Protein Science最新文献

{"title":"Mining unique cysteine synthetases and computational study on thoroughly eliminating feedback inhibition through tunnel engineering","authors":"Shuai Xu, Zong‐Lin Li, Zhi‐Min Li, Hong‐Lai Liu","doi":"10.1002/pro.5160","DOIUrl":"https://doi.org/10.1002/pro.5160","url":null,"abstract":"L‐cysteine is an essential component in pharmaceutical and agricultural industries, and synthetic biology has made strides in developing new metabolic pathways for its production, particularly in archaea with unique O‐phosphoserine sulfhydrylases (OPSS) as key enzymes. In this study, we employed database mining to identify a highly catalytic activity OPSS from <jats:italic>Acetobacterium</jats:italic> sp. (AsOPSS). However, it was observed that the enzymatic activity of AsOPSS suffered significant feedback inhibition from the product L‐cysteine, exhibiting an IC<jats:sub>50</jats:sub> value of merely 1.2 mM. A semi‐rational design combined with tunnel analysis strategy was conducted to engineer AsOPSS. The best variant, AsOPSS<jats:sup>A218R</jats:sup> was achieved, totally eliminating product inhibition without sacrificing catalytic efficiency. Molecular docking and molecular dynamic simulations indicated that the binding conformation of AsOPSS<jats:sup>A218R</jats:sup> with L‐cys was altered, leading to a reduced affinity between L‐cysteine and the active pocket. Tunnel analysis revealed that the AsOPSS<jats:sup>A218R</jats:sup> variant reshaped the landscape of the tunnel, resulting in the construction of a new tunnel. Furthermore, random acceleration molecular dynamics simulation and umbrella sampling simulation demonstrated that the novel tunnel improved the suitability for product release and effectively separated the interference between the product release and substrate binding processes. Finally, more than 45 mM of L‐cysteine was produced in vitro within 2 h using the AsOPSS<jats:sup>A218R</jats:sup> variant. Our findings emphasize the potential for relieving feedback inhibition by artificially generating new product release channels, while also laying an enzymatic foundation for efficient L‐cysteine production.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"82 1","pages":""},"PeriodicalIF":8.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253089","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Benchmarking reverse docking through AlphaFold2 human proteome","authors":"Qing Luo, Sheng Wang, Hoi Yeung Li, Liangzhen Zheng, Yuguang Mu, Jingjing Guo","doi":"10.1002/pro.5167","DOIUrl":"https://doi.org/10.1002/pro.5167","url":null,"abstract":"Predicting the binding of ligands to the human proteome via reverse‐docking methods enables the understanding of ligand's interactions with potential protein targets in the human body, thereby facilitating drug repositioning and the evaluation of potential off‐target effects or toxic side effects of drugs. In this study, we constructed 11 reverse docking pipelines by integrating site prediction tools (PointSite and SiteMap), docking programs (Glide and AutoDock Vina), and scoring functions (Glide, Autodock Vina, RTMScore, DeepRMSD, and OnionNet‐SFCT), and then thoroughly benchmarked their predictive capabilities. The results show that the Glide_SFCT (PS) pipeline exhibited the best target prediction performance based on the atomic structure models in AlphaFold2 human proteome. It achieved a success rate of 27.8% when considering the top 100 ranked prediction. This pipeline effectively narrows the range of potential targets within the human proteome, laying a foundation for drug target prediction, off‐target assessment, and toxicity prediction, ultimately boosting drug development. By facilitating these critical aspects of drug discovery and development, our work has the potential to ultimately accelerate the identification of new therapeutic agents and improve drug safety.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"5 1","pages":""},"PeriodicalIF":8.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253094","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"AlzDiscovery: A computational tool to identify Alzheimer's disease‐causing missense mutations using protein structure information","authors":"Qisheng Pan, Georgina Becerra Parra, Yoochan Myung, Stephanie Portelli, Thanh Binh Nguyen, David B. Ascher","doi":"10.1002/pro.5147","DOIUrl":"https://doi.org/10.1002/pro.5147","url":null,"abstract":"Alzheimer's disease (AD) is one of the most common forms of dementia and neurodegenerative diseases, characterized by the formation of neuritic plaques and neurofibrillary tangles. Many different proteins participate in this complicated pathogenic mechanism, and missense mutations can alter the folding and functions of these proteins, significantly increasing the risk of AD. However, many methods to identify AD‐causing variants did not consider the effect of mutations from the perspective of a protein three‐dimensional environment. Here, we present a machine learning‐based analysis to classify the AD‐causing mutations from