Protein Science最新文献

{"title":"Decoding the allosteric grammar of protein kinases: A dual-stream framework integrating protein language models and energy landscape frustration analysis.","authors":"Will Gatlin, Max Ludwick, Lucas Turano, Brandon Foley, Kamila Riedlová, Vít Škrhák, Marian Novotný, David Hoksza, Gennady M Verkhivker","doi":"10.1002/pro.70714","DOIUrl":"10.1002/pro.70714","url":null,"abstract":"<p><p>The spatial and energetic encoding of allosteric regulatory sites remains a major challenge in structural biology, frequently representing a \"blind spot\" for sequence-based artificial intelligence (AI) models. We present a protein language model (PLM)-guided approach complemented by the energy landscape frustration analysis as a dual-stream framework to investigate the relationship between AI prediction of binding sites and biophysical organization of regulatory pockets across the human kinome. By probing a fine-tuned residue-level PLM classifier across 453 kinase structures, a clear performance gap is discovered between highly predictable orthosteric pockets (Types I, I.5, and II) and poorly resolved distal allosteric sites (Type IV). Rather than attempting to interpret this blind spot through internal AI attributions alone, we use independent local frustration profiles to analyze the underlying physics of these sites. We determine that the detectability of orthosteric and allosteric binding sites reflects their energetic embedding within the protein energy landscape. Orthosteric catalytic sites reside within minimally frustrated, optimized energetic regions that are consistently detected with high confidence. In contrast, allosteric sites are enriched in neutrally frustrated zones, producing diffuse and context-dependent predictions. We demonstrate that this neutral frustration of functional regions acts as a biophysical lubricant, facilitating the conformational plasticity required for regulatory transitions while simultaneously eroding the coevolutionary signals exploited by PLMs. Atomic-resolution analysis of abelson murine leukemia (ABL) kinase spanning multiple conformational states and complexes bound to diverse ligands provides mechanistic validation of this principle. The myristoyl allosteric pocket in ABL remains neutrally frustrated across complexes with physiological ligands, chemically diverse modulators, from allosteric inhibitors to activators, and conformations engaged with SH2-SH3 regulatory domains. We propose that allosteric sites are encoded in persistent neutrally frustrated regions optimized for context-dependent regulatory modulation. This study reveals how the organization of the protein energy landscape shapes universal \"allosteric grammar\" and algorithmic detectability of regulatory binding sites.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"35 8","pages":"e70714"},"PeriodicalIF":5.6,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13347374/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148412643","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":"Applications and limitations of AI tools in enzyme design.","authors":"Rosa Teijeiro-Juiz, Nina Egeler, Grzegorz Jamróg, Marion Ringel, Thomas Brück, Bruno Di Geronimo, Bernhard Loll","doi":"10.1002/pro.70698","DOIUrl":"10.1002/pro.70698","url":null,"abstract":"<p><p>Enzymes catalyze various different chemical reactions often with high efficiency and selectivity compared to synthetic catalysts. Advances in protein engineering over the past decades have allowed researchers to design enzymes, improving their catalytic performance and adapting them for specific or entirely novel chemical reactions. However, the need for the experimental validation of thousands of computational designs remains one of the major bottlenecks. Yet another restriction is our still limited knowledge about transition state architectures, effects of mutations, active-site dynamics just to name a few. To overcome this, the combination of experimental and computational methods is essential, yet many experimentalists are facing significant obstacles when entering the field of computational enzyme design. To address these obstacles, this review offers a comprehensive introduction and overview of several current artificial intelligence (AI)-driven methods available for enzyme design, with a focus on reaction-to-sequence design, structure prediction, substrate scope prediction, engineering of stable variants, design of enzymes with non-canonical amino acids, and de novo design. Subsequently, this work serves as an accessible guide for experimental researchers with interest in learning how to use AI-based computational methods in enzyme engineering.