Protein Science最新文献_第4页

Role of vinculin in the structural dynamics of cadherin-catenin complexes and its implications for F-actin binding. 血管蛋白在钙粘蛋白-连环蛋白复合物结构动力学中的作用及其对f -肌动蛋白结合的影响。

IF 5.2 3区生物学

Protein Science Pub Date : 2025-09-01 DOI: 10.1002/pro.70259

Benedict Hui, Zimei Bu, Xiaolin Cheng

{"title":"Role of vinculin in the structural dynamics of cadherin-catenin complexes and its implications for F-actin binding.","authors":"Benedict Hui, Zimei Bu, Xiaolin Cheng","doi":"10.1002/pro.70259","DOIUrl":"10.1002/pro.70259","url":null,"abstract":"Adherens junctions (AJs) are essential for maintaining tissue integrity and regulating intercellular signaling and tumor progression. At the core of AJs is the cadherin-catenin (ABE) complex, which links to the cytoskeletal actin filament (F-actin). Vinculin, a cytoskeletal protein, is recruited to AJs under recurrently high tensions to modulate cell adhesion. However, the molecular mechanisms underlying vinculin recruitment and activation remain elusive due to the highly dynamic and heterogeneous nature of the cadherin-catenin-vinculin (VABE) complex. In this study, we performed molecular dynamics (MD) simulations to probe the structure, dynamics, and domain interactions within the VABE complex. Our simulations reveal that vinculin binding enhances the conformational flexibility of α-catenin and expands the configurational space sampled by its actin-binding domain (ABD). This is consistent with an increase in configurational entropy upon complex assembly, suggesting that an entropic trap mechanism-previously proposed for ABE-may also underlie force-sensitive binding in the VABE complex. Furthermore, we provide detailed structural insights into α-catenin/vinculin and α-catenin/β-catenin interactions, elucidating how vinculin recruitment impacts the dynamics of the ABE complex. Interestingly, while vinculin binding increases overall structural fluctuations, ABD exposure remains comparable to that of the ABE complex alone. This is likely due to ABD's interaction with the M1 subdomain, which emerges from α-catenin unfolding upon vinculin binding. Together, these findings deepen our understanding of vinculin-mediated mechanotransduction at AJs and its role in modulating cytoskeletal dynamics.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 9","pages":"e70259"},"PeriodicalIF":5.2,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12355968/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144856119","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}

引用次数: 0

Computational modeling and experimental validation of the interaction between tumor biomarker mesothelin and an engineered targeting protein with therapeutic activity. 肿瘤生物标志物间皮素与具有治疗活性的工程靶向蛋白之间相互作用的计算建模和实验验证。

IF 5.2 3区生物学

Protein Science Pub Date : 2025-09-01 DOI: 10.1002/pro.70263

Margherita Piccardi, Valeria Butera, Ignazio Sardo, Stefano Landi, Federica Gemignani, Giampaolo Barone, Angelo Spinello, Sarah J Moore

{"title":"Computational modeling and experimental validation of the interaction between tumor biomarker mesothelin and an engineered targeting protein with therapeutic activity.","authors":"Margherita Piccardi, Valeria Butera, Ignazio Sardo, Stefano Landi, Federica Gemignani, Giampaolo Barone, Angelo Spinello, Sarah J Moore","doi":"10.1002/pro.70263","DOIUrl":"10.1002/pro.70263","url":null,"abstract":"Mesothelin (MSLN) is a cell surface glycoprotein overexpressed in many solid tumors, which is known to interact with cancer antigen CA125/MUC16, promoting cancer cell adhesion and metastasis. MSLN has been used as a target of multiple antibody-based therapeutic strategies, but their efficacy remains limited, potentially due to inherent pharmacokinetics conferred by the structure of antibodies (~150 kDa). To provide an alternative targeting molecule, we engineered a small scaffold protein derived from the tenth domain of human fibronectin type III (Fn3, 12.8 kDa) to bind MSLN with nanomolar affinity as a theranostic agent for MSLN-positive cancers. In this study, we explored the Fn3-MSLN interaction site through computational modeling and experimentally validated the model through domain-level and fine epitope mapping. Fn3-MSLN binding was predicted by a consensus approach, comparing multiple protein-protein docking software, the deep-learning-based algorithm AlphaFold3, and performing molecular dynamics (MD) simulations. To validate the prediction, full-length MSLN, single MSLN domains, or combinations of domains were expressed on the yeast surface, and Fn3 binding to displayed MSLN domains was measured by flow cytometry. The employed algorithms predicted two distinct binding modes for Fn3. Overall, experimental data agreed with our in silico prediction resulting from the AlphaFold3 model, confirming that MSLN domains B and C are predominantly involved in the interaction.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 9","pages":"e70263"},"PeriodicalIF":5.2,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12355969/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144856113","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}