their benign counterparts in 21 AD‐related proteins leveraging both sequence‐ and structure‐based features. Using computational tools to estimate the effect of mutations on protein stability, we first observed a bias of the pathogenic mutations with significant destabilizing effects on family AD‐related proteins. Combining this insight, we built a generic predictive model, and improved the performance by tuning the sample weights in the training process. Our final model achieved the performance on area under the receiver operating characteristic curve up to 0.95 in the blind test and 0.70 in an independent clinical validation, outperforming all the state‐of‐the‐art methods. Feature interpretation indicated that the hydrophobic environment and polar interaction contacts were crucial to the decision on pathogenic phenotypes of missense mutations. Finally, we presented a user‐friendly web server, AlzDiscovery, for researchers to browse the predicted phenotypes of all possible missense mutations on these 21 AD‐related proteins. Our study will be a valuable resource for AD screening and the development of personalized treatment.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"22 1","pages":""},"PeriodicalIF":8.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253091","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of artificial photosystems based on designed proteins for mechanistic insights into photosynthesis","authors":"Gonzalo Pérez Serrano, Claudia F. Echavarría, Sara H. Mejias","doi":"10.1002/pro.5164","DOIUrl":"https://doi.org/10.1002/pro.5164","url":null,"abstract":"This review aims to provide an overview of the progress in protein‐based artificial photosystem design and their potential to uncover the underlying principles governing light‐harvesting in photosynthesis. While significant advances have been made in this area, a gap persists in reviewing these advances. This review provides a perspective of the field, pinpointing knowledge gaps and unresolved challenges that warrant further inquiry. In particular, it delves into the key considerations when designing photosystems based on the chromophore and protein scaffold characteristics, presents the established strategies for artificial photosystems engineering with their advantages and disadvantages, and underscores the recent breakthroughs in understanding the molecular mechanisms governing light‐harvesting, charge separation, and the role of the protein motions in the chromophore's excited state relaxation. By disseminating this knowledge, this article provides a foundational resource for defining the field of bio‐hybrid photosystems and aims to inspire the continued exploration of artificial photosystems using protein design.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"2 1","pages":""},"PeriodicalIF":8.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253120","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cryo‐EM and solid state NMR together provide a more comprehensive structural investigation of protein fibrils","authors":"Blake D. Fonda, Masato Kato, Yang Li, Dylan T. Murray","doi":"10.1002/pro.5168","DOIUrl":"https://doi.org/10.1002/pro.5168","url":null,"abstract":"The tropomyosin 1 isoform I/C C‐terminal domain (Tm1‐LC) fibril structure is studied jointly with cryogenic electron microscopy (cryo‐EM) and solid state nuclear magnetic resonance (NMR). This study demonstrates the complementary nature of these two structural biology techniques. Chemical shift assignments from solid state NMR are used to determine the secondary structure at the level of individual amino acids, which is faithfully seen in cryo‐EM reconstructions. Additionally, solid state NMR demonstrates that the region not observed in the reconstructed cryo‐EM density is primarily in a highly mobile random coil conformation rather than adopting multiple rigid conformations. Overall, this study illustrates the benefit of investigations combining cryo‐EM and solid state NMR to investigate protein fibril structure.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"66 1","pages":""},"PeriodicalIF":8.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253066","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Peptides inhibiting the assembly of monomeric human l‐lactate dehydrogenase into catalytically active homotetramer decrease the synthesis of lactate in cultured cells","authors":"Alessandra Stefan, Luca Gentilucci, Francesca Ruffolo, Valentina Rossi, Sofia Sordi, Tingting He, Giuseppina di Stefano, Federica Santino, Maurizio Brigotti, Claudia Scotti, Luisa Iamele, Hugo de Jonge, Fabrizio Dal Piaz, Danilo Rocco Santarcangelo, Alejandro Hochkoeppler","doi":"10.1002/pro.5161","DOIUrl":"https://doi.org/10.1002/pro.5161","url":null,"abstract":"The energetic metabolism of cancer cells relies on a substantial commitment of pyruvate to the catalytic action of lactate‐generating dehydrogenases. This coupling mainly depends on lactate dehydrogenase A (LDH‐A), which is overexpressed in different types of cancers, and therefore represents