Proteins-Structure Function and Bioinformatics最新文献

The omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) characterized by 30 mutations in its spike protein, has rapidly spread worldwide since November 2021, significantly exacerbating the ongoing COVID-19 pandemic. In order to investigate the relationship between these mutations and the variant's high transmissibility, we conducted a systematic analysis of the mutational effect on spike-angiotensin-converting enzyme-2 (ACE2) interactions and explored the structural/energy correlation of key mutations, utilizing a reliable coarse-grained model. Our study extended beyond the receptor-binding domain (RBD) of spike trimer through comprehensive modeling of the full-length spike trimer rather than just the RBD. Our free-energy calculation revealed that the enhanced binding affinity between the spike protein and the ACE2 receptor is correlated with the increased structural stability of the isolated spike protein, thus explaining the omicron variant's heightened transmissibility. The conclusion was supported by our experimental analyses involving the expression and purification of the full-length spike trimer. Furthermore, the energy decomposition analysis established those electrostatic interactions make major contributions to this effect. We categorized the mutations into four groups and established an analytical framework that can be employed in studying future mutations. Additionally, our calculations rationalized the reduced affinity of the omicron variant towards most available therapeutic neutralizing antibodies, when compared with the wild type. By providing concrete experimental data and offering a solid explanation, this study contributes to a better understanding of the relationship between theories and observations and lays the foundation for future investigations.

Understanding the emergence and structural characteristics of de novo and random proteins is crucial for unraveling protein evolution and designing novel enzymes. However, experimental determination of their structures remains challenging. Recent advancements in protein structure prediction, particularly with AlphaFold2 (AF2), have expanded our knowledge of protein structures, but their applicability to de novo and random proteins is unclear. In this study, we investigate the structural predictions and confidence scores of AF2 and protein language model-based predictor ESMFold for de novo and conserved proteins from Drosophila and a dataset of comparable random proteins. We find that the structural predictions for de novo and random proteins differ significantly from conserved proteins. Interestingly, a positive correlation between disorder and confidence scores (pLDDT) is observed for de novo and random proteins, in contrast to the negative correlation observed for conserved proteins. Furthermore, the performance of structure predictors for de novo and random proteins is hampered by the lack of sequence identity. We also observe fluctuating median predicted disorder among different sequence length quartiles for random proteins, suggesting an influence of sequence length on disorder predictions. In conclusion, while structure predictors provide initial insights into the structural composition of de novo and random proteins, their accuracy and applicability to such proteins remain limited. Experimental determination of their structures is necessary for a comprehensive understanding. The positive correlation between disorder and pLDDT could imply a potential for conditional folding and transient binding interactions of de novo and random proteins.

Three-dimensional (3D) structure information, now available at the proteome scale, may facilitate the detection of remote evolutionary relationships in protein superfamilies. Here, we illustrate this with the identification of a novel family of protein domains related to the ferredoxin-like superfold, by combining (i) transitive sequence similarity searches, (ii) clustering approaches, and (iii) the use of AlphaFold2 3D structure models. Domains of this family were initially identified in relation with the intracellular biomineralization of calcium carbonates by Cyanobacteria. They are part of the large heavy-metal-associated (HMA) superfamily, departing from the latter by specific sequence and structural features. In particular, most of them share conserved basic amino acids  (hence their name CoBaHMA for Conserved Basic residues HMA), forming a positively charged surface, which is likely to interact with anionic partners. CoBaHMA domains are found in diverse modular organizations in bacteria, existing in the form of monodomain proteins or as part of larger proteins, some of which are membrane proteins involved in transport or lipid metabolism. This suggests that the CoBaHMA domains may exert a regulatory function, involving interactions with anionic lipids. This hypothesis might have a particular resonance in the context of the compartmentalization observed for cyanobacterial intracellular calcium carbonates.

