Protein Science最新文献

{"title":"Dynamic networks connect the USP14 active site region with the proteasome interaction surface.","authors":"Johannes Salomonsson, Linda Sjöstrand, Arvid Eskilson, Dean Derbyshire, Pádraig D'Arcy, Maria Sunnerhagen, Alexandra Ahlner","doi":"10.1002/pro.70077","DOIUrl":"10.1002/pro.70077","url":null,"abstract":"<p><p>Ubiquitin-specific protease 14 (USP14) is a member of the USP family responsible for the catalytic removal of ubiquitin (Ub) from proteins directed to the proteasome, implicated in the pathogenesis of neurodegeneration and cancer. Crystallography and cryo-EM analysis have identified loop regions crucial for the deubiquitinase activity of USP14, specifically those involved in Ub and proteasome binding. However, the structural changes in USP14 upon ligand binding to these regions are minimal, indicating significant yet uncharacterized dynamic contributions to its function. In this study, through structural and dynamical NMR experiments and functional evaluation, we demonstrate that small mutations designed to impact Ub binding and catalytic activity without disturbing the USP structure display both local and long-range effects. The affected residues connect the catalytic site and the Ub binding region with the proteasome interaction surface through a network of loops, which show varied dynamics on the ps-ms time scale. Collectively, our findings experimentally reveal different aspects of dynamic connections within USP14, suggesting the presence of allosteric networks that link enzyme activity with regulatory function. The identification of coupled clusters of possible allostery participants in the free USP domain provides new insights into the dynamic regulation of USP14, with potential implications for understanding its role in cellular processes.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 4","pages":"e70077"},"PeriodicalIF":4.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11912437/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143650295","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":"Deciphering opening mechanisms of 14-3-3 proteins.","authors":"Exequiel E Barrera, Rostislav Skrabana, Diego M Bustos","doi":"10.1002/pro.70108","DOIUrl":"10.1002/pro.70108","url":null,"abstract":"<p><p>The 14-3-3 proteins are a highly conserved family of regulatory molecules that play crucial roles in various cellular processes. They are known for their ability to bind to phosphorylated serine and threonine residues on target proteins, which allows them to modulate their activity, localization, and stability. In mammals, there are seven known paralogs of 14-3-3 proteins, designated as β, ε, ζ, η, σ, τ, and γ. Each paralog has distinct biological functions and tissue distributions, which allow a diverse range of regulatory roles in cellular processes. The conformational plasticity of 14-3-3s regulates their interaction with protein partners but has not yet been thoroughly characterized. We investigated this topic by classical molecular dynamics simulations and observed how the γ, ε, and ζ paralogs exhibit different opening rates. A PCA analysis identified the main modes of these opening-conformational variations. Using correlation-based tools and simulations with single amino acid substitutions, we have recognized how the amphipathic 14-3-3 groove opening is triggered by a distally located aliphatic-π interaction. The identified residues form a partially conserved small cavity between helices H6, H7, and H8, representing a potential paralog-specific drug site.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 4","pages":"e70108"},"PeriodicalIF":4.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11934215/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143701393","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 new view of missense mutations in α-mannosidosis using molecular dynamics conformational ensembles.","authors":"Špela Mandl, Bruno Di Geronimo, Santiago Alonso-Gil, Christoph Grininger, Gibu George, Ulrika Ferstl, Sereina Annik Herzog, Bojan Žagrović, Christoph Nusshold, Tea Pavkov-Keller, Pedro A Sánchez-Murcia","doi":"10.1002/pro.70080","DOIUrl":"10.1002/pro.70080","url":null,"abstract":"<p><p>The mutation of remote positions on enzyme scaffolds and how these residue changes can affect enzyme catalysis is still far from being fully understood. One paradigmatic example is the group of lysosomal storage disorders, where the enzyme activity of a lysosomal enzyme is abolished or severely reduced. In this work, we analyze molecular dynamics simulation conformational ensembles to unveil the molecular features controlling the deleterious effects of the 43 reported missense mutations in the human lysosomal α-mannosidase. Using residue descriptors for protein dynamics, their coupling with the active site, and their impact on protein stability, we have assigned the contribution of each of the missense mutations into protein stability, protein dynamics, and their connectivity with the active site. We demonstrate here that the use of conformational ensembles is a powerful approach not only to better understand missense mutations at the molecular level but also to revisit the missense mutations reported in lysosomal storage disorders in order to aid the treatment of these diseases.