Sirtuin 2 (SIRT2) is a class III histone deacetylase that is highly conserved from bacteria to mammals. We prepared and characterized the wild‐type (WT) and mutant forms of the histone deacetylase (HDAC) domain of human SIRT2 (hSIRT2) using various biophysical methods and evaluated their deacetylation activity. We found that WT hSIRT2 HDAC (residues 52–357) forms a homodimer in a concentration‐dependent manner with a dimer–monomer dissociation constant of 8.3 ± 0.5 μM, which was determined by mass spectrometry. The dimer was disrupted into two monomers by binding to the HDAC inhibitors SirReal1 and SirReal2. We also confirmed dimer formation of hSIRT2 HDAC in living cells using a NanoLuc complementation reporter system. Examination of the relationship between dimer formation and deacetylation activity using several mutants of hSIRT2 HDAC revealed that some non‐dimerizing mutants exhibited deacetylation activity for the N‐terminal peptide of histone H3, similar to the wild type. The hSIRT2 HDAC mutant Δ292–306, which lacks a SIRT2‐specific disordered loop region, was identified to exist as a monomer with slightly reduced deacetylation activity; the X‐ray structure of the mutant Δ292–306 was almost identical to that of the WT hSIRT2 HDAC bound to an inhibitor. These results indicate that hSIRT2 HDAC forms a dimer, but this is independent of deacetylation activity. Herein, we discuss insights into the dimer formation of hSIRT2 based on our biophysical experimental results.
{"title":"Biophysical insights into the dimer formation of human Sirtuin 2","authors":"Noa Suzuki, Tsuyoshi Konuma, Takahisa Ikegami, Satoko Akashi","doi":"10.1002/pro.4994","DOIUrl":"https://doi.org/10.1002/pro.4994","url":null,"abstract":"Sirtuin 2 (SIRT2) is a class III histone deacetylase that is highly conserved from bacteria to mammals. We prepared and characterized the wild‐type (WT) and mutant forms of the histone deacetylase (HDAC) domain of human SIRT2 (<jats:italic>h</jats:italic>SIRT2) using various biophysical methods and evaluated their deacetylation activity. We found that WT <jats:italic>h</jats:italic>SIRT2 HDAC (residues 52–357) forms a homodimer in a concentration‐dependent manner with a dimer–monomer dissociation constant of 8.3 ± 0.5 μM, which was determined by mass spectrometry. The dimer was disrupted into two monomers by binding to the HDAC inhibitors SirReal1 and SirReal2. We also confirmed dimer formation of <jats:italic>h</jats:italic>SIRT2 HDAC in living cells using a NanoLuc complementation reporter system. Examination of the relationship between dimer formation and deacetylation activity using several mutants of <jats:italic>h</jats:italic>SIRT2 HDAC revealed that some non‐dimerizing mutants exhibited deacetylation activity for the N‐terminal peptide of histone H3, similar to the wild type. The <jats:italic>h</jats:italic>SIRT2 HDAC mutant <jats:italic>Δ</jats:italic>292–306, which lacks a SIRT2‐specific disordered loop region, was identified to exist as a monomer with slightly reduced deacetylation activity; the X‐ray structure of the mutant <jats:italic>Δ</jats:italic>292–306 was almost identical to that of the WT <jats:italic>h</jats:italic>SIRT2 HDAC bound to an inhibitor. These results indicate that <jats:italic>h</jats:italic>SIRT2 HDAC forms a dimer, but this is independent of deacetylation activity. Herein, we discuss insights into the dimer formation of <jats:italic>h</jats:italic>SIRT2 based on our biophysical experimental results.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":null,"pages":null},"PeriodicalIF":8.0,"publicationDate":"2024-04-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140634229","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}
Michael C. Baxa, Xiaoxuan Lin, Cedrick D. Mukinay, Srinivas Chakravarthy, Joseph R. Sachleben, Sarah Antilla, Nina Hartrampf, Joshua A. Riback, Isabelle A. Gagnon, Bradley L. Pentelute, Patricia L. Clark, Tobin R. Sosnick
Despite the generally accepted role of the hydrophobic effect as the driving force for folding, many intrinsically disordered proteins (IDPs), including those with hydrophobic content typical of foldable proteins, behave nearly as self‐avoiding random walks (SARWs) under physiological conditions. Here, we tested how temperature and ionic conditions influence the dimensions of the N‐terminal domain of pertactin (PNt), an IDP with an amino acid composition typical of folded proteins. While PNt contracts somewhat with temperature, it nevertheless remains expanded over 10–58°C, with a Flory exponent, ν, >0.50. Both low and high ionic strength also produce contraction in PNt, but this contraction is mitigated by reducing charge segregation. With 46% glycine and low hydrophobicity, the reduced form of snow flea anti‐freeze protein (red‐sfAFP) is unaffected by temperature and ionic strength and persists as a near‐SARW, ν ~ 0.54, arguing that the thermal contraction of PNt is due to stronger interactions between hydrophobic side chains. Additionally, red‐sfAFP is a proxy for the polypeptide backbone, which has been thought to collapse in water. Increasing the glycine segregation in red‐sfAFP had minimal effect on ν. Water remained a good solvent even with 21 consecutive glycine residues (ν > 0.5), and red‐sfAFP variants lacked stable backbone hydrogen bonds according to hydrogen exchange. Similarly, changing glycine segregation has little impact on ν in other glycine‐rich proteins. These findings underscore the generality that many disordered states can be expanded and unstructured, and that the hydrophobic effect alone is insufficient to drive significant chain collapse for typical protein sequences.
