Protein Science最新文献

{"title":"Recognizing amino acid sidechains in a medium-resolution cryo-electron density map.","authors":"Dibyendu Mondal, Vipul Kumar, Tadej Satler, Rakesh Ramachandran, Daniel Saltzberg, Ilan Chemmama, Kala Bharath Pilla, Ignacia Echeverria, Benjamin M Webb, Meghna Gupta, Klim Verba, Andrej Sali","doi":"10.1002/pro.70217","DOIUrl":"10.1002/pro.70217","url":null,"abstract":"<p><p>Building an accurate atomic structure model of a protein into a cryo-electron microscopy (cryo-EM) map at worse than 3 Å resolution is difficult. To facilitate this task, we devised a method for assigning the amino acid residue sequence to the backbone fragments traced in an input cryo-EM map (EMSequenceFinder). EMSequenceFinder relies on a Bayesian scoring function for ranking 20 standard amino acid residue types at a given backbone position, based on the fit to a density map, map resolution, and secondary structure propensity. The fit to a density is quantified by a convolutional neural network that was trained on ~5.56 million amino acid residue densities extracted from cryo-EM maps at 3-10 Å resolution and corresponding atomic structure models deposited in the Electron Microscopy Data Bank (EMDB). We benchmarked EMSequenceFinder by predicting the sequences of 58,044 distinct ɑ-helix and β-strand fragments, given the fragment backbone coordinates fitted in their density maps. EMSequenceFinder identifies the correct sequence as the best-scoring sequence in 77.8% of these cases. We also assessed EMSequenceFinder on separate datasets of cryo-EM maps at resolutions from 4 to 6 Å. The accuracy of EMSequenceFinder (58%) was better than that of three tested state-of-the-art methods, including findMysequence (45%), ModelAngelo (27%), and sequence_from_map in Phenix (12.9%). We further illustrate EMSequenceFinder by threading the Severe Acute Respiratory Syndrome Coronavirus 2 Non-Structural Protein 2 sequence into eight cryo-EM maps at resolutions from 3.7 to 7.0 Å. EMSequenceFinder is implemented in our open-source Integrative Modeling Platform (IMP) program. Thus, it is expected to be helpful for integrative structure modeling based on a cryo-EM map and other information, such as models of protein complex components and chemical crosslinks between them. EMSequenceFinder is available as part of our open-source IMP distribution at https://integrativemodeling.org/.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 8","pages":"e70217"},"PeriodicalIF":5.2,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12302279/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144732858","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":"Mechanism of (-)-Englerin A and calcium binding on the human TRPC5 channel.","authors":"Yikun Chen, Kangcheng Song, Wenjun Guo, Miao Wei, Lei Chen","doi":"10.1002/pro.70218","DOIUrl":"10.1002/pro.70218","url":null,"abstract":"<p><p>The natural product (-)-Englerin A (EA) selectively inhibits renal cancer cell growth by potently activating TRPC4 and TRPC5-containing ion channels. However, its binding site on these channels has remained elusive. In this study, we present two cryo-EM structures of human TRPC5 in complex with EA at 2.5 and 2.6 Å resolution, which reveal the EA-binding site and identify two major conformations influenced by calcium. EA binds between the pore helix and S5/S6 helices of hTRPC5, forming critical hydrophobic and polar interactions that underscore its specificity. Calcium binding at the intracellular domain of TRPC5 induces structural changes that stabilize the domain in a compact conformation. These findings expand our understanding of the structural pharmacology of TRPC5 and provide a framework for investigating calcium regulation in TRPC channels.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 8","pages":"e70218"},"PeriodicalIF":5.2,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12267661/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144650216","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":"Extracellular catalysis of environmental substrates by Shewanella oneidensis MR-1 occurs via active sites on the C-terminal domains of MtrC.","authors":"Alejandro Morales-Florez, Colin W J Lockwood, Benjamin W Nash, Marcus J Edwards, Jessica H van Wonderen, Amit Sachdeva, Julea N Butt, Thomas A Clarke","doi":"10.1002/pro.70243","DOIUrl":"10.1002/pro.70243","url":null,"abstract":"<p><p>The Gram-negative Shewanellaceae family is well known for its ability to transfer catabolically derived electrons to extracellular terminal electron acceptors through electron conduits that permeate the outer membrane. The primary conduit is MtrCAB, a trimeric porin-cytochrome complex that contains the cell surface exposed decaheme cytochrome MtrC. This donates electrons to extracellular substrates, including OmcA, soluble metals, organic electron shuttles, and insoluble metal oxides. However, it is not clear whether this broad substrate specificity requires specific sites for binding and reduction, or whether reduction occurs through non-specific interactions near exposed hemes on the cytochrome surface. Shewanella oneidensis