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 4","pages":"e70080"},"PeriodicalIF":4.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11931667/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143693129","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":"AlphaFold2's training set powers its predictions of some fold-switched conformations.","authors":"Joseph W Schafer, Lauren L Porter","doi":"10.1002/pro.70105","DOIUrl":"10.1002/pro.70105","url":null,"abstract":"<p><p>AlphaFold2 (AF2), a deep-learning-based model that predicts protein structures from their amino acid sequences, has recently been used to predict multiple protein conformations. In some cases, AF2 has successfully predicted both dominant and alternative conformations of fold-switching proteins, which remodel their secondary and/or tertiary structures in response to cellular stimuli. Whether AF2 has learned enough protein folding principles to reliably predict alternative conformations outside of its training set is unclear. Previous work suggests that AF2 predicted these alternative conformations by memorizing them during training. Here, we use CFold-an implementation of the AF2 network trained on a more limited subset of experimentally determined protein structures-to directly test how well the AF2 architecture predicts alternative conformations of fold switchers outside of its training set. We tested CFold on eight fold switchers from six protein families. These proteins-whose secondary structures switch between α-helix and β-sheet and/or whose hydrogen bonding networks are reconfigured dramatically-had not been tested previously, and only one of their alternative conformations was in CFold's training set. Successful CFold predictions would indicate that the AF2 architecture can predict disparate alternative conformations of fold-switched conformations outside of its training set, while unsuccessful predictions would suggest that AF2 predictions of these alternative conformations likely arise from association with structures learned during training. Despite sampling 1300-4300 structures/protein with various sequence sampling techniques, CFold predicted only one alternative structure outside of its training set accurately and with high confidence while also generating experimentally inconsistent structures with higher confidence. Though these results indicate that AF2's current success in predicting alternative conformations of fold switchers stems largely from its training data, results from a sequence pruning technique suggest developments that could lead to a more reliable generative model in the future.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 4","pages":"e70105"},"PeriodicalIF":4.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11934219/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143701391","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":"Fine-tuned interactions between globular and disordered regions of single-stranded DNA binding (SSB) protein are required for dynamic condensation under physiological conditions.","authors":"Zoltán J Kovács, Péter Ecsédi, Gábor M Harami, János Pálinkás, Mina Botros, Lamiya Mahmudova, Viktoria Katran, Dávid Érfalvy, Miklós Cervenak, László Smeller, Mihály Kovács","doi":"10.1002/pro.70109","DOIUrl":"10.1002/pro.70109","url":null,"abstract":"<p><p>Increasing evidence points to the importance of liquid-liquid phase separation (LLPS)-driven protein condensation in both eukaryotic and bacterial cell physiology. The formation of condensates may involve interactions between both structured (globular) domains and intrinsically disordered protein regions and requires multivalency that is often brought about by oligomerization. Here we dissect such contributions by assessing engineered variants of bacterial (Escherichia coli) single-stranded DNA binding (SSB) protein whose condensation has recently been implicated in bacterial genome metabolism. A truncated SSB variant (SSBdC, lacking the conserved C-terminal peptide (CTP)) was used to assess the importance of interactions between SSB's globular oligonucleotide/oligosaccharide binding (OB) domain and the CTP. We show that OB-CTP interactions are essential for dynamic condensation in physiological (crowded, glutamate-rich) environments. Via assessment of a protein variant (SSB^H55Y) from the known thermosensitive ssb-1 mutant, we also show that the perturbation of OB-OB contacts significantly impairs the stability of SSB tetramers and results in thermally induced protein aggregation, underscoring the importance of multivalence brought about by stereospecific contacts. Our data point to adaptive fine-tuning of SSB interactions to physiological condensation and demonstrate that SSB represents a versatile system for selective engineering of condensation-driving interactions between globular and disordered regions.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 4","pages":"e70109"},"PeriodicalIF":4.