{"title":"How hydrophobicity, side chains, and salt affect the dimensions of disordered proteins","authors":"Michael C. Baxa, Xiaoxuan Lin, Cedrick D. Mukinay, Srinivas Chakravarthy, Joseph R. Sachleben, Sarah Antilla, Nina Hartrampf, Joshua A. Riback, Isabelle A. Gagnon, Bradley L. Pentelute, Patricia L. Clark, Tobin R. Sosnick","doi":"10.1002/pro.4986","DOIUrl":"https://doi.org/10.1002/pro.4986","url":null,"abstract":"Despite the generally accepted role of the hydrophobic effect as the driving force for folding, many intrinsically disordered proteins (IDPs), including those with hydrophobic content typical of foldable proteins, behave nearly as self‐avoiding random walks (SARWs) under physiological conditions. Here, we tested how temperature and ionic conditions influence the dimensions of the N‐terminal domain of pertactin (PNt), an IDP with an amino acid composition typical of folded proteins. While PNt contracts somewhat with temperature, it nevertheless remains expanded over 10–58°C, with a Flory exponent, <jats:italic>ν</jats:italic>, >0.50. Both low and high ionic strength also produce contraction in PNt, but this contraction is mitigated by reducing charge segregation. With 46% glycine and low hydrophobicity, the reduced form of snow flea anti‐freeze protein (red‐sfAFP) is unaffected by temperature and ionic strength and persists as a near‐SARW, <jats:italic>ν</jats:italic> ~ 0.54, arguing that the thermal contraction of PNt is due to stronger interactions between hydrophobic side chains. Additionally, red‐sfAFP is a proxy for the polypeptide backbone, which has been thought to collapse in water. Increasing the glycine segregation in red‐sfAFP had minimal effect on <jats:italic>ν</jats:italic>. Water remained a good solvent even with 21 consecutive glycine residues (<jats:italic>ν</jats:italic> > 0.5), and red‐sfAFP variants lacked stable backbone hydrogen bonds according to hydrogen exchange. Similarly, changing glycine segregation has little impact on <jats:italic>ν</jats:italic> in other glycine‐rich proteins. These findings underscore the generality that many disordered states can be expanded and unstructured, and that the hydrophobic effect alone is insufficient to drive significant chain collapse for typical protein sequences.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":null,"pages":null},"PeriodicalIF":8.0,"publicationDate":"2024-04-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140572555","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}
Yubexi Correa, Mathilde Ravel, Marie Imbert, Sarah Waldie, Luke Clifton, Ann Terry, Felix Roosen‐Runge, Jens O. Lagerstedt, Michael Moir, Tamim Darwish, Marité Cárdenas, Rita Del Giudice
High‐density lipoproteins (HDLs) are responsible for removing cholesterol from arterial walls, through a process known as reverse cholesterol transport. The main protein in HDL, apolipoprotein A‐I (ApoA‐I), is essential to this process, and changes in its sequence significantly alter HDL structure and functions. ApoA‐I amyloidogenic variants, associated with a particular hereditary degenerative disease, are particularly effective at facilitating cholesterol removal, thus protecting carriers from cardiovascular disease. Thus, it is conceivable that reconstituted HDL (rHDL) formulations containing ApoA‐I proteins with functional/structural features similar to those of amyloidogenic variants hold potential as a promising therapeutic approach. Here we explored the effect of protein cargo and lipid composition on the function of rHDL containing one of the ApoA‐I amyloidogenic variants G26R or L174S by Fourier transformed infrared spectroscopy and neutron reflectometry. Moreover, small‐angle x‐ray scattering uncovered the structural and functional differences between rHDL particles, which could help to comprehend higher cholesterol efflux activity and apparent lower phospholipid (PL) affinity. Our findings indicate distinct trends in lipid exchange (removal vs. deposition) capacities of various rHDL particles, with the rHDL containing the ApoA‐I amyloidogenic variants showing a markedly lower ability to remove lipids from artificial membranes compared to the rHDL containing the native protein. This effect strongly depends on the level of PL unsaturation and on the particles' ultrastructure. The study highlights the importance of the protein cargo, along with lipid composition, in shaping rHDL structure, contributing to our understanding of lipid–protein interactions and their behavior.