an appealing therapeutic target. Taking into account that the activity of LDHs is exclusively exerted by their tetrameric forms, it was recently shown that peptides perturbing the monomers‐to‐tetramer assembly inhibit human LDH‐A (hLDH‐A). However, to identify these peptides, tetrameric hLDH‐A was transiently exposed to strongly acidic conditions inducing its dissociation into monomers, which were tested as a target for peptides at low pH. Nevertheless, the availability of native monomeric hLDH‐A would allow performing similar screenings under physiological conditions. Here we report on the unprecedented isolation of recombinant monomeric hLDH‐A at neutral pH, and on its use to identify peptides inhibiting the assembly of the tetrameric enzyme. Remarkably, the GQNGISDL octapeptide, mimicking the 296–303 portion of hLDH‐A C‐terminal region, was observed to effectively inhibit the target enzyme. Moreover, by dissecting the action of this octapeptide, the cGQND cyclic tetrapeptide was found to act as the parental compound. Furthermore, we performed assays using MCF7 and BxPC3 cultured cells, exclusively expressing hLDH‐A and hLDH‐B, respectively. By means of these assays we detected a selective action of linear and cyclic GQND tetrapeptides, inhibiting lactate secretion in MCF7 cells only. Overall, our observations suggest that peptides mimicking the C‐terminal region of hLDH‐A effectively interfere with protein–protein interactions responsible for the assembly of the tetrameric enzyme.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"8 1","pages":""},"PeriodicalIF":8.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253067","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Substrate selectivity and inhibition of the human lysyl hydroxylase JMJD7","authors":"Nurgül Bilgin, Anthony Tumber, Siddhant Dhingra, Eidarus Salah, Aziza Al‐Salmy, Sandra Pinzón Martín, Yicheng Wang, Christopher J. Schofield, Jasmin Mecinović","doi":"10.1002/pro.5162","DOIUrl":"https://doi.org/10.1002/pro.5162","url":null,"abstract":"Jumonji‐C (JmjC) domain‐containing protein 7 (JMJD7) is a human Fe(II) and 2‐oxoglutarate dependent oxygenase that catalyzes stereospecific C3‐hydroxylation of lysyl‐residues in developmentally regulated GTP binding proteins 1 and 2 (DRG1/2). We report studies exploring a diverse set of lysine derivatives incorporated into the DRG1 peptides as potential human JMJD7 substrates and inhibitors. The results indicate that human JMJD7 has a relatively narrow substrate scope beyond lysine compared to some other JmjC hydroxylases and lysine‐modifying enzymes. The geometrically constrained (<jats:italic>E</jats:italic>)‐dehydrolysine is an efficient alternative to lysine for JMJD7‐catalyzed C3‐hydroxylation. γ‐Thialysine and γ‐azalysine undergo C3‐hydroxylation, followed by degradation to formylglycine. JMJD7 also catalyzes the S‐oxidation of DRG1‐derived peptides possessing methionine and homomethionine residues in place of lysine. Inhibition assays show that DRG1 variants possessing cysteine/selenocysteine instead of the lysine residue efficiently inhibit JMJD7 via cross‐linking. The overall results inform on the substrate selectivity and inhibition of human JMJD7, which will help enable the rational design of selective small‐molecule and peptidomimetic inhibitors of JMJD7.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"15 1","pages":""},"PeriodicalIF":8.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253070","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mutational analysis reveals the importance of residues of the access tunnel inhibitor site to human P-glycoprotein (ABCB1)-mediated transport.","authors":"Paula B Salazar, Megumi Murakami, Nandhini Ranganathan, Stewart R Durell, Suresh V Ambudkar","doi":"10.1002/pro.5155","DOIUrl":"10.1002/pro.5155","url":null,"abstract":"<p><p>Human P-glycoprotein (P-gp) utilizes energy from ATP hydrolysis for the efflux of chemically dissimilar amphipathic small molecules and plays an important role in the development of resistance to chemotherapeutic agents in most cancers. Efforts to overcome drug resistance have focused on inhibiting P-gp-mediated drug efflux. Understanding the features distinguishing P-gp inhibitors from substrates is critical. Cryo-electron microscopy has revealed distinct binding patterns, emphasizing the role of the L-site or access tunnel in inhibition. We substituted 5-9 residues of the L-site with alanine to investigate whether the binding of a second inhibitor molecule to the L-site is required for inhibiting drug efflux. We reveal, for the first time, that mutations in the L-site affect the drug efflux activity of P-gp, despite their distance from the substrate-binding pocket (SBP). Surprisingly, after the mutations were introduced, inhibitors such as tariquidar and zosuquidar still inhibited drug efflux by mutant P-gps. Communication between the transmembrane helices (TMHs) and nucleotide-binding domains (NBDs) was evaluated using the ATPase assay, revealing distinct modulation patterns by inhibitors for the mutants, with zosuquidar exhibiting substrate-like stimulation of ATPase. Furthermore, L-site mutations abolished ATP-dependent thermal stabilization. In silico molecular docking studies corroborated the altered inhibitor binding due to mutations in the L-site residues, shedding light on their critical role in substrate transport and inhibitor interactions with P-gp. These findings suggest that inhibitors bind either to the SBP alone, and/or to alternate site(s) when the L-site is disabled by mutagenesis.