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"35 8","pages":"e70698"},"PeriodicalIF":5.6,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13351933/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148423291","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":"Pocket restraints guided by B-cell epitope prediction improve Chai-1 antibody-antigen structure modeling.","authors":"Joakim Nøddeskov Clifford, Morten Nielsen","doi":"10.1002/pro.70730","DOIUrl":"10.1002/pro.70730","url":null,"abstract":"<p><p>The accurate prediction of antibody-antigen (AbAg) complexes is a key challenge for computational immunology, with applications in therapeutic antibody design and diagnostics. Current deep learning methods have the potential to generate high-confidence AbAg structures. However, these methods often fail to predict the correct AbAg structure, placing the antibody incorrectly on the antigen and converge on repeatedly predicting the same redundant binding mode. Here, we present BepiPocket and DiscoPocket, two simple approaches that integrate B-cell epitope prediction tools to guide antibody-epitope restraints during Chai-1 structure prediction. On a dataset of 1628 AbAg complexes, we demonstrate that using the sequence-based predictor BepiPred-3.0 (BepiPocket) and the structure-based predictor DiscoTope-3.0 (DiscoPocket) substantially improved both the accuracy and diversity of predicted AbAg complexes compared to standard Chai-1 modeling with random seed variation. The software for both BepiPocket and DiscoPocket algorithms is freely available at https://github.com/mnielLab/BepiPocket.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"35 8","pages":"e70730"},"PeriodicalIF":5.6,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13393073/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148562828","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":"Sequence determinants of the hypomobility of intrinsically disordered proteins in SDS-PAGE.","authors":"Ankush Garg, Maciej B Gielnik, Magnus Kjaergaard","doi":"10.1002/pro.70725","DOIUrl":"10.1002/pro.70725","url":null,"abstract":"<p><p>Proteins with intrinsically disordered regions (IDRs) migrate at a higher apparent molecular weight in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, complicating their analysis and identification. Here, we investigate the sequence determinants of the hypomobility of IDRs using a series of synthetic low-complexity domains. We find that negative charge increases the apparent molecular weight, but neutral polar tracts also have abnormally slow migration. Positive charge and hydrophobic residues decrease the apparent molecular weight, although lysine residues show a biphasic effect with decreased migration at high fractional contents. Combinations of residues show that different sequence contributions to the apparent molecular weight are not additive. The results can be rationalized by the protein-decorated micelle model by considering both SDS binding and the compaction of protein SDS-complexes.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"35 8","pages":"e70725"},"PeriodicalIF":5.6,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13371092/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148448872","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":"Critical amino acid residues in the N-terminal domain of NADPH-dependent assimilatory sulfite reductase flavoprotein mediate octameric assembly.","authors":"Nidhi Walia, Thais Pedrete, Fozieh Ahmadizadeh, Ekhtiar Rahman, Yashika Garg, Brian Washburn, Cheryl Pye, Fanny C Liu, Peter Randolph, Kevin L Weiss, Gergely Nagy, Christian Bleiholder, M Elizabeth Stroupe","doi":"10.1002/pro.70709","DOIUrl":"10.1002/pro.70709","url":null,"abstract":"<p><p>How large, flexible enzymes assemble into defined oligomeric architectures remains a central question in biology. NADPH-dependent assimilatory sulfite reductase (SiR) forms a heterododecamer built on an octameric flavoprotein (SiRFP) core, yet the molecular basis for this assembly has been unresolved because of its disordered N-terminus. Here, we use ion mobility mass spectrometry, small-angle neutron scattering, and mutagenesis to define the mechanism of SiRFP oligomerization. We show that SiRFP forms a discrete, stable octamer in solution. We also report that its N-terminal 52-residue segment is necessary and sufficient to mediate assembly, also mediating oligomerization when fused to a heterologous protein. Structure-guided mutagenesis identifies four residues (Gln22, Tyr39, Phe40, and Gln47) whose substitution disrupts the octamer, producing concentration-dependent lower-order species while retaining catalytic activity. These findings define the determinants of SiRFP assembly with broader implications for engineering homomeric protein complexes.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"35 8","pages":"e70709"},"PeriodicalIF":5.6,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13355859/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148423398","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":"Mitochondrial membrane remodeling in stress adaptation: Lipid control of organelle quality.","authors":"Lena J Reichert, F-Nora Vögtle, Carlotta Peselj","doi":"10.1002/pro.70703","DOIUrl":"10.1002/pro.70703","url":null,"abstract":"<p><p>Mitochondria respond to proteotoxic stress through the mitochondrial unfolded protein response, traditionally viewed as a transcriptional program that restores proteostasis by inducing chaperones and proteases. Emerging evidence indicates that mitochondrial membrane remodeling