高密度脂蛋白（HDL）负责通过一种被称为胆固醇逆向运输的过程将胆固醇从动脉壁中清除。高密度脂蛋白中的主要蛋白质载脂蛋白 A-I（ApoA-I）对这一过程至关重要，其序列的变化会显著改变高密度脂蛋白的结构和功能。载脂蛋白 A-I 淀粉样变体与一种特殊的遗传性退行性疾病有关，在促进胆固醇清除方面特别有效，从而保护携带者免受心血管疾病的侵害。因此，可以想象，含有载脂蛋白 I 蛋白的重组高密度脂蛋白（rHDL）制剂具有与淀粉样蛋白变体相似的功能/结构特征，有望成为一种有前景的治疗方法。在此，我们通过傅立叶变换红外光谱和中子反射仪，探讨了蛋白质载体和脂质组成对含有一种载脂蛋白A-I淀粉样蛋白变体G26R或L174S的rHDL功能的影响。此外，小角 X 射线散射揭示了 rHDL 颗粒之间的结构和功能差异，这有助于理解更高的胆固醇外排活性和明显较低的磷脂（PL）亲和力。我们的研究结果表明，不同的rHDL颗粒在脂质交换（清除与沉积）能力方面有不同的趋势，与含有原生蛋白的rHDL相比，含有ApoA-I淀粉样蛋白变体的rHDL从人工膜上清除脂质的能力明显较低。这种效应在很大程度上取决于聚乳酸的不饱和程度和颗粒的超微结构。这项研究强调了蛋白质货物以及脂质成分在塑造 rHDL 结构方面的重要性，有助于我们了解脂质与蛋白质之间的相互作用及其行为。
{"title":"Lipid exchange of apolipoprotein A‐I amyloidogenic variants in reconstituted high‐density lipoprotein with artificial membranes","authors":"Yubexi Correa, Mathilde Ravel, Marie Imbert, Sarah Waldie, Luke Clifton, Ann Terry, Felix Roosen‐Runge, Jens O. Lagerstedt, Michael Moir, Tamim Darwish, Marité Cárdenas, Rita Del Giudice","doi":"10.1002/pro.4987","DOIUrl":"https://doi.org/10.1002/pro.4987","url":null,"abstract":"High‐density lipoproteins (HDLs) are responsible for removing cholesterol from arterial walls, through a process known as reverse cholesterol transport. The main protein in HDL, apolipoprotein A‐I (ApoA‐I), is essential to this process, and changes in its sequence significantly alter HDL structure and functions. ApoA‐I amyloidogenic variants, associated with a particular hereditary degenerative disease, are particularly effective at facilitating cholesterol removal, thus protecting carriers from cardiovascular disease. Thus, it is conceivable that reconstituted HDL (rHDL) formulations containing ApoA‐I proteins with functional/structural features similar to those of amyloidogenic variants hold potential as a promising therapeutic approach. Here we explored the effect of protein cargo and lipid composition on the function of rHDL containing one of the ApoA‐I amyloidogenic variants G26R or L174S by Fourier transformed infrared spectroscopy and neutron reflectometry. Moreover, small‐angle x‐ray scattering uncovered the structural and functional differences between rHDL particles, which could help to comprehend higher cholesterol efflux activity and apparent lower phospholipid (PL) affinity. Our findings indicate distinct trends in lipid exchange (removal vs. deposition) capacities of various rHDL particles, with the rHDL containing the ApoA‐I amyloidogenic variants showing a markedly lower ability to remove lipids from artificial membranes compared to the rHDL containing the native protein. This effect strongly depends on the level of PL unsaturation and on the particles' ultrastructure. The study highlights the importance of the protein cargo, along with lipid composition, in shaping rHDL structure, contributing to our understanding of lipid–protein interactions and their behavior.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":null,"pages":null},"PeriodicalIF":8.0,"publicationDate":"2024-04-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140572671","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}
Miguel Romano‐Moreno, Elsa N. Astorga‐Simón, Adriana L. Rojas, Aitor Hierro
Endosomal trafficking ensures the proper distribution of lipids and proteins to various cellular compartments, facilitating intracellular communication, nutrient transport, waste disposal, and the maintenance of cell structure. Retromer, a peripheral membrane protein complex, plays an important role in this process by recruiting the associated actin‐polymerizing WASH complex to establish distinct sorting domains. The WASH complex is recruited through the interaction of the VPS35 subunit of retromer with the WASH complex subunit FAM21. Here, we report the identification of two separate fragments of FAM21 that interact with VPS35, along with a third fragment that binds to the VPS29 subunit of retromer. The crystal structure of VPS29 bound to a peptide derived from FAM21 shows a distinctive sharp bend that inserts into a conserved hydrophobic pocket with a binding mode similar to that adopted by other VPS29 effectors. Interestingly, despite the network of interactions between FAM21 and retromer occurring near the Parkinson's disease‐linked mutation (D620N) in VPS35, this mutation does not significantly impair the direct association with FAM21 in vitro.