MtrC is composed of four domains, with the hemes closely packed and distributed evenly between domains II and IV. The domains are arranged to allow electron transport across the cytochrome via interdomain electron transfer, but the significance of this conserved feature is not understood. Here we use site-directed mutagenesis to generate an MtrC variant that is comprised only of domains I and II (MtrC_DI,II). The properties of this MtrC_DI,II are effectively identical to domains I and II of full-length MtrC. Whole-cell assays revealed that S. oneidensis cells replacing full-length MtrC with MtrC_DI,II had significantly lower rates of OmcA, flavin mononucleotide, and Fe(III) citrate reduction. Our results demonstrate that MtrC domains III and IV contain sites for association of specific substrates, enabling the reduction of extracellular electron acceptors in S. oneidensis.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 8","pages":"e70243"},"PeriodicalIF":5.2,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12304082/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144732857","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":"Deep sequencing combined with high-throughput screening enables efficient development of a pH-dependent high-affinity binding domain targeting HER3.","authors":"Marit Möller, Malin Jönsson, Magnus Lundqvist, Johan Rockberg, John Löfblom, Hanna Tegel, Sophia Hober","doi":"10.1002/pro.70247","DOIUrl":"10.1002/pro.70247","url":null,"abstract":"<p><p>In vitro methods for developing binding domains have been well-established for many years, owing to the cost-efficient synthesis of DNA and high-throughput selection and screening technologies. However, generating high-affinity binding domains often requires the development of focused maturation libraries for a second selection, which typically demands a detailed understanding of the binding surfaces from the initial selection, a process that can be time-consuming. In this study, we accelerated this process by using deep sequencing data from the first selection to guide the design of the maturation library. Additionally, we employed a high-throughput screening system using flow cytometry based on Escherichia coli display to identify conditional binding domains from the selection output. This approach enabled the development of a high-affinity binder targeting the cancer biomarker HER3, with a binding affinity of 3.3 nM at extracellular pH 7.4, 100 times higher than the first-generation binding domain. Notably, the binding domain features a pH-dependent release mechanism, enabling rapid release in slightly acidic environments (pH ≈6), which resemble endosomal conditions. When conjugated to the cytotoxin mertansine (DM1), the binding domain demonstrated specific cytotoxic activity against HER3-expressing cell lines, with an IC50 of 2-5 nM. The presented approach enables the efficient development of conditional binding domains which hold promise for therapeutic applications.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 8","pages":"e70247"},"PeriodicalIF":5.2,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144732856","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":"Navigating infection by pathogenic spirochetes: The host-bacteria interface at the atomic level.","authors":"Libor Hejduk, Norbert Müller, Adriana Rathner, Ján Štěrba, Shang-Cheng Hung, Chia-Lin Chyan, Ryan O M Rego, Martin Strnad","doi":"10.1002/pro.70185","DOIUrl":"10.1002/pro.70185","url":null,"abstract":"<p><p>Pathogenic spirochetes bind and interact with various host structures and molecules throughout the course of infection. By utilizing their outer surface molecules, spirochetes can effectively modulate their dissemination, interact with immune system regulators, and select specific destination niches within the host. The three-dimensional structures of multiple spirochetal surface proteins have been elucidated, providing insight into their modus operandi. This review focuses on the structural characteristics of these sticky molecules and their functional implications, highlighting how these features contribute to the pathogenicity of spirochetes and their ability to persist in the host and vector environments. Recognizing the structural motifs and ligands to which these important virulence determinants bind could open new avenues for developing strategies to block colonization by spirochetal pathogens.