引用次数: 0

RNA binding and coacervation promote preservation of peptide form and function across the heterochiral-homochiral divide. RNA的结合和凝聚促进了多肽形式和功能在异手性-同手性分裂中的保存。

IF 5.2 3区生物学

Protein Science Pub Date : 2025-09-01 DOI: 10.1002/pro.70273

Manas Seal, Ilan Edelstein, Yosef Scolnik, Orit Weil-Ktorza, Norman Metanis, Yaakov Levy, Liam M Longo, Daniella Goldfarb

{"title":"RNA binding and coacervation promote preservation of peptide form and function across the heterochiral-homochiral divide.","authors":"Manas Seal, Ilan Edelstein, Yosef Scolnik, Orit Weil-Ktorza, Norman Metanis, Yaakov Levy, Liam M Longo, Daniella Goldfarb","doi":"10.1002/pro.70273","DOIUrl":"https://doi.org/10.1002/pro.70273","url":null,"abstract":"Recent evidence suggests that peptide-RNA coacervates may have buffered the emergence of folded domains from flexible peptides. As primitive peptides were likely composed of both L- and D-amino acids, we hypothesized that coacervates may have also supported the emergence of chiral control. To test this hypothesis, we compared the coacervation propensities of an isotactic (homochiral) peptide and a syndiotactic (alternating chirality) peptide, both with an identical sequence derived from the ancient helix-hairpin-helix (HhH) motif. Using electron paramagnetic resonance (EPR) spectroscopy and atomistic molecular dynamics (MD) simulations, we found that the syndiotactic peptide does not form stable dimers with high α-helicity in solution, unlike the isotactic peptide. However, both peptides do coacervate with RNA, albeit with distinct reentrant phase behaviors. Coacervation in each case is facilitated by oligomer formation, likely dimerization, upon RNA binding that promotes RNA cross-linking. Additionally, RNA cross-linking and coacervation of the syndiotactic peptide may involve α-helical conformations, according to atomistic MD simulations. Coarse-grained MD simulations indicate that the differences in reentrant phase behavior of isotactic and syndiotactic peptides are associated with differences in dimer flexibility and stability, which modulate the strength of peptide-peptide and peptide-RNA interactions and, consequently, the effectiveness of RNA cross-linking. These results illustrate how RNA binding and/or coacervation by early proteins could have promoted the transition of flexible, heterochiral peptides into folded, homochiral domains.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 9","pages":"e70273"},"PeriodicalIF":5.2,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12375978/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144966521","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}

引用次数: 0

AlphaFold model quality self-assessment improvement via deep graph learning. 通过深度图学习改进AlphaFold模型质量自评估。

IF 5.2 3区生物学

Protein Science Pub Date : 2025-09-01 DOI: 10.1002/pro.70274

Jacob Verburgt, Zicong Zhang, Daisuke Kihara

引用次数: 0

An intrinsically disordered region of Ubp10 regulates its binding and activity on ubiquitinated histone substrates. Ubp10的内在紊乱区域调节其在泛素化组蛋白底物上的结合和活性。

IF 5.2 3区生物学

Protein Science Pub Date : 2025-08-01 DOI: 10.1002/pro.70237

Anneliese M Faustino, Melesse Nune, Raquel Merino-Urteaga, Edgar Manriquez-Sandoval, Matthew Poyton, Taekjip Ha, Cynthia Wolberger, Stephen D Fried