{"title":"Retromer‐mediated recruitment of the WASH complex involves discrete interactions between VPS35, VPS29, and FAM21","authors":"Miguel Romano‐Moreno, Elsa N. Astorga‐Simón, Adriana L. Rojas, Aitor Hierro","doi":"10.1002/pro.4980","DOIUrl":"https://doi.org/10.1002/pro.4980","url":null,"abstract":"Endosomal trafficking ensures the proper distribution of lipids and proteins to various cellular compartments, facilitating intracellular communication, nutrient transport, waste disposal, and the maintenance of cell structure. Retromer, a peripheral membrane protein complex, plays an important role in this process by recruiting the associated actin‐polymerizing WASH complex to establish distinct sorting domains. The WASH complex is recruited through the interaction of the VPS35 subunit of retromer with the WASH complex subunit FAM21. Here, we report the identification of two separate fragments of FAM21 that interact with VPS35, along with a third fragment that binds to the VPS29 subunit of retromer. The crystal structure of VPS29 bound to a peptide derived from FAM21 shows a distinctive sharp bend that inserts into a conserved hydrophobic pocket with a binding mode similar to that adopted by other VPS29 effectors. Interestingly, despite the network of interactions between FAM21 and retromer occurring near the Parkinson's disease‐linked mutation (D620N) in VPS35, this mutation does not significantly impair the direct association with FAM21 in vitro.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":null,"pages":null},"PeriodicalIF":8.0,"publicationDate":"2024-04-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140572659","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}
Johanna Trommer, Florian Lesniowski, Johannes Buchner, Hristo L. Svilenov
The antigen‐binding sites in conventional antibodies are formed by hypervariable complementarity‐determining regions (CDRs) from both heavy chains (HCs) and light chains (LCs). A deviation from this paradigm is found in a subset of bovine antibodies that bind antigens via an ultra‐long CDR. The HCs bearing ultra‐long CDRs pair with a restricted set of highly conserved LCs that convey stability to the antibody. Despite the importance of these LCs, their specific features remained unknown. Here, we show that the conserved bovine LC found in antibodies with ultra‐long CDRs exhibits a distinct combination of favorable physicochemical properties such as good secretion from mammalian cells, strong dimerization, high stability, and resistance to aggregation. These physicochemical traits of the LCs arise from a combination of the specific sequences in the germline CDRs and a lambda LC framework. In addition to understanding the molecular architecture of antibodies with ultra‐long CDRs, our findings reveal fundamental insights into LC characteristics that can guide the design of antibodies with improved properties.
{"title":"Specific features of a scaffolding antibody light chain","authors":"Johanna Trommer, Florian Lesniowski, Johannes Buchner, Hristo L. Svilenov","doi":"10.1002/pro.4990","DOIUrl":"https://doi.org/10.1002/pro.4990","url":null,"abstract":"The antigen‐binding sites in conventional antibodies are formed by hypervariable complementarity‐determining regions (CDRs) from both heavy chains (HCs) and light chains (LCs). A deviation from this paradigm is found in a subset of bovine antibodies that bind antigens via an ultra‐long CDR. The HCs bearing ultra‐long CDRs pair with a restricted set of highly conserved LCs that convey stability to the antibody. Despite the importance of these LCs, their specific features remained unknown. Here, we show that the conserved bovine LC found in antibodies with ultra‐long CDRs exhibits a distinct combination of favorable physicochemical properties such as good secretion from mammalian cells, strong dimerization, high stability, and resistance to aggregation. These physicochemical traits of the LCs arise from a combination of the specific sequences in the germline CDRs and a lambda LC framework. In addition to understanding the molecular architecture of antibodies with ultra‐long CDRs, our findings reveal fundamental insights into LC characteristics that can guide the design of antibodies with improved properties.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":null,"pages":null},"PeriodicalIF":8.0,"publicationDate":"2024-04-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140572665","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}
Alba Ledesma‐Fernandez, Susana Velasco‐Lozano, Pedro Campos‐Muelas, Ricardo Madrid, Fernando López‐Gallego, Aitziber L. Cortajarena
Enzyme scaffolding is an emerging approach for enhancing the catalytic efficiency of multi‐enzymatic cascades by controlling their spatial organization and stoichiometry. This study introduces a novel family of engineered SCAffolding Bricks, named SCABs, utilizing the consensus tetratricopeptide repeat (CTPR) domain for organized multi‐enzyme systems. Two SCAB systems are developed, one employing head‐to‐tail interactions with reversible covalent disulfide bonds, the other relying on non‐covalent metal‐driven assembly via engineered metal coordinating interfaces. Enzymes are directly fused to SCAB modules, triggering assembly in a non‐reducing environment or by metal presence. A proof‐of‐concept with formate dehydrogenase (FDH) and L‐alanine dehydrogenase (AlaDH) shows enhanced specific productivity by 3.6‐fold compared to free enzymes, with the covalent stapling outperforming the metal‐driven assembly. This enhancement likely stems from higher‐order supramolecular assembly and improved NADH cofactor regeneration, resulting in more efficient cascades. This study underscores the potential of protein engineering to tailor scaffolds, leveraging supramolecular spatial‐organizing tools, for more efficient enzymatic cascade reactions.