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 7","pages":"e70185"},"PeriodicalIF":4.5,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12183334/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144369174","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":"A high throughput assay for measuring secreted protein based on a de novo fluorescent reporter reveals regulatory and structural insights in Salmonella type three secretion system.","authors":"Samuel Alexander Leach, Jordan Scott Summers, Edward Wen, Danielle Tullman-Ercek","doi":"10.1002/pro.70183","DOIUrl":"10.1002/pro.70183","url":null,"abstract":"<p><p>Intracellular protein production in bacteria is limited by the need for lysis and costly purification. A promising alternative is to engineer the host organism for protein secretion. While the Salmonella enterica serovar Typhimurium (Salmonella typhimurium) Type 3 Secretion System (T3SS) has been utilized for protein secretion, its study and eventual applicability for recombinant protein production are constrained by the lack of high-throughput assays to quantitatively measure secretion titer. Developing such assays is challenging, as proteins must remain unfolded for secretion, limiting the use of several common reporter proteins. In this work, we develop a high-throughput secretion assay using mini-fluorescence activating protein (mFAP). mFAP forms a chromophore only upon addition of an exogenous substrate, allowing secretion and subsequent fluorescence detection. We demonstrate mFAP secretion via the T3SS with an N-terminal secretion tag and show that the fluorescent signal in the secreted fraction is rapid and linear over three orders of magnitude. Using this assay, we screen S. typhimurium strains with secretion-enhancing mutations, identifying a constitutively active strain and revealing temporally controlled secretion dynamics. We also show that this assay may be applicable to other secretion systems, providing a universal tool for tracking heterologous protein secretion.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 7","pages":"e70183"},"PeriodicalIF":4.5,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12198052/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144497891","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":"Uncovering protein conformational dynamics within two-component viral biomolecular condensates.","authors":"Alice Colyer, Julia Acker, Alexander Borodavka, Antonio N Calabrese","doi":"10.1002/pro.70181","DOIUrl":"10.1002/pro.70181","url":null,"abstract":"<p><p>Biomolecular condensates selectively compartmentalize and organize biomolecules within the crowded cellular milieu and are instrumental in some disease mechanisms. Upon infection, many RNA viruses form biomolecular condensates that are often referred to as viral factories. The assembly mechanism of these viral factories remains poorly defined but involves transient, non-stoichiometric protein/RNA interactions, making their structural characterization challenging. Here, we sought to investigate the structural dynamics and intermolecular interactions of the key proteins responsible for condensate formation upon rotavirus infection, namely NSP2 (an RNA chaperone) and NSP5 (an intrinsically disordered protein [IDP]), using a combination of hydrogen-deuterium exchange mass spectrometry (HDX-MS), native MS, and biophysical tools. Our data reveal key structural features of intrinsically disordered NSP5 that are vital for condensate assembly and highlight inter/intra-protein interactions involved in condensate assembly. Moreover, we demonstrate that within a condensate there are altered conformational dynamics within the C-terminal region of NSP2, which has previously been shown to play a role in regulating its RNA chaperoning activity, and in the disordered regions of NSP5. We propose that altered conformational dynamics in NSP2 and NSP5 are critical for regulation of RNA annealing within a biomolecular condensate and for condensate assembly/client recruitment, respectively. Combined, our data demonstrate that the unique environment within a biomolecular condensate can tune functionally important protein conformational dynamics, which may play a crucial role in the replication of rotaviruses.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 7","pages":"e70181"},"PeriodicalIF":4.5,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12168090/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144302720","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":"Unraveling antibody-induced mechanical stability of antigen: Insights from single-molecule studies.","authors":"Soham Chakraborty, Shivam Pandit, Krishnendu Sinha, Madhu Bhatt, Debojyoti Chowdhury, Suman Chakrabarty, Shubhasis Haldar","doi":"10.1002/pro.70201","DOIUrl":"10.1002/pro.70201","url":null,"abstract":"<p><p>Antigen-antibody interaction, as a prominent ligand-receptor reaction, plays a crucial role in immunological responses. Notably, antigens can contain multiple ligand binding sites that define their intermolecular interactions more intricately and thereby make them context-dependent. Here, we have investigated the binding-induced effect of the largest antibody isotype, IgM, on protein L mechanical stability using single-molecule magnetic tweezers. Our results showed that IgM elevates the protein L mechanical stability by increasing its unfolding time. Interestingly, we were able to resolve distinct IgM-bound states of protein L by characterizing their unfolding dwell time: while the IgM-unbound state has the lowest dwell time, it increases with the IgM concentration via binding to either one or both of its binding sites, reconciling the IgM-induced protein L mechanical stability. To delve into the plausible mechanism of such intricate phenomena, we performed steered a molecular dynamic simulation of protein L and determined its unfolding rupture force at those multiple IgM-bound states, their corresponding molecular insights, and interaction gymnastics through binding interfaces. Additionally, we unraveled the mechanical response of these binding interfaces to be different; and during dimer IgM complex formation, these binding interfaces synergistically increase the mechanical stability of the complex. This provides the underlying principles of IgM-induced protein L stability under mechanical constraints. Overall, this study provides an in-depth understanding of a generic mechanism of antibody-induced mechanical stability of antigenic substrate under physiological sheer stress.