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11947617/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143721199","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":"Visualizing and analyzing 3D biomolecular structures using Mol* at RCSB.org: Influenza A H5N1 virus proteome case study.","authors":"Sebastian Bittrich, Alexander S Rose, David Sehnal, Jose M Duarte, Yana Rose, Joan Segura, Dennis W Piehl, Brinda Vallat, Chenghua Shao, Charmi Bhikadiya, Jesse Liang, Mark Ma, David S Goodsell, Stephen K Burley, Shuchismita Dutta","doi":"10.1002/pro.70093","DOIUrl":"10.1002/pro.70093","url":null,"abstract":"<p><p>The easiest and often most useful way to work with experimentally determined or computationally predicted structures of biomolecules is by viewing their three-dimensional (3D) shapes using a molecular visualization tool. Mol* was collaboratively developed by RCSB Protein Data Bank (RCSB PDB, RCSB.org) and Protein Data Bank in Europe (PDBe, PDBe.org) as an open-source, web-based, 3D visualization software suite for examination and analyses of biostructures. It is capable of displaying atomic coordinates and related experimental data of biomolecular structures together with a variety of annotations, facilitating basic and applied research, training, education, and information dissemination. Across RCSB.org, the RCSB PDB research-focused web portal, Mol* has been implemented to support single-mouse-click atomic-level visualization of biomolecules (e.g., proteins, nucleic acids, carbohydrates) with bound cofactors, small-molecule ligands, ions, water molecules, or other macromolecules. RCSB.org Mol* can seamlessly display 3D structures from various sources, allowing structure interrogation, superimposition, and comparison. Using influenza A H5N1 virus as a topical case study of an important pathogen, we exemplify how Mol* has been embedded within various RCSB.org tools-allowing users to view polymer sequence and structure-based annotations integrated from trusted bioinformatics data resources, assess patterns and trends in groups of structures, and view structures of any size and compositional complexity. In addition to being linked to every experimentally determined biostructure and Computed Structure Model made available at RCSB.org, Standalone Mol* is freely available for visualizing any atomic-level or multi-scale biostructure at rcsb.org/3d-view.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 4","pages":"e70093"},"PeriodicalIF":4.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11915458/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143658318","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 novel protein for bioremediation of gadolinium waste.","authors":"Harvey D Lee, Connor J Grady, Katie Krell, Cooper Strebeck, Aimen Al-Hilfi, Brianna Ricker, Melanie Linn, Nicole Y Xin, Nathan M Good, N Cecilia Martinez-Gomez, Assaf A Gilad","doi":"10.1002/pro.70101","DOIUrl":"10.1002/pro.70101","url":null,"abstract":"<p><p>Several hundreds of tons of gadolinium-based contrast agents (GBCAs) are being dumped into the environment every year. Although macrocyclic GBCAs exhibit superior stability compared to their linear counterparts, we have found that the structural integrity of chelates is susceptible to ultraviolet light, regardless of configuration. In this study, we present a synthetic protein termed GLamouR that binds and reports gadolinium in an intensiometric manner. We then explore the extraction of gadolinium from MRI patient urine as a preventative measure for gadolinium pollution and investigate the viability of employing cost-effective bioremediation techniques for treating contaminated water bodies. Based on promising results, we anticipate proteins such as GLamouR can be used for detecting and mining rare earth elements beyond gadolinium and hope to expand the biological toolbox for such applications.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 4","pages":"e70101"},"PeriodicalIF":4.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11915603/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143658390","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":"Solution structure of the Z0 domain from transcription repressor BCL11A sheds light on the sequence properties of protein-binding zinc fingers.","authors":"Rilee E Harris, Richard D Whitehead, Andrei T Alexandrescu","doi":"10.1002/pro.70097","DOIUrl":"10.1002/pro.70097","url":null,"abstract":"<p><p>The transcription repressor BCL11A governs the switch from fetal to adult hemoglobin during development. By targeting BCL11A, fetal hemoglobin expression can be de-repressed to substitute for defective adult hemoglobin in inherited diseases including beta-thalassemia and sickle-cell anemia. BCL11A has six CCHH-type zinc fingers, of which domains 4-6 are necessary and sufficient for dsDNA binding. Here, we focus on a putative ZNF at the N-terminus of BCL11A (residues 46-72), Z0, thought to modulate oligomerization of the transcription repressor. Using NMR and CD spectroscopy at low concentrations that favor the monomer, Z0 is shown to be a thermostable CCHC zinc finger with a pM dissociation constant for zinc. The NMR structure of Z0 has a prototypical beta-beta-alpha fold, with a hydrophobic knob