{"title":"Engineering bio‐brick protein scaffolds for organizing enzyme assemblies","authors":"Alba Ledesma‐Fernandez, Susana Velasco‐Lozano, Pedro Campos‐Muelas, Ricardo Madrid, Fernando López‐Gallego, Aitziber L. Cortajarena","doi":"10.1002/pro.4984","DOIUrl":"https://doi.org/10.1002/pro.4984","url":null,"abstract":"Enzyme scaffolding is an emerging approach for enhancing the catalytic efficiency of multi‐enzymatic cascades by controlling their spatial organization and stoichiometry. This study introduces a novel family of engineered SCAffolding Bricks, named SCABs, utilizing the consensus tetratricopeptide repeat (CTPR) domain for organized multi‐enzyme systems. Two SCAB systems are developed, one employing head‐to‐tail interactions with reversible covalent disulfide bonds, the other relying on non‐covalent metal‐driven assembly via engineered metal coordinating interfaces. Enzymes are directly fused to SCAB modules, triggering assembly in a non‐reducing environment or by metal presence. A proof‐of‐concept with formate dehydrogenase (FDH) and L‐alanine dehydrogenase (AlaDH) shows enhanced specific productivity by 3.6‐fold compared to free enzymes, with the covalent stapling outperforming the metal‐driven assembly. This enhancement likely stems from higher‐order supramolecular assembly and improved NADH cofactor regeneration, resulting in more efficient cascades. This study underscores the potential of protein engineering to tailor scaffolds, leveraging supramolecular spatial‐organizing tools, for more efficient enzymatic cascade reactions.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":null,"pages":null},"PeriodicalIF":8.0,"publicationDate":"2024-04-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140572552","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}
Louise Thines, Hyunbum Jang, Zhigang Li, Samar Sayedyahossein, Ryan Maloney, Ruth Nussinov, David B. Sacks
KSR1, a key scaffold protein for the MAPK pathway, facilitates ERK activation upon growth factor stimulation. We recently demonstrated that KSR1 binds the Ca2+‐binding protein calmodulin (CaM), thereby providing an intersection between KSR1‐mediated and Ca2+ signaling. In this study, we set out to generate a KSR1 point mutant with reduced Ca2+/CaM binding in order to unravel the functional implications of their interaction. To do so, we solved the structural determinants of complex formation. Using purified fragments of KSR1, we showed that Ca2+/CaM binds to the CA3 domain of KSR1. We then used in silico molecular modeling to predict contact residues for binding. This approach identified two possible modes of interaction: (1) binding of extended Ca2+/CaM to a globular conformation of KSR1‐CA3 via electrostatic interactions or (2) binding of collapsed Ca2+/CaM to α‐helical KSR1‐CA3 via hydrophobic interactions. Experimentally, site‐directed mutagenesis of the predicted contact residues for the two binding models favored that where collapsed Ca2+/CaM binds to the α‐helical conformation of KSR1‐CA3. Importantly, replacing KSR1‐Phe355 with Asp reduces Ca2+/CaM binding by 76%. The KSR1‐F355D mutation also significantly impairs the ability of EGF to activate ERK, which reveals that Ca2+/CaM binding promotes KSR1‐mediated MAPK signaling. This work, by uncovering structural insight into the binding of KSR1 to Ca2+/CaM, identifies a KSR1 single‐point mutant as a bioreagent to selectively study the crosstalk between Ca2+ and KSR1‐mediated signaling.
