Current Research in Structural Biology最新文献

Binding of nucleotides and their derivatives is one of the most ancient elementary functions dating back to the Origin of Life. We review here the works considering one of the key elements in binding of (di)nucleotide-containing ligands – phosphate binding. We start from a brief discussion of major participants, conditions, and events in prebiotic evolution that resulted in the Origin of Life. Tracing back to the basic functions, including metal and phosphate binding, and, potentially, formation of primitive protein-protein interactions, we focus here on the phosphate binding. Critically assessing works on the structural, functional, and evolutionary aspects of phosphate binding, we perform a simple computational experiment reconstructing its most ancient and generic sequence prototype. The profiles of the phosphate binding signatures have been derived in form of position-specific scoring matrices (PSSMs), their peculiarities depending on the type of the ligands have been analyzed, and evolutionary connections between them have been delineated. Then, the apparent prototype that gave rise to all relevant phosphate-binding signatures had also been reconstructed. We show that two major signatures of the phosphate binding that discriminate between the binding of dinucleotide- and nucleotide-containing ligands are GxGxxG and GxxGxG, respectively. It appears that the signature archetypal for dinucleotide-containing ligands is more generic, and it can frequently bind phosphate groups in nucleotide-containing ligands as well. The reconstructed prototype's key signature GxGGxG underlies the role of glycine residues in providing flexibility and interactions necessary for binding the phosphate groups. The prototype also contains other ancient amino acids, valine, and alanine, showing versatility towards evolutionary design and functional diversification.

The ever-changing environmental conditions and pollution are the prime reasons for the onset of several emerging and re-merging diseases. This demands the faster designing of new drugs to curb the deadly diseases in less waiting time to cure the animals and humans. Drug molecules interact with only protein surface on specific locations termed as ligand binding sites (LBS). Therefore, the knowledge of LBS is required for rational drug designing. Existing geometrical LBS prediction methods rely on search of cavities based on the fact that 83% of the LBS found in deep cavities, however, these methods usually fail where LBS localize outside deep cavities. To overcome this challenge, the present work provides an artificial neural network (ANN) based method to predict LBS outside deep cavities in animal proteins including human to facilitate drug designing. In the present work a feed-forward backpropagation neural network was trained by utilizing 38 structural, atomic, physiochemical, and evolutionary discriminant features of LBS and non-LBS residues localized in the extracted roughest patch on protein surface. The performance of this ANN based prediction method was found 76% better for those proteins where cavity subspace (extracted by MetaPocket 2.0, a consensus method) failed to predict LBS due to their localization outside the deep cavities. The prediction of LBS outside deep cavities will facilitate in drug designing for the proteins where it is not possible due to lack of LBS information as the geometrical LBS prediction methods rely on extraction of deep cavities.

Sortase proteins play a crucial role as integral membrane proteins in anchoring bacterial surface proteins by recognizing them through a Cell-Wall Sorting (CWS) motif and cleaving them at specific sites before initiating pilus assembly. Both sortases and their substrate proteins are major virulence factors in numerous Gram-positive pathogens, making them attractive targets for antimicrobial intervention. Recognizing the significance of virulence proteins, a comprehensive exploration of their structural and functional characteristics is essential to enhance our understanding of pilus assembly in diverse Gram-positive bacteria. Therefore, this review article discusses the structural features of different classes of sortases and pilin proteins, primarily serving as substrates for sortase-assembled pili. Moreover, it thoroughly examines the molecular-level interactions between sortases and their inhibitors, providing insights from both structural and functional perspectives. In essence, this review article will provide a contemporary and complete understanding of both sortase pathways and various strategies to target them effectively to counteract the virulence.

CD8⁺ T cells are crucial for viral elimination and recovery from viral infection. Nonetheless, the current understanding of the T cell response to SARS-CoV-2 at the antigen level remains limited. The Spike protein is an external structural protein that is prone to mutations, threatening the efficacy of current vaccines. Therefore, we have characterised the immune response towards the immunogenic Spike-derived peptide (S_976-984, VLNDILSRL), restricted to the HLA-A*02:01 molecule, which is mutated in both Alpha (S982A) and Omicron BA.1 (L981F) variants of concern. We determined that the mutation in the Alpha variant (S982A) impacted both the stability and conformation of the peptide, bound to HLA-A*02:01, in comparison to the original S_976-984. We identified a longer and overlapping immunogenic peptide (S_975-984, SVLNDILSRL) that could be presented by HLA-A*02:01, HLA-A*11:01 and HLA-B*13:01 allomorphs. We showed that S975-specific CD8⁺ T cells were weakly cross-reactive to the mutant peptides despite their similar conformations when presented by HLA-A*11:01. Altogether, our results show that the impact of SARS-CoV-2 mutations on peptide presentation is HLA allomorph-specific, and that post vaccination there are T cells able to react and cross-react towards the variant of concern peptides.