comprising about half the structure. The unusual proportion of hydrophobic residues in Z0 led us to investigate if this is a more general feature of zinc fingers that do not bind dsDNA. We used the ZF and WebLogo servers to examine sequences of zinc fingers with demonstrated DNA-binding function, non-DNA-binders, and the CCHC-type family of protein-binders. DNA-binders are distinguished by contiguous stretches of high-scoring zinc fingers. Non-DNA-binders show a depletion of polar residues at the positions expected to contact nucleotides and increased sequence divergence, making these domains more likely to be annotated as atypical, degenerate, or to be missed as zinc fingers. We anticipate these sequence patterns will help distinguish DNA-binders from non-binders, an open problem in the functional understanding of zinc-finger motifs.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 4","pages":"e70097"},"PeriodicalIF":4.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11915622/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143658308","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":"Crowding beyond excluded volume: A tale of two dimers.","authors":"Gil I Olgenblum, Claire J Stewart, Thomas W Redvanly, Owen M Young, Francis Lauzier, Sophia Hazlett, Shikun Wang, David A Rockcliffe, Stuart Parnham, Gary J Pielak, Daniel Harries","doi":"10.1002/pro.70062","DOIUrl":"10.1002/pro.70062","url":null,"abstract":"<p><p>Protein-protein interactions are modulated by their environment. High macromolecular solute concentrations crowd proteins and shift equilibria between protein monomers and their assemblies. We aim to understand the mechanism of crowding by elucidating the molecular-level interactions that determine dimer stability. Using ¹⁹F-NMR spectroscopy, we studied the effects of various polyethylene glycols (PEGs) on the equilibrium thermodynamics of two protein complexes: a side-by-side and a domain-swap dimer. Analysis using our mean-field crowding model shows that, contrary to classic crowding theories, PEGs destabilize both dimers through enthalpic interactions between PEG and the monomers. The enthalpic destabilization becomes more dominant with increasing PEG concentration because the reduction in PEG mesh size with concentration diminishes the stabilizing effect of excluded volume interactions. Additionally, the partially folded domain-swap monomers fold in the presence of PEG, contributing to dimer stabilization at low PEG concentrations. Our results reveal that polymers crowd protein complexes through multiple conjoined mechanisms, impacting both their stability and oligomeric state.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 4","pages":"e70062"},"PeriodicalIF":4.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11912439/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143650327","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":"Structural insights into the fusion of annexin A5 and fluorescent proteins generating hundredfold differentiated binding affinities to phosphatidylserine.","authors":"Mengyue Gao, Wei Tang, Shihui Wang, Yunke Wang, Minjin Hu, Zichun Hua","doi":"10.1002/pro.70086","DOIUrl":"10.1002/pro.70086","url":null,"abstract":"<p><p>Fluorescent proteins (FPs) are an indispensable part of modern biology. Numerous studies utilize FPs for protein labeling and cell tracking purposes. They are commonly fused with proteins to aid in their visualization. It is generally assumed that these FP tags have minimal impact on the properties of the fusion proteins. Do the FP types affect the function and characteristics of target proteins on earth? So far, there is no definite answer. Fluorescent annexin A5 (AnxA5) has been extensively employed as apoptosis probes. However, except for chemically labeled AnxA5, there are few developed FP-based AnxA5 probes. Therefore, it is essential to screen out suitable FPs for developing high-affinity AnxA5 probes. Here, various fusion proteins (AnxA5-FPs) were developed. The fusion of AnxA5 did not change the chromophore environments of FPs, while the fusion of FPs led to over a 100-fold difference in AnxA5's affinity for phosphatidylserine (PS). We found that polymeric AnxA5-FPs had higher PS-affinity. Remarkably, although the structures of FPs were similar, they fused with AnxA5 in different modes, generating fusion proteins with different spatial conformations. The difference in conformation resulted in variations in the PS-binding pattern of AnxA5, leading to differing levels of PS-affinity. More importantly, we found five high-affinity (Kd > 10^-7 M) FP-based AnxA5 probes with different excitation wavelengths. Together, these observations suggested that differences in the fusion modes of AnxA5 and FPs provided a robust mechanism for modulating PS-affinity of AnxA5. We anticipate that our findings can provide a guideline to develop highly sensitive AnxA5 probes.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 4","pages":"e70086"},"PeriodicalIF":4.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11917116/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143658313","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}