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"33 9","pages":"e5155"},"PeriodicalIF":4.5,"publicationDate":"2024-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11350596/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142081380","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Clustering protein functional families at large scale with hierarchical approaches.","authors":"Nicola Bordin, Harry Scholes, Clemens Rauer, Joel Roca-Martínez, Ian Sillitoe, Christine Orengo","doi":"10.1002/pro.5140","DOIUrl":"10.1002/pro.5140","url":null,"abstract":"<p><p>Proteins, fundamental to cellular activities, reveal their function and evolution through their structure and sequence. CATH functional families (FunFams) are coherent clusters of protein domain sequences in which the function is conserved across their members. The increasing volume and complexity of protein data enabled by large-scale repositories like MGnify or AlphaFold Database requires more powerful approaches that can scale to the size of these new resources. In this work, we introduce MARC and FRAN, two algorithms developed to build upon and address limitations of GeMMA/FunFHMMER, our original methods developed to classify proteins with related functions using a hierarchical approach. We also present CATH-eMMA, which uses embeddings or Foldseek distances to form relationship trees from distance matrices, reducing computational demands and handling various data types effectively. CATH-eMMA offers a highly robust and much faster tool for clustering protein functions on a large scale, providing a new tool for future studies in protein function and evolution.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"33 9","pages":"e5140"},"PeriodicalIF":4.5,"publicationDate":"2024-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11325189/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141983082","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Integrating molecular dynamics simulation with small- and wide-angle X-ray scattering to unravel the flexibility, antigen-blocking, and protease-restoring functions in a hindrance-based pro-antibody.","authors":"Jun Min Liao, Shih-Ting Hong, Yeng-Tseng Wang, Yi-An Cheng, Kai-Wen Ho, Shu-Ing Toh, Orion Shih, U-Ser Jeng, Ping-Chiang Lyu, I-Chen Hu, Ming-Yii Huang, Chin-Yuan Chang, Tian-Lu Cheng","doi":"10.1002/pro.5124","DOIUrl":"10.1002/pro.5124","url":null,"abstract":"<p><p>Spatial hindrance-based pro-antibodies (pro-Abs) are engineered antibodies to reduce monoclonal antibodies' (mAbs) on-target toxicity using universal designed blocking segments that mask mAb antigen-binding sites through spatial hindrance. By linking through protease substrates and linkers, these blocking segments can be removed site-specifically. Although many types of blocking segments have been developed, such as coiled-coil and hinge-based Ab locks, the molecular structure of the pro-Ab, particularly the region showing how the blocking fragment blocks the mAb, has not been elucidated by X-ray crystallography or cryo-EM. To achieve maximal effect, a pro-Ab must have high antigen-blocking and protease-restoring efficiencies, but the unclear structure limits its further optimization. Here, we utilized molecular dynamics (MD) simulations to study the dynamic structures of a hinge-based Ab lock pro-Ab, pro-Nivolumab, and validated the simulated structures with small- and wide-angle X-ray scattering (SWAXS). The MD results were closely consistent with SWAXS data (χ² _best-fit = 1.845, χ² _allMD = 3.080). The further analysis shows a pronounced flexibility of the Ab lock (root-mean-square deviation = 10.90 Å), yet it still masks the important antigen-binding residues by 57.3%-88.4%, explaining its 250-folded antigen-blocking efficiency. The introduced protease accessible surface area method affirmed better protease efficiency for light chain (33.03 Ų) over heavy chain (5.06 Ų), which aligns with the experiments. Overall, we developed MD-SWAXS validation method to study the dynamics of flexible blocking segments and introduced methodologies to estimate their antigen-blocking and protease-restoring efficiencies, which would potentially be advancing the clinical applications of any spatial hindrance-based pro-Ab.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"33 9","pages":"e5124"},"PeriodicalIF":4.5,"publicationDate":"2024-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11325194/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141983083","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}