{"title":"An intrinsically disordered region of Ubp10 regulates its binding and activity on ubiquitinated histone substrates.","authors":"Anneliese M Faustino, Melesse Nune, Raquel Merino-Urteaga, Edgar Manriquez-Sandoval, Matthew Poyton, Taekjip Ha, Cynthia Wolberger, Stephen D Fried","doi":"10.1002/pro.70237","DOIUrl":"10.1002/pro.70237","url":null,"abstract":"Monoubiquitinated histone H2B at K123 in yeast (K120 in humans) is a transient modification that is both attached and removed during transcription. H2B is ubiquitinated in yeast by the E2/E3 pair, Rad6/Bre1, and deubiquitinated by two enzymes, Ubp8 and Ubp10. Previous studies had shown that Ubp10 has higher activity on ubiquitinated H2A/H2B dimers than on intact nucleosomes, but that activity on nucleosomes is higher in the presence of the histone chaperone, FACT. By contrast, the Ubp8 complex has equal activity on both histone substrates and is unaffected by FACT. We report here the results of single-molecule FRET experiments showing that FACT unwraps DNA and evicts ubiquitinated H2A/H2B dimers, the preferred substrate of Ubp10. To explore the basis for the differing activity of Ubp10 on ubiquitinated H2A/H2B dimers and nucleosomes, we employed crosslinking mass spectrometry combined with structural modeling. These studies revealed that Ubp10 forms a different set of interactions with H2A/H2B in free versus nucleosomal states. Acidic stretches within the N-terminal intrinsically disordered region (IDR) of Ubp10 interact extensively with H2A/H2B heterodimers, whereas this portion of Ubp10 interacts more with the tails of histones H3 and H4 in the nucleosome. The importance of these interactions for affinity is consistent with binding studies showing the IDR is necessary for substrate interactions. Structural modeling using the crosslinks as constraints suggests that the complex formed by Ubp10 with free H2A/H2B dimers could not be formed within a nucleosome due to steric clash with the DNA, H3, and H4, thereby explaining its low activity on ubiquitinated nucleosomes.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 8","pages":"e70237"},"PeriodicalIF":5.2,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144732923","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}

引用次数: 0

Structural investigations connect the disordered N-terminal extension of HypB to the activities of HypB and SlyD in E. coli. 结构研究将大肠杆菌中HypB的无序n端延伸与HypB和sld的活性联系起来。

IF 5.2 3区生物学

Protein Science Pub Date : 2025-08-01 DOI: 10.1002/pro.70231

Wayne W H Law, Dmitry Pichugin, Ranjith Muhandiram, Deborah B Zamble, Voula Kanelis

{"title":"Structural investigations connect the disordered N-terminal extension of HypB to the activities of HypB and SlyD in E. coli.","authors":"Wayne W H Law, Dmitry Pichugin, Ranjith Muhandiram, Deborah B Zamble, Voula Kanelis","doi":"10.1002/pro.70231","DOIUrl":"10.1002/pro.70231","url":null,"abstract":"The activities of [NiFe]-hydrogenase enzymes, which are critical to many microbes, require insertion of a Ni(II) ion into the bimetallic catalytic center. Delivery of Ni(II) to [NiFe]-hydrogenases depends, in part, on the metallochaperone HypB, which lies at the center of a Ni(II) transfer pathway that includes the metal storage protein SlyD and the metallochaperone HypA. SlyD is a source of Ni(II) ions for HypB, whereas Ni(II) from HypB is transferred to HypA. In this work, we examine how the intrinsically disordered N-terminal extension (NTE) of HypB modulates the action of the HypB GTPase domain (G-domain). The NTE contains a high-affinity Ni(II) binding site, while the G-domain contains a lower affinity Ni(II) binding site that is affected by binding of guanine nucleotides. The HypB G-domain is also affected by SlyD and provides Ni(II) to HypA. Our NMR data show that, although disordered, the HypB NTE possesses residual structure and makes transient interactions with the HypB G-domain and with SlyD. A set of common residues in the center of the NTE are affected by SlyD and G-domain binding, and also by binding of Ni(II) to the high-affinity site located at the N terminus of the protein. The NTE interacts with residues in or near the Ni(II)- and GDP-binding sites in the G-domain, which are also affected when SlyD binds the NTE. Thus, the data showcase a complex interaction network between HypB and SlyD, and provide molecular details regarding how the HypB NTE links the activities of the HypB G-domain and SlyD.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 8","pages":"e70231"},"PeriodicalIF":5.2,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12284835/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144691296","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}

引用次数: 0

Barrel expansion of outer membrane protein G nanopore through β-hairpin duplication. 通过β-发夹复制外膜蛋白G纳米孔的桶状扩张。