Anchoring of coagulation factors to anionic regions of the membrane involves the C2 domain as a key player. The rate of enzymatic reactions of the coagulation factors is increased by several orders of magnitude upon membrane binding. However, the precise mechanisms behind the rate acceleration remain unclear, primarily because of a lack of understanding of the conformational dynamics of the C2-containing factors and corresponding complexes. We elucidate the membrane-bound form of the C2 domain from human coagulation factor V (FV–C2) by characterizing its membrane binding the specific lipid-protein interactions. Employing all-atom molecular dynamics simulations and leveraging the highly mobile membrane-mimetic (HMMM) model, we observed spontaneous binding of FV-C2 to a phosphatidylserine (PS)-containing membrane within 2–25 ns across twelve independent simulations. FV-C2 interacted with the membrane through three loops (spikes 1–3), achieving a converged, stable orientation. Multiple HMMM trajectories of the spontaneous membrane binding provided extensive sampling and ample data to examine the membrane-induced effects on the conformational dynamics of C2 as well as specific lipid-protein interactions. Despite existing crystal structures representing presumed “open” and “closed” states of FV-C2, our results revealed a continuous distribution of structures between these states, with the most populated structures differing from both “open” and “closed” states observed in crystal environments. Lastly, we characterized a putative PS-specific binding site formed by K23, Q48, and S78 located in the groove enclosed by spikes 1–3 (PS-specificity pocket), suggesting a different orientation of a bound headgroup moiety compared to previous proposals based upon analysis of static crystal structures.

KvAP is a prokaryotic Kv channel, which has been widely used as a model system to understand voltage- and lipid-dependent gating mechanisms. In phospholipid membranes, the KvAP-VSD adopts the activated/‘Up’ conformation, whereas the presence of non-phospholipids in membranes favours the structural transition to resting/‘Down’ state. The S3b-S4 paddle motif loop of KvAP-VSD is functionally important as this participates in protein-protein interactions and is the target for animal toxins. In this study, we have monitored the modulatory role of cholesterol – the physiologically-relevant non-phospholipid – on the organization and dynamics of the S3b-S4 loop of the isolated KvAP-VSD in membranes by site-directed fluorescence approaches using the environmental sensitivity of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-ethylenediamine (NBD) fluorescence. Our results show that cholesterol alters the dynamic nature (rotational and hydration dynamics) of S3b-S4 loop in a segmental fashion, i.e., the residues 110 to 114 and 115 to 117 behave differently in the presence of cholesterol, which is accompanied by considerable change in conformational heterogeneity. Further, quantitative depth measurements using the parallax quenching method reveal that the sensor loop is located at the shallow interfacial region of cholesterol-containing membranes, suggesting that the sensor loop organization is not directly correlated with S4 helix movement. Our results clearly show that cholesterol-induced changes in bilayer properties may not be the predominant factor for the sensor loop's altered structural dynamics, but can be attributed to the conformational change of the KvAP-VSD in cholesterol-containing membranes. Overall, these results are relevant for gating mechanisms, particularly the lipid-dependent gating, of Kv channels in membranes.

Extradenticle (EXD) is a partner protein of the HOX transcription factors and plays an important role in the development of Drosophila. It confers increased affinity and specificity of DNA-binding to the HOX proteins. However, the DNA-binding homeodomain of EXD has a significantly weaker affinity to DNA compared to the HOX homeodomains. Here, we show that a glycine residue (G290) in the middle of the EXD DNA-binding helix primarily results in this weaker binding. Glycine destabilizes helices. To probe its role in the stability and function of the protein, G290 was mutated to alanine. The intrinsic stability of the DNA-binding helix increased in the G290A mutant as observed by NMR studies and molecular dynamics (MD) simulation. Also, NMR dynamics and MD simulation show that dynamic motions present in the wild-type protein are quenched in the mutant. This in turn resulted in increased stability of the entire homeodomain (ΔΔG_G→A of −2.6 kcal/mol). Increased protein stability resulted in three-fold better DNA-binding affinity of the mutant as compared to the wild-type protein. Molecular mechanics with generalized Born and surface area solvation (MMGBSA) analysis of our MD simulation on DNA-bound models of both wild-type and mutant proteins shows that the contribution to binding is enhanced for most of the interface residues in the mutant compared to the wild-type. Interestingly, the flexible N-terminal arm makes more stable contact with the DNA minor groove in the mutant. We found that the two interaction sites i.e. the DNA-binding helix and the unstructured N-terminal arm influence each other via the bound DNA. These results provide an interesting conundrum: alanine at position 290 enhances both the stability and the DNA-binding affinity of the protein, however, evolution prefers glycine at this position. We have provided several plausible explanations for this apparent conundrum. The function of the EXD as a HOX co-factor requires its ability to discriminate similar DNA sequences, which is most likely comprom

The pervasive presence of plastic in the environment has reached a concerning scale, being identified in many ecosystems. Bioremediation is the cheapest and most eco-friendly alternative to remove this polymer from affected areas. Recent work described that a novel cold-active esterase enzyme extracted from the bacteria Kaistella jeonii could promiscuously degrade PET. Compared to the well-known PETase from Ideonella sakaiensis, this novel esterase presents a low sequence identity yet has a remarkably similar folding. However, enzymatic assays demonstrated a lower catalytic efficiency. In this work, we employed a strict computational approach to investigate the binding mechanism between the esterase and PET. Understanding the underlying mechanism of binding can shed light on the evolutive mechanism of how enzymes have been evolving to degrade these artificial molecules and help develop rational engineering approaches to improve PETase-like enzymes. Our results indicate that this esterase misses a disulfide bridge, keeping the catalytic residues closer and possibly influencing its catalytic efficiency. Moreover, we describe the structural response to the interaction between enzyme and PET, indicating local and global effects. Our results aid in deepening the knowledge behind the mechanism of biological catalysis of PET degradation and as a base for the engineering of novel PETases.