constitutes an additional adaptive component of this response. Regulated changes in mitochondrial lipid composition, particularly involving the signature phospholipid cardiolipin, support mitochondrial function during stress by stabilizing protein import machineries, promoting mitochondrial protein biogenesis, and facilitating recovery from dysfunction. In addition, stress originating in other organelles, especially the endoplasmic reticulum, reshapes mitochondrial membranes through altered lipid biosynthesis, inter-organelle lipid trafficking, and stress signaling pathways. These findings suggest that mitochondrial membrane remodeling represents a regulatory layer of organelle quality control integrated within interconnected stress response networks and may provide new opportunities to enhance mitochondrial resilience in disease.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"35 8","pages":"e70703"},"PeriodicalIF":5.6,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13347175/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148412772","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":"Caspase-mediated cleavage events hidden by secondary proteolysis during apoptosis.","authors":"Fatemeh Fotouhi, Erik Gomez-Cardona, Jainilkumar Patel, Olivier Julien, Howard Fearnhead","doi":"10.1002/pro.70724","DOIUrl":"10.1002/pro.70724","url":null,"abstract":"<p><p>Caspases are cysteine proteases that cleave specific proteins to control a range of cellular processes including cell death, inflammation, and differentiation. Proteomic approaches, like N-terminomics, have been central to identifying both cleaved proteins and where they are cleaved. We recently reported N-terminomics on differentiating and dying C2C12 myoblasts, identifying many new caspase substrates and cleavage sites. Here, we present a further analysis of this dataset demonstrating that there are caspase substrates \"hidden\" in our and in other N-terminomics datasets. We propose that for a subset of proteins, caspase cleavage is followed by removal of two amino acids from the neo-N-terminus by a dipeptidyl peptidase (DPP). As the peptide sequence of the neo-N-terminus is used to identify the cleavage site, this hides the caspase-mediated cleavage event. The implications of this are that N-terminomics datasets can be used to reveal hitherto unidentified caspase substrates and provide more information on cell-fate decisions. In C2C12 myoblasts, we have shown that cleavage of \"hidden\" substrates in apoptotic myoblasts that is absent from differentiating myoblasts (which also contain active caspase-3), suggesting that cleavage may identify apoptotic cells. A better understanding of the interplay between caspases and other proteases and their sequential action on caspase substrates may allow further elucidation of cell fate decisions.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"35 8","pages":"e70724"},"PeriodicalIF":5.6,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13385206/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148536657","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":"Evolution of N-terminal mechanical lability as a determinant for Type III secretion.","authors":"Katherine E DaPron, Alexandre M Plastow, Morgan R Fink, Brandon Dzuba, Marc-Andre LeBlanc, Thomas T Perkins, Emad Tajkhorshid, Marcelo C Sousa","doi":"10.1002/pro.70708","DOIUrl":"10.1002/pro.70708","url":null,"abstract":"<p><p>Many Gram-negative pathogens critically depend on the Type III secretion system (T3SS) to inject effector proteins into host cells for colonization. Because the channel of the T3SS is narrow (~2 nm), effectors must be unfolded for secretion. However, the T3SS cannot unfold mechanically robust substrates (GFP, ubiquitin, and dihydrofolate reductase), severely impairing their secretion. Consistent with this, effectors are exceptionally mechanically labile, unfolding at low forces. Thus, secretion competency is correlated with mechanical properties. Effector sequences have significantly diverged from non-effectors, suggesting that secretion exerts evolutionary pressure selecting mechanical lability. Here, using atomic-force-microscopy-based force spectroscopy, we show that effector NleC is mechanically labile (F_unfold = 13.5 pN at 100 nm/s) and mechanically compliant, as characterized by a large distance to the transition state (Δx^‡ = 2.7 nm). In contrast, the non-effector homolog protealysin is mechanically stable (F_unfold = 50.7 pN at 100 nm/s) and brittle (Δx^‡ = 0.7 nm), comparable to proteins known to impair secretion (F_unfold >80 pN; Δx^‡ <0.4 nm). Denaturant-induced unfolding assays demonstrate that effectors exhibit rates typical of their fold, further reinforcing mechanical properties rather than fast unfolding kinetics (k₀) predicts secretion. Steered molecular dynamic simulations revealed NleC unfolding initiates at the N-terminus, consistent with current secretion models, whereas protealysin unfolding initiates at the C-terminus. Notably, the NleC N-terminus is primarily α-helical while non-effector homologs contain β-sheets, which may account for the distinct unfolding pathway. Together, these results support the notion that mechanical lability is an evolved, structurally encoded feature underlying effector secretion.