Human acyl protein thioesterases (APTs) catalyze the depalmitoylation of S-acylated proteins attached to the plasma membrane, facilitating reversible cycles of membrane anchoring and detachment. We previously showed that a bacterial APT homologue, FTT258 from the gram-negative pathogen Francisella tularensis, exists in equilibrium between a closed and open state based on the structural dynamics of a flexible loop overlapping its active site. Although the structural dynamics of this loop are not conserved in human APTs, the amino acid sequence of this loop is highly conserved, indicating essential but divergent functions for this loop in human APTs. Herein, we investigated the role of this loop in regulating the catalytic activity, ligand binding, and protein folding of human APT1, a depalmitoylase connected with cancer, immune, and neurological signaling. Using a combination of substitutional analysis with kinetic, structural, and biophysical characterization, we show that even in its divergent structural location in human APT1 that this loop still regulates the catalytic activity of APT1 through contributions to ligand binding and substrate positioning. We confirmed previously known roles for multiple residues (Phe72 and Ile74) in substrate binding and catalysis while adding new roles in substrate selectivity (Pro69), in catalytic stabilization (Asp73 and Ile75), and in transitioning between the membrane binding β-tongue and substrate-binding loops (Trp71). Even conservative substitution of this tryptophan (Trp71) fulcrum led to complete loss of catalytic activity, a 13°C decrease in total protein stability, and drastic drops in ligand affinity, indicating that the combination of the size, shape, and aromaticity of Trp71 are essential to the proper structure of APT1. Mixing buried hydrophobic surface area with contributions to an exposed secondary surface pocket, Trp71 represents a previously unidentified class of essential tryptophans within α/β hydrolase structure and a potential allosteric binding site within human APTs.

The biosynthesis pathways of coenzyme A (CoA) in most archaea involve several unique enzymes including dephospho-CoA kinase (DPCK) that converts dephospho-CoA to CoA in the final step of CoA biosynthesis in all domains of life. The archaeal DPCK is unrelated to the analogous bacterial and eukaryotic enzymes and shows no significant sequence similarity to any proteins with known structures. Unusually, the archaeal DPCK utilizes GTP as the phosphate donor although the analogous bacterial and eukaryotic enzymes are ATP-dependent kinases. Here, we report the crystal structure of DPCK and its complex with GTP and a magnesium ion from the archaeal hyperthermophile Thermococcus kodakarensis. The crystal structure demonstrates why GTP is the preferred substrate of this kinase. We also report the activity analyses of site-directed mutants of crucial residues determined based on sequence conservation and the crystal structure. From these results, the key residues involved in the reaction of phosphoryl transfer and the possible dephospho-CoA binding site are inferred.

Mitochondria play a central role in energy production and cellular metabolism. Mitochondria contain their own small genome (mitochondrial DNA, mtDNA) that carries the genetic instructions for proteins required for ATP synthesis. The mitochondrial proteome, including the mitochondrial transcriptional machinery, is subject to post-translational modifications (PTMs), including acetylation and phosphorylation. We set out to determine whether PTMs of proteins associated with mtDNA may provide a potential mechanism for the regulation of mitochondrial gene expression. Here, we focus on mitochondrial ribosomal protein L12 (MRPL12), which is thought to stabilize mitochondrial RNA polymerase (POLRMT) and promote transcription. Numerous acetylation sites of MRPL12 were identified by mass spectrometry. We employed amino acid mimics of the acetylated (lysine to glutamine mutants) and deacetylated (lysine to arginine mutants) versions of MRPL12 to interrogate the role of lysine acetylation in transcription initiation in vitro and mitochondrial gene expression in HeLa cells. MRPL12 acetyl and deacetyl protein mimics were purified and assessed for their ability to impact mtDNA promoter binding of POLRMT. We analyzed mtDNA content and mitochondrial transcript levels in HeLa cells upon overexpression of acetyl and deacetyl mimics of MRPL12. Our results suggest that MRPL12 single-site acetyl mimics do not change the mtDNA promoter binding ability of POLRMT or mtDNA content in HeLa cells. Individual acetyl mimics may have modest effects on mitochondrial transcript levels. We found that the mitochondrial deacetylase, Sirtuin 3, is capable of deacetylating MRPL12 in vitro, suggesting a potential role for dynamic acetylation controlling MRPL12 function in a role outside of the regulation of gene expression.

