Current Research in Structural Biology最新文献

The SARS-CoV-2 spike protein presents a surface with enormous membrane binding potential to host tissues and organelles of infected cells. Its exposed trimeric head binds not only the angiotensin-converting enzyme 2 (ACE2), but also host phospholipids which are missing from all existing structures. Hence, the membrane interaction surfaces that mediate viral fusion, entry, assembly and egress remain unclear. Here the spike:membrane docking sites are identified based on membrane optimal docking area (MODA) analysis of 3D structures of spike proteins in closed and open conformations at endocytic and neutral pH levels as well as ligand complexes. This reveals multiple membrane binding sites in the closed spike head that together prefer convex membranes and are modulated by pH, fatty acids and post-translational modifications including glycosylation. The exposure of the various membrane interaction sites adjusts upon domain repositioning within the trimer, allowing formation of intermediate bilayer complexes that lead to the prefusion state while also enabling ACE2 receptor recognition. In contrast, all antibodies that target the spike head would block the membrane docking process that precedes ACE2 recognition. Together this illuminates the engagements of the spike protein with plasma, endocytic, ER or exocytic vesicle membranes that help to drive the cycle of viral infection, and offers novel sites for intervention.

Intercellular adhesion (IcaADBC) operon is necessary for PNAG (Polyβ-1,6-N-acetyl-D-glucosamine) biosynthesis of biofilm formation in Staphylococcus epidermidis. IcaC protein has a wide range of functions in terms of growth phase variation, migration, transposon insertion, PNAG modification, biofilm formation. Unusual TTTA signature motifs were identified from nucleotide sequence. Asparagine-linked glycosylation consensus motifs were identified at position 169 and 240. S. epidermidis was a close evolutionary association with S. haemolyticus and other Staphylococcus spp. Due to the non-availability of crystal structure, protein threading procedure was selected for constructing a full length IcaC three-dimensional structure. QMEANBrane structure quality assessment with model scores −100000 range within predicted integral membrane structure. IcaC motif constitutes 18 transmembrane helix, 37 helix-helix interaction, 8 beta turn, 2 gamma turn. Binding free energy was calculated with their succinate ligand docking form hydrogen bond with critical amino acids showed ΔG score −2.574 ​kJ/mol using Schrödinger. Serine (Ser96), Glutamic acid (Glu99), Tryptophan (Trp191) were active site amino acids form the catalytic core required for O-succinyltransferase function. Molecular dynamics simulation (MDS) was performed to evaluate the stability of IcaC protein and IcaC-Succinate binding complexes with the active site amino acids throughout trajectories captured with time scale 100 ns simulation period using GROMACS 4.5.

Genetic mutations in p53 are frequently associated with many types of cancers that affect its stability and activity through multiple ways. The Ser46 residue present in the transactivation domain2 (TAD2) domain of p53 undergoes phosphorylation that blocks its degradation by MDM2 and leads to cell cycle arrest/apoptosis/necrosis upon intrinsic or extrinsic stresses. On the other hand, unphosphorylated p53 mutants escape cell arrest or death triggered by these molecular signaling axes and lead to carcinogenesis. Phosphorylation of Ser in the TAD2 domain of p53 mediates its interactions with transcription factor p62, yielding transcriptional activation of downstream pro-apoptotic genes. The p53 phosphorylation causes string-like elongated conformation that increases its binding affinity with the PH domain of p62. On the other hand, lack of phosphorylation causes helix-like motifs and low binding affinity to p62. We undertook molecular simulation analyses to investigate the potential of some natural small molecules (Withanone (Wi-N) & Withaferin-A (Wi-A) from Ashwagandha; Cucurbitacin-B (Cuc-B) from bitter Cucumber; and Caffeic acid phenethyl ester (CAPE) and Artepillin C (ARC) from honeybee propolis) to interact with p62-binding region of p53 and restore its wild-type activity. We found that Wi-N, Wi-A, and Cuc-B have the potential to restore p53-p62 interaction for phosphorylation-deficient p53 mutants. Wi-N, in particular, caused a reversal of the α-helical structure into an elongated string-like conformation similar to the wild-type p53. These data suggested the use of these natural compounds for the treatment of p53^Ser46 mutant harbouring cancers. We also compared the efficiency of Wi-N, Wi-A, Cuc-B, CAPE, and ARC to abrogate Mortalin-p53 binding resulting in nuclear translocation and reactivation of p53 function and provide experimental evidence to the computational analysis. Taken together, the use of these small molecules for reactivation of p53 in cancer cells is suggested.

The human equilibrative nucleoside transporter 1 (hENT1) is an effective controller of adenosine signaling by regulating its extracellular and intracellular concentration, and has become a solid drug target of clinical used adenosine reuptake inhibitors (AdoRIs). Currently, the mechanisms of adenosine transport and inhibition for hENT1 remain unclear, which greatly limits the in-depth understanding of its inner workings as well as the development of novel inhibitors. In this work, the dynamic details of hENT1 underlie adenosine transport and the inhibition mechanism of the non-nucleoside AdoRIs dilazep both were investigated by comparative long-time unbiased molecular dynamics simulations. The calculation results show that the conformational transitions of hENT1 from the outward open to metastable occluded state are mainly driven by TM1, TM2, TM7 and TM9. One of the trimethoxyphenyl rings in dilazep serves as the adenosyl moiety of the endogenous adenosine substrate to competitively occupy the orthosteric site of hENT1. Due to extensive and various VDW interactions with N30, M33, M84, P308 and F334, the other trimethoxyphenyl ring is stuck in the opportunistic site near the extracellular side preventing the complete occlusion of thin gate simultaneously. Obviously, dilazep shows significant inhibitory activity by disrupting the local induce-fit action in substrate binding cavity and blocking the transport cycle of whole protein. This study not only reveals the nucleoside transport mechanism by hENT1 at atomic level, but also provides structural guidance for the subsequent design of novel non-nucleoside AdoRIs with enhanced pharmacologic properties.