IF 5.2 3区生物学

Protein Science Pub Date : 2025-08-01 DOI: 10.1002/pro.70203

Joshua C Foster, Bach Pham, Ryan Pham, Patrick Ryan, Nhu Tong, Jacqueline Sharp, Satomi Inaba-Inoue, Jie Liang, Konstantinos Beis, Min Chen

{"title":"Barrel expansion of outer membrane protein G nanopore through β-hairpin duplication.","authors":"Joshua C Foster, Bach Pham, Ryan Pham, Patrick Ryan, Nhu Tong, Jacqueline Sharp, Satomi Inaba-Inoue, Jie Liang, Konstantinos Beis, Min Chen","doi":"10.1002/pro.70203","DOIUrl":"https://doi.org/10.1002/pro.70203","url":null,"abstract":"Outer membrane β-barrel proteins (OMPs) are channels found in the outer membranes of Gram-negative bacteria characterized by a stable and diverse barrel architecture, which has made them attractive for nanopore sensing applications. Here, we systematically investigated the feasibility of expanding outer membrane protein G (OmpG) from its native 14-stranded β-barrel to an enhanced conductance variant by independently duplicating each of its seven hairpin units and inserting them downstream of their endogenous positions. Most combinations did not increase pore diameter, but duplication of the terminal seventh hairpin exhibited a rare population of pores with enhanced conductance, suggesting barrel enlargement. Further engineering efforts to optimize the terminal β-turn sequence have resulted in up to 50% of pores with increased conductance. Importantly, the enlarged pores retained the sensing functionality of the original scaffold, highlighting the potential of this approach for developing β-barrel OMP sensors with tunable dimensions.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 8","pages":"e70203"},"PeriodicalIF":5.2,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12267663/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144966515","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}

引用次数: 0

An alternative pocket for binding the N-degrons by the UBR1 and UBR2 ubiquitin E3 ligases. 通过UBR1和UBR2泛素E3连接连接N-degrons的替代口袋。

IF 5.2 3区生物学

Protein Science Pub Date : 2025-08-01 DOI: 10.1002/pro.70248

Shih-Ting Huang, Dai-Hua Chen, Tianchen Ren, Nicole Thomas, Jian Wu, Banumathi Sankaran, Renee Jones, Susan Taylor, Yuan Chen

{"title":"An alternative pocket for binding the N-degrons by the UBR1 and UBR2 ubiquitin E3 ligases.","authors":"Shih-Ting Huang, Dai-Hua Chen, Tianchen Ren, Nicole Thomas, Jian Wu, Banumathi Sankaran, Renee Jones, Susan Taylor, Yuan Chen","doi":"10.1002/pro.70248","DOIUrl":"https://doi.org/10.1002/pro.70248","url":null,"abstract":"The UBR family of ubiquitin ligases binds to N-termini of their targets (known as N-degron) to induce their ubiquitination and degradation via a conserved domain known as UBR-box. UBR1 and UBR2 share the highest sequence homology among the family, and substantial structural studies were previously performed for substrate binding by the UBR-boxes of UBR1 and UBR2. Here, we describe a new pocket in the UBR-boxes of UBR1 and UBR2 for binding the second residues of N-degrons through determining five co-crystal structures of the UBR-boxes with various N-degron peptides. Together with binding affinities measured by fluorescence polarization, we show that the two highly homologous UBR-boxes can interact with the second residue of an N-degron differently. In addition, the UBR-boxes undergo different conformational changes when binding N-degrons. Furthermore, we demonstrate that the sidechain of the third amino acid of an N-degron has no contribution to binding the UBR-boxes. These findings represent a new conceptual advancement for the UBR E3 ligases and the new insights described here can be leveraged for developing their selective ligands for research and potential therapies.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 8","pages":"e70248"},"PeriodicalIF":5.2,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12304086/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144966494","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}

引用次数: 0

Resolving the structure and assembly of the honeybee silk heterotetrameric coiled coil. 解决了蜜蜂丝异质四聚体线圈的结构和装配问题。

IF 5.2 3区生物学

Protein Science Pub Date : 2025-08-01 DOI: 10.1002/pro.70230

Caitlin L Johnston, Chacko Jobichen, Lyndall J Briggs, Michelle Michie, Jian-Wei Liu, Craig J Morton, Andrew C Warden, Tara D Sutherland

