\| PubMed ID: 31845803	2020
Ikarugamycin inhibits pancreatic cancer cell glycolysis by targeting hexokinase 2. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 31944405	2020
Key structural motifs in Thymine DNA glycosylase responsible for recognizing certain DNA bent conformation revealed by atomic simulations. Biochemical and biophysical research communications\| PubMed ID: 32291075	2020
DeepAntigen: a novel method for neoantigen prioritization via 3D genome and deep sparse learning. Bioinformatics (Oxford, England)\| PubMed ID: 32592462	2020
A novel neoantigen discovery approach based on chromatin high order conformation. BMC medical genomics\| PubMed ID: 32854726	2020
Atomic resolution of short-range sliding dynamics of thymine DNA glycosylase along DNA minor-groove for lesion recognition. Nucleic acids research\| PubMed ID: 33469643	2021
Mechanism of REST/NRSF regulation of clustered protocadherin α genes. Nucleic acids research\| PubMed ID: 33849071	2021
RQC helical hairpin in Bloom's syndrome helicase regulates DNA unwinding by dynamically intercepting nascent nucleotides. iScience\| PubMed ID: 35005551	2022
Early aggregation mechanism of Aβ revealed by Markov state models. International journal of biological macromolecules\| PubMed ID: 35134456	2022

Lin-Tai Da

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