{"title":"Disruption of Ca2+/calmodulin:KSR1 interaction lowers ERK activation","authors":"Louise Thines, Hyunbum Jang, Zhigang Li, Samar Sayedyahossein, Ryan Maloney, Ruth Nussinov, David B. Sacks","doi":"10.1002/pro.4982","DOIUrl":"https://doi.org/10.1002/pro.4982","url":null,"abstract":"KSR1, a key scaffold protein for the MAPK pathway, facilitates ERK activation upon growth factor stimulation. We recently demonstrated that KSR1 binds the Ca<jats:sup>2+</jats:sup>‐binding protein calmodulin (CaM), thereby providing an intersection between KSR1‐mediated and Ca<jats:sup>2+</jats:sup> signaling. In this study, we set out to generate a KSR1 point mutant with reduced Ca<jats:sup>2+</jats:sup>/CaM binding in order to unravel the functional implications of their interaction. To do so, we solved the structural determinants of complex formation. Using purified fragments of KSR1, we showed that Ca<jats:sup>2+</jats:sup>/CaM binds to the CA3 domain of KSR1. We then used in silico molecular modeling to predict contact residues for binding. This approach identified two possible modes of interaction: (1) binding of extended Ca<jats:sup>2+</jats:sup>/CaM to a globular conformation of KSR1‐CA3 via electrostatic interactions or (2) binding of collapsed Ca<jats:sup>2+</jats:sup>/CaM to α‐helical KSR1‐CA3 via hydrophobic interactions. Experimentally, site‐directed mutagenesis of the predicted contact residues for the two binding models favored that where collapsed Ca<jats:sup>2+</jats:sup>/CaM binds to the α‐helical conformation of KSR1‐CA3. Importantly, replacing KSR1‐Phe<jats:sup>355</jats:sup> with Asp reduces Ca<jats:sup>2+</jats:sup>/CaM binding by 76%. The KSR1‐F<jats:sup>355</jats:sup>D mutation also significantly impairs the ability of EGF to activate ERK, which reveals that Ca<jats:sup>2+</jats:sup>/CaM binding promotes KSR1‐mediated MAPK signaling. This work, by uncovering structural insight into the binding of KSR1 to Ca<jats:sup>2+</jats:sup>/CaM, identifies a KSR1 single‐point mutant as a bioreagent to selectively study the crosstalk between Ca<jats:sup>2+</jats:sup> and KSR1‐mediated signaling.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":null,"pages":null},"PeriodicalIF":8.0,"publicationDate":"2024-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140572651","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}
Yogesh Narkhede, Roopashi Saxena, Tej Sharma, Jacob P. Conarty, Valentina Toro Ramirez, Balindile B. Motsa, Souad Amiar, Sheng Li, Prem P. Chapagain, Olaf Wiest, Robert V. Stahelin
The Ebola virus (EBOV) is a lipid‐enveloped virus with a negative sense RNA genome that can cause severe and often fatal viral hemorrhagic fever. The assembly and budding of EBOV is regulated by the matrix protein, VP40, which is a peripheral protein that associates with anionic lipids at the inner leaflet of the plasma membrane. VP40 is sufficient to form virus‐like particles (VLPs) from cells, which are nearly indistinguishable from authentic virions. Due to the restrictions of studying EBOV in BSL‐4 facilities, VP40 has served as a surrogate in cellular studies to examine the EBOV assembly and budding process from the host cell plasma membrane. VP40 is a dimer where inhibition of dimer formation halts budding and formation of new VLPs as well as VP40 localization to the plasma membrane inner leaflet. To better understand VP40 dimer stability and critical amino acids to VP40 dimer formation, we integrated computational approaches with experimental validation. Site saturation/alanine scanning calculation, combined with molecular mechanics‐based generalized Born with Poisson‐Boltzmann surface area (MM‐GB/PBSA) method and molecular dynamics simulations were used to predict the energetic contribution of amino acids to VP40 dimer stability and the hydrogen bonding network across the dimer interface. These studies revealed several previously unknown interactions and critical residues predicted to impact VP40 dimer formation. In vitro and cellular studies were then pursued for a subset of VP40 mutations demonstrating reduction in dimer formation (in vitro) or plasma membrane localization (in cells). Together, the computational and experimental approaches revealed critical residues for VP40 dimer stability in an alpha‐helical interface (between residues 106–117) as well as in a loop region (between residues 52–61) below this alpha‐helical region. This study sheds light on the structural origins of VP40 dimer formation and may inform the design of a small molecule that can disrupt VP40 dimer stability.