{"title":"Resolving the structure and assembly of the honeybee silk heterotetrameric coiled coil.","authors":"Caitlin L Johnston, Chacko Jobichen, Lyndall J Briggs, Michelle Michie, Jian-Wei Liu, Craig J Morton, Andrew C Warden, Tara D Sutherland","doi":"10.1002/pro.70230","DOIUrl":"10.1002/pro.70230","url":null,"abstract":"Coiled coil structures, first proposed by Crick in the 1950s, are protein structural motifs found across diverse biological systems. Honeybee silk was among the earliest identified coiled coils, with X-ray diffraction studies in the 1960s revealing its characteristic helical packing. Decades of research have provided insights into silk composition and formation, yet the molecular details of its coiled coil assembly and final structure remained unresolved. In this study, we generated a structural model of the tetrameric coiled coil using AlphaFold and validated it with crosslinking mass spectrometry and medium-resolution cryo-electron microscopy. The model reveals that the four proteins (F1-F4) adopt an antiparallel configuration in a defined clockwise arrangement (F1-F3-F2-F4). Furthermore, we experimentally investigated the formation of this coiled coil complex using biochemical techniques, including blue-native PAGE and circular dichroism spectroscopy. The sum of these experimental results and the structural predictions has allowed for the elucidation of key transitional steps in the assembly pathway, suggesting molecular interactions that may drive tetramer formation. These findings support a stepwise assembly model in which F2 and F4 form a stable core, F3 binds to the complex, and F1 initiates formation of the final, highly ordered structure. These structural insights establish a framework for understanding and directing coiled coil assembly, the fundamental building block of honeybee silk. By resolving this structure and its assembly process, this work lays the foundation for future rational design of functional sequences and materials with tailored properties.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 8","pages":"e70230"},"PeriodicalIF":5.2,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12284834/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144691295","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}

引用次数: 0

Two cysteines control Tse1 secretion by H1-T6SS in Pseudomonas aeruginosa. 两种半胱氨酸控制铜绿假单胞菌H1-T6SS分泌Tse1。

IF 5.2 3区生物学

Protein Science Pub Date : 2025-08-01 DOI: 10.1002/pro.70226

Marie M Grandjean, Jean-Pierre Duneau, Edwige B Garcin, Laetitia Houot, Olivier Bornet, Christophe Bordi, Latifa Elantak, Corinne Sebban-Kreuzer

{"title":"Two cysteines control Tse1 secretion by H1-T6SS in Pseudomonas aeruginosa.","authors":"Marie M Grandjean, Jean-Pierre Duneau, Edwige B Garcin, Laetitia Houot, Olivier Bornet, Christophe Bordi, Latifa Elantak, Corinne Sebban-Kreuzer","doi":"10.1002/pro.70226","DOIUrl":"10.1002/pro.70226","url":null,"abstract":"Type Six Secretion Systems (T6SS) are molecular machines that export toxic effector proteins into bacterial competitors or eukaryotic cells. Pseudomonas aeruginosa's H1-T6SS secretes Tse1, which contains a disulfide bond between cysteines at positions 7 and 148, linking its N- and C-terminal regions. The role of this disulfide bond in Tse1 activity and mechanism of action during bacterial competition is unknown. In this study, we investigated the role of the C7-C148 disulfide bond within Tse1. First, NMR spectroscopy experiments suggest a redox-active instead of a structural disulfide bond. Moreover, while the presence of this bond did not alter Tse1's amidase activity or toxicity in Escherichia coli, substituting cysteines C7 or C148 in P. aeruginosa strains affected the bacterium's capacity to lyse prey cells. Secretome analysis showed that the Tse1C148S variant was not secreted via the H1-T6SS, whereas the Tse1C7S variant was secreted. These findings suggest that cysteine 148 is likely important for Tse1's assembly with the T6SS machinery, while cysteine 7 appears to be involved in its disassembly, potentially through the formation of the disulfide bond. This study points to a potential redox regulation mechanism during the assembly and disassembly of Tse1 with Hcp1, consistent with a \"bridge of delivery\" model.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 8","pages":"e70226"},"PeriodicalIF":5.2,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12302283/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144732859","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}

引用次数: 0