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 7","pages":"e70201"},"PeriodicalIF":4.5,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12168486/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144302721","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":"Biomolecular condensates-Prerequisites for anhydrobiosis?","authors":"Charles A Elder, Hannah M Skaggs, Lynnette M A Dirk, David F Grimm, Clinton J Belott, Willem F Wolkers, Harriëtte Oldenhof, Vladimir N Uversky, A Bruce Downie, Michael A Menze","doi":"10.1002/pro.70192","DOIUrl":"10.1002/pro.70192","url":null,"abstract":"<p><p>It is often underappreciated that despite water being a requirement for life on Earth, organisms belonging to all taxonomic kingdoms have developed mechanisms to survive desiccation. These organisms, referred to as anhydrobiotes, accumulate specific biomolecules during or before drying that facilitate the survival of desiccation stress. Compounds utilized by a wide variety of anhydrobiotes during desiccation include metabolites such as sugars and amino acids, as well as proteins with extensive intrinsically disordered regions. Intrinsically disordered proteins that are constitutively expressed or upregulated during the onset or in preparation for desiccation include late embryogenesis abundant proteins, tardigrade disordered proteins, hydrophilins, some small heat shock proteins, and prion-like proteins. Some of these proteins form biomolecular condensates in the cellular environment. We hypothesize that phase transitions driven by anhydrobiosis-related intrinsically disordered proteins play a substantial role in enabling anhydrobiosis by (1) contributing to the downregulation of metabolic and developmental processes, (2) selectively sequestering desiccation-sensitive molecules into a \"protective compartment\" during drying, (3) interfering with programmed cell death signaling pathways to confer optimal time for the cell to repair after rehydration, (4) resisting intracellular volume changes to aid in membrane stabilization during desiccation, and (5) changing the biophysical properties of water to reduce desiccation-induced cellular damage. Biochemical strategies in anhydrobiotes are certainly multifaceted and may differ among systems. Nevertheless, a better understanding of the relevance of phase transitions in anhydrobiosis may allow us to get one step closer to unraveling the enigmatic phenomenon of life without water.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 7","pages":"e70192"},"PeriodicalIF":4.5,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12168135/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144302748","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":"Apo-state structure of the metabotropic glutamate receptor 5 transmembrane domain obtained using a photoswitchable ligand.","authors":"Yasushi Kondo, Caitlin Hatton, Robert Cheng, Matilde Trabuco, Hannah Glover, Quentin Bertrand, Fabienne Stierli, Hans-Peter Seidel, Thomas Mason, Sivathmika Sarma, Friedjof Tellkamp, Michal Kepa, Florian Dworkowski, Pedram Mehrabi, Michael Hennig, Joerg Standfuss","doi":"10.1002/pro.70104","DOIUrl":"10.1002/pro.70104","url":null,"abstract":"<p><p>Metabotropic glutamate receptor 5 (mGlu5) is implicated in various neurodegenerative disorders, making it an attractive drug target. Although several ligand-bound crystal structures of mGlu5 exist, their apo-state crystal structure remains unknown. Here, we study mGlu5 structural changes using the photochemical affinity switch, alloswitch-1, in combination with time-resolved freeze-trapping methods. By X-ray crystallography, we demonstrated that isomerizing alloswitch-1 leads to its release from the binding pocket within a few seconds. The apo structure, determined at a resolution of 2.9 Å, is more comparable to the inactive state than to the active state. Our approach presents an accessible alternative to time-resolved serial crystallography for capturing thermodynamically stable transient intermediates. The mGlu5 apo-structure provides molecular insights into the ligand-free allosteric pocket, which can guide the design of new allosteric modulators.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 7","pages":"e70104"},"PeriodicalIF":5.2,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12168131/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144302747","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}