This study focuses on investigating the effects of an oncogenic mutation (G12V) on the stability and interactions within the KRAS-RGL1 protein complex. The KRAS-RGL1 complex is of particular interest due to its relevance to KRAS-associated cancers and the potential for developing targeted drugs against the KRAS system. The stability of the complex and the allosteric effects of specific residues are examined to understand their roles as modulators of complex stability and function. Using molecular dynamics simulations, we calculate the mutual information, MI, between two neighboring residues at the interface of the KRAS-RGL1 complex, and employ the concept of interaction information, II, to measure the contribution of a third residue to the interaction between interface residue pairs. Negative II indicates synergy, where the presence of the third residue strengthens the interaction, while positive II suggests anti-cooperativity. Our findings reveal that MI serves as a dominant factor in determining the results, with the G12V mutation increasing the MI between interface residues, indicating enhanced correlations due to the formation of a more compact structure in the complex. Interestingly, although II plays a role in understanding three-body interactions and the impact of distant residues, it is not significant enough to outweigh the influence of MI in determining the overall stability of the complex. Nevertheless, II may nonetheless be a relevant factor to consider in future drug design efforts. This study provides valuable insights into the mechanisms of complex stability and function, highlighting the significance of three-body interactions and the impact of distant residues on the binding stability of the complex. Additionally, our findings demonstrate that constraining the fluctuations of a third residue consistently increases the stability of the G12V variant, making it challenging to weaken complex formation of the mutated species through allosteric manipulation. The novel perspective offered by this approach on protein dynamics, function, and allostery has potential implications for understanding and targeting other protein complexes involved in vital cellular processes. The results contribute to our understanding of the effects of oncogenic mutations on protein-protein interactions and provide a foundation for future therapeutic interventions in the context of KRAS-associated cancers and beyond.

Glial fibrillary acidic protein (GFAP) is a promising biomarker for brain and spinal cord disorders. Recent studies have highlighted the differences in the reliability of GFAP measurements in different biological matrices. The reason for these discrepancies is poorly understood as our knowledge of the protein's 3-dimensional conformation, proteoforms, and aggregation remains limited. Here, we investigate the structural properties of GFAP under different conditions. For this, we characterized recombinant GFAP proteins from various suppliers and applied hydrogen-deuterium exchange mass spectrometry (HDX-MS) to provide a snapshot of the conformational dynamics of GFAP in artificial cerebrospinal fluid (aCSF) compared to the phosphate buffer. Our findings indicate that recombinant GFAP exists in various conformational species. Furthermore, we show that GFAP dimers remained intact under denaturing conditions. HDX-MS experiments show an overall decrease in H-bonding and an increase in solvent accessibility of GFAP in aCSF compared to the phosphate buffer, with clear indications of mixed EX2 and EX1 kinetics. To understand possible structural interface regions and the evolutionary conservation profiles, we combined HDX-MS results with the predicted GFAP-dimer structure by AlphaFold-Multimer. We found that deprotected regions with high structural flexibility in aCSF overlap with predicted conserved dimeric 1B and 2B domain interfaces. Structural property predictions combined with the HDX data show an overall deprotection and signatures of aggregation in aCSF. We anticipate that the outcomes of this research will contribute to a deeper understanding of the structural flexibility of GFAP and ultimately shed light on its behavior in different biological matrices.

Bacteriophages are the natural predators of bacteria and are available abundantly everywhere in nature. Lytic phages can specifically infect their bacterial host (through attachment to the receptor) and use their host replication machinery to replicate rapidly, a feature that enables them to kill a disease-causing bacteria. Hence, phage attachment to the host bacteria is the first important step of the infection process. It is reported in this study that the receptor could be an LPS which is responsible for the attachment of the Sfk20 phage to its host (Shigella flexneri 2a). Phage Sfk20 bacteriolytic activity was examined for preliminary optimization of phage titer. The phage Sfk20 viability at different saline conditions was conducted. The LC-MS/MS technique used here for detecting and identifying 40 Sfk20 phage proteins helped us to get an initial understanding of the structural landscape of phage Sfk20. From the identified proteins, six structurally significant proteins were selected for structure prediction using two neural network systems: AlphaFold2 and ESMFold, and one homology modeling software: Phyre2. Later the performance of these modeling systems was compared using various metrics. We conclude from the available and generated information that AlphaFold2 and Phyre2 perform better than ESMFold for predicting Sfk20 phage protein structures.