{"title":"Computational and experimental identification of keystone interactions in Ebola virus matrix protein VP40 dimer formation","authors":"Yogesh Narkhede, Roopashi Saxena, Tej Sharma, Jacob P. Conarty, Valentina Toro Ramirez, Balindile B. Motsa, Souad Amiar, Sheng Li, Prem P. Chapagain, Olaf Wiest, Robert V. Stahelin","doi":"10.1002/pro.4978","DOIUrl":"https://doi.org/10.1002/pro.4978","url":null,"abstract":"The Ebola virus (EBOV) is a lipid‐enveloped virus with a negative sense RNA genome that can cause severe and often fatal viral hemorrhagic fever. The assembly and budding of EBOV is regulated by the matrix protein, VP40, which is a peripheral protein that associates with anionic lipids at the inner leaflet of the plasma membrane. VP40 is sufficient to form virus‐like particles (VLPs) from cells, which are nearly indistinguishable from authentic virions. Due to the restrictions of studying EBOV in BSL‐4 facilities, VP40 has served as a surrogate in cellular studies to examine the EBOV assembly and budding process from the host cell plasma membrane. VP40 is a dimer where inhibition of dimer formation halts budding and formation of new VLPs as well as VP40 localization to the plasma membrane inner leaflet. To better understand VP40 dimer stability and critical amino acids to VP40 dimer formation, we integrated computational approaches with experimental validation. Site saturation/alanine scanning calculation, combined with molecular mechanics‐based generalized Born with Poisson‐Boltzmann surface area (MM‐GB/PBSA) method and molecular dynamics simulations were used to predict the energetic contribution of amino acids to VP40 dimer stability and the hydrogen bonding network across the dimer interface. These studies revealed several previously unknown interactions and critical residues predicted to impact VP40 dimer formation. In vitro and cellular studies were then pursued for a subset of VP40 mutations demonstrating reduction in dimer formation (in vitro) or plasma membrane localization (in cells). Together, the computational and experimental approaches revealed critical residues for VP40 dimer stability in an alpha‐helical interface (between residues 106–117) as well as in a loop region (between residues 52–61) below this alpha‐helical region. This study sheds light on the structural origins of VP40 dimer formation and may inform the design of a small molecule that can disrupt VP40 dimer stability.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":null,"pages":null},"PeriodicalIF":8.0,"publicationDate":"2024-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140572788","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}
Kristi Lichimo, Dana J. Sowa, Andriana Tetenych, Monica M. Warner, Caitlin Doubleday, Harman S. Dev, Catie Luck, Sara N. Andres
Translesion DNA synthesis pathways are necessary to ensure bacterial replication in the presence of DNA damage. Translesion DNA synthesis carried out by the PolV mutasome is well‐studied in Escherichia coli, but ~one third of bacteria use a functionally homologous protein complex, consisting of ImuA, ImuB, and ImuC (also called DnaE2). Numerous in vivo studies have shown that all three proteins are required for translesion DNA synthesis and that ImuC is the error‐prone polymerase, but the roles of ImuA and ImuB are unclear. Here we carry out biochemical characterization of ImuA and a truncation of ImuB from Myxococcus xanthus. We find that ImuA is an ATPase, with ATPase activity enhanced in the presence of DNA. The ATPase activity is likely regulated by the C‐terminus, as loss of the ImuA C‐terminus results in DNA‐independent ATP hydrolysis. We also find that ImuA binds a variety of DNA substrates, with DNA binding affinity affected by the addition of ADP or adenylyl‐imidodiphosphate. An ImuB truncation also binds DNA, with lower affinity than ImuA. In the absence of DNA, ImuA directly binds ImuB with moderate affinity. Finally, we show that ImuA and ImuB self‐interact, but that ImuA is predominantly a monomer, while truncated ImuB is a trimer in vitro. Together, with our findings and the current literature in the field, we suggest a model for translesion DNA synthesis, where a trimeric ImuB would provide sufficient binding sites for DNA, the β‐clamp, ImuC, and ImuA, and where ImuA ATPase activity may regulate assembly and disassembly of the translesion DNA synthesis complex.