Protein oligomerization has two notable aspects: it is crucial for the performing cellular and molecular processes accurately, and it produces amyloid fibril precursors. Although a clear explanation for amyloidosis as a whole is lacking, most studies have emphasized the importance of protein misfolding followed by formation of cytotoxic oligomer structures, which are responsible for disorders as diverse as neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases, and metabolic disorders, such as type 2 diabetes. Constant surveillance by oligomeric protein structures known as molecular chaperones enables cells to overcome the challenge of misfolded proteins and their harmful assemblies. These molecular chaperones encounter proteins in cells, and benefit cell survival as long as they perform correctly. Thus, this review highlights the roles of structural aspects of chaperone protein oligomers in determining cell fate—either succumbing to amyloid oligomers or survival—as well as experimental approaches used to investigate these entities.

Significant advances in the past decade have enabled high-resolution structure determination of a vast variety of proteins by cryogenic electron microscopy single particle analysis. Despite improved sample preparation, next-generation imaging hardware, and advanced single particle analysis algorithms, small proteins remain elusive for reconstruction due to low signal-to-noise and lack of distinctive structural features. Multiple efforts have therefore been directed at the development of size-increase techniques for small proteins. Here we review the latest methods for increasing effective molecular weight of proteins <100 ​kDa through target protein binding or target protein fusion - specifically by using nanobody-based assemblies, fusion tags, and symmetric scaffolds. Finally, we summarize these state-of-the-art techniques into a decision-tree to facilitate the design of tailored future approaches, and thus for further exploration of ever-smaller proteins that make up the largest part of the human genome.

Proteins are innately dynamic, which is important for their functions, but which also poses significant challenges when studying their structures. Gas-phase techniques can utilise separation and a range of sample manipulations to transcend some of the limitations of conventional techniques for structural biology in crystalline or solution phase, and isolate different states for separate interrogation. However, the transfer from solution to the gas phase risks affecting the structures, and it is unclear to what extent different conformations remain distinct in the gas phase, and if resolution in silico can recover the native conformations and their differences. Here, we use extensive molecular dynamics simulations to study the two distinct conformations of dimeric capsid protein of the MS2 bacteriophage. The protein undergoes notable restructuring of its peripheral parts in the gas phase, but subsequent simulation in solvent largely recovers the native structure. Our results suggest that despite some structural loss due to the experimental conditions, gas-phase structural biology techniques provide meaningful data that inform not only about the structures but also conformational dynamics of proteins.

The comparison of 303,250 human SARS-CoV-2 spike protein sequences with the reference protein sequence Wuhan-Hu-1, showed ∼96.5% of the spike protein sequence has undergone the mutations till date, since outbreak of the COVID-19 pandemic disease that was first reported in December 2019. A total of 1,269,629 mutations were detected corresponding to 1,229 distinct mutation sites in the spike proteins comprising 1,273 amino acid residues. Thereby, ∼3.5% of the human SARS-CoV-2 spike protein sequence has remained invariant in the past two years. Considering different mutations occur at the same mutation site, a total of 4,729 distinct mutations were observed and are catalogued in the present work. The WHO/CDC, U.S.A., classification and definitions for the current variants being monitored (VBM) and variant of concern (VOC) are assigned to the SARS-CoV-2 spike protein mutations identified in the present work along with a list of other amino acid substitutions observed for the variants. All 195 amino acid residues in receptor binding domain (Thr333-Pro527) were associated with mutations in SARS-CoV-2 spike protein sequence including Lys417, Tyr449, Tyr453, Ala475, Asn487, Thr500, Asn501 and Gly502 that make interactions with the ACE-2 receptor ≤3.2 ​Å distance as observed in the crystal structure complex available in the Protein Data Bank (PDB code:6LZG). However, not all these residues were mutated in the same spike protein. Especially, Gly502 mutated only in two spike protein sequences and Tyr449 mutated only in seven spike protein sequences among the spike protein sequences analysed constitute potential sites for the design of suitable inhibitors/drugs. Further, forty-four invariant residues were observed that correspond to ten domains/regions in the SARS-CoV-2 spike protein and some of the residues exposed to the protein surface amongst these may serve as epitope targets to develop monoclonal antibodies.

Translation initiation in eukaryotes relies on a complex network of interactions that are continuously reorganized throughout the process. As more information becomes available about the structure of the ribosomal preinitiation complex (PIC) at various points in translation initiation, new questions arise about which interactions occur when, their roles, and regulation. The eukaryotic translation factor (eIF) 5 is the GTPase-activating protein (GAP) for the GTPase eIF2, which brings the initiator Met-tRNA_i to the PIC. eIF5 also plays a central role in PIC assembly and remodeling through interactions with other proteins, including eIFs 1, 1A, and 3c. Phosphorylation by casein kinase 2 (CK2) significantly increases the eIF5 affinity for eIF2. The interaction between eIF5 and eIF1A was reported to be mediated by the eIF5 C-terminal domain (CTD) and the eIF1A N-terminal tail. Here, we report a new contact interface, between eIF5-CTD and the oligonucleotide/oligosaccharide-binding fold (OB) domain of eIF1A, which contributes to the overall affinity between the two proteins. We also show that the interaction is modulated by dynamic intramolecular interactions within both eIF5 and eIF1A. CK2 phosphorylation of eIF5 increases its affinity for eIF1A, offering new insights into the mechanisms by which CK2 stimulates protein synthesis and cell proliferation.