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"35 8","pages":"e70708"},"PeriodicalIF":5.6,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13351618/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148423458","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":"MIF-MAPMS: Enhancing identification of myelin autoantigenic peptides in multiple sclerosis through multimodal information fusion.","authors":"Watshara Shoombuatong, Nalini Schaduangrat, Pramote Chumnanpuen, Lawankorn Mookdarsanit, Pakpoom Mookdarsanit","doi":"10.1002/pro.70695","DOIUrl":"10.1002/pro.70695","url":null,"abstract":"<p><p>Multiple sclerosis (MS) arises from an autoimmune response in which the immune system erroneously targets myelin autoantigens within the central nervous system, leading to myelin degradation and subsequent neurological dysfunction. Identifying myelin autoantigenic peptides (MAPs) is therefore critical for understanding MS pathogenesis and developing targeted therapies; however, conventional experimental approaches remain time-consuming and costly. Thus, computational methods that can perform in silico screening of T cell-specific MAP in MS (MAPMSs) using only peptide sequences are highly desirable. Existing computational methods primarily rely on a single modality, which often fails to capture key information of MAPMSs, leading to limited sequence representation and generalization ability. To address this limitation, we propose MIF-MAPMS, a novel multimodal information fusion framework that leverages multimodal information, including peptide format and SMILEs notation, for accurate MAPMS identification. This novel framework processes different modalities of compositional descriptors, molecular fingerprints, ESM-2 embeddings, and Mol2V embeddings using specific deep learning methods, leading to enriched MAPMS representation. Subsequently, the extracted embeddings are fused and passed through a multilayer perceptron (MLP), followed by a fully connected neural network for MAPMS identification. Both cross-validation and independent test results show that MIF-MAPMS attains significant improvements in MAPMS identification over the benchmark main and alternative datasets, with Matthew's correlation coefficient (MCC) of 0.931-0.968 and 0.812-0.928, providing 5.78%-8.04% and 1.22%-2.98% increases, respectively, compared to the existing method. Ablation studies further confirm the necessity of multimodal information fusion in improving MAPMS representation and the model's predictive performance. All codes and datasets are freely available online at https://github.com/lawankorn-m/MIF-MAPMS.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"35 8","pages":"e70695"},"PeriodicalIF":5.6,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13347377/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148412696","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":"Immunoglobulin binding protein (BiP) forms two types of dimers.","authors":"Karina New, Miguel I A Lagos-Espinoza, Nathalie Casanova-Morales, Roi Asor, John William Young, Zahra Alavi, Christian A M Wilson","doi":"10.1002/pro.70690","DOIUrl":"10.1002/pro.70690","url":null,"abstract":"<p><p>Immunoglobulin binding protein (BiP) is a chaperone protein that plays crucial roles in protein folding and transport by binding unfolded proteins in the substrate binding domain (SBD), an interaction allosterically linked to the nucleotide occupancy of the nucleotide binding domain (NBD). BiP also forms oligomers that influence its activity, particularly in binding polypeptide clients. Using single-molecule approaches and mechanical and enzymatic activity assays, BiP monomer stability and oligomerization were analyzed both alone and in response to nucleotides and peptides. We find that the formation of BiP dimers exhibits biphasic behavior as a function of BiP concentration, suggesting concentration-dependent changes in dimer dissociation constants. At low BiP concentrations, dimers were disrupted by peptide substrate and adenosine triphosphate (ATP) but remained unaffected by ADP or Adenosine 5'-O-(3-thio)triphosphate (ATPγS). At high concentrations, dimers are unaffected by peptides, but their assembly is inhibited by ATP and ATPγS to the same degree. These results suggest the formation of two distinct BiP dimers exhibiting unique binding affinities, kinetics, and potentially structures. We propose the existence of a high-affinity dimer binding site within the SBD of BiP, while the formation of a low-affinity dimer involves interactions between the lid and NBD of each protomer. Our findings demonstrate the importance of considering single-molecule characteristics when interpreting bulk studies on protein function and regulation.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"35 8","pages":"e70690"},"PeriodicalIF":5.6,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13347316/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148412704","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}