转座DNA合成途径是确保细菌在DNA损伤情况下进行复制的必要条件。在大肠杆菌中，由 PolV 突变体进行的转座子 DNA 合成已被充分研究，但约三分之一的细菌使用功能上同源的蛋白复合物，由 ImuA、ImuB 和 ImuC（也称为 DnaE2）组成。大量体内研究表明，转座子 DNA 合成需要这三种蛋白，ImuC 是易出错的聚合酶，但 ImuA 和 ImuB 的作用尚不清楚。在这里，我们对黄曲霉毒素中的 ImuA 和 ImuB 的截短部分进行了生化鉴定。我们发现，ImuA 是一种 ATPase，在 DNA 存在的情况下 ATPase 活性增强。ATPase 活性可能受 C 端调节，因为 ImuA C 端缺失会导致不依赖 DNA 的 ATP 水解。我们还发现，ImuA 与多种 DNA 底物结合，DNA 结合亲和力受添加 ADP 或腺苷酰亚胺二磷酸的影响。ImuB 截短体也能与 DNA 结合，但亲和力低于 ImuA。在没有 DNA 的情况下，ImuA 直接与 ImuB 结合，亲和力适中。最后，我们证明了 ImuA 和 ImuB 的自我相互作用，但在体外，ImuA 主要是单体，而截短的 ImuB 是三聚体。结合我们的研究结果和该领域的现有文献，我们提出了一个转座子 DNA 合成模型，在该模型中，三聚体 ImuB 将为 DNA、β-夹、ImuC 和 ImuA 提供足够的结合位点，而 ImuA 的 ATPase 活性可能会调节转座子 DNA 合成复合物的组装和解体。
{"title":"Myxococcus xanthus translesion DNA synthesis protein ImuA is an ATPase enhanced by DNA","authors":"Kristi Lichimo, Dana J. Sowa, Andriana Tetenych, Monica M. Warner, Caitlin Doubleday, Harman S. Dev, Catie Luck, Sara N. Andres","doi":"10.1002/pro.4981","DOIUrl":"https://doi.org/10.1002/pro.4981","url":null,"abstract":"Translesion DNA synthesis pathways are necessary to ensure bacterial replication in the presence of DNA damage. Translesion DNA synthesis carried out by the PolV mutasome is well‐studied in <jats:italic>Escherichia coli</jats:italic>, but ~one third of bacteria use a functionally homologous protein complex, consisting of ImuA, ImuB, and ImuC (also called DnaE2). Numerous in vivo studies have shown that all three proteins are required for translesion DNA synthesis and that ImuC is the error‐prone polymerase, but the roles of ImuA and ImuB are unclear. Here we carry out biochemical characterization of ImuA and a truncation of ImuB from <jats:italic>Myxococcus xanthus</jats:italic>. We find that ImuA is an ATPase, with ATPase activity enhanced in the presence of DNA. The ATPase activity is likely regulated by the C‐terminus, as loss of the ImuA C‐terminus results in DNA‐independent ATP hydrolysis. We also find that ImuA binds a variety of DNA substrates, with DNA binding affinity affected by the addition of ADP or adenylyl‐imidodiphosphate. An ImuB truncation also binds DNA, with lower affinity than ImuA. In the absence of DNA, ImuA directly binds ImuB with moderate affinity. Finally, we show that ImuA and ImuB self‐interact, but that ImuA is predominantly a monomer, while truncated ImuB is a trimer in vitro. Together, with our findings and the current literature in the field, we suggest a model for translesion DNA synthesis, where a trimeric ImuB would provide sufficient binding sites for DNA, the β‐clamp, ImuC, and ImuA, and where ImuA ATPase activity may regulate assembly and disassembly of the translesion DNA synthesis complex.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":null,"pages":null},"PeriodicalIF":8.0,"publicationDate":"2024-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140572652","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}
Mantas Liutkus, Ivan R. Sasselli, Adriana L. Rojas, Aitziber L. Cortajarena
As protein crystals are increasingly finding diverse applications as scaffolds, controlled crystal polymorphism presents a facile strategy to form crystalline assemblies with controllable porosity with minimal to no protein engineering. Polymorphs of consensus tetratricopeptide repeat proteins with varying porosity were obtained through co‐crystallization with metal salts, exploiting the innate metal ion geometric requirements. A single structurally exposed negative amino acid cluster was responsible for metal coordination, despite the abundance of negatively charged residues. Density functional theory calculations showed that while most of the crystals were the most thermodynamically stable assemblies, some were kinetically trapped states. Thus, crystalline porosity diversity is achieved and controlled with metal coordination, opening a new scope in the application of proteins as biocompatible protein‐metal‐organic frameworks (POFs). In addition, metal‐dependent polymorphic crystals allow direct comparison of metal coordination preferences.
{"title":"Diverse crystalline protein scaffolds through metal‐dependent polymorphism","authors":"Mantas Liutkus, Ivan R. Sasselli, Adriana L. Rojas, Aitziber L. Cortajarena","doi":"10.1002/pro.4971","DOIUrl":"https://doi.org/10.1002/pro.4971","url":null,"abstract":"As protein crystals are increasingly finding diverse applications as scaffolds, controlled crystal polymorphism presents a facile strategy to form crystalline assemblies with controllable porosity with minimal to no protein engineering. Polymorphs of consensus tetratricopeptide repeat proteins with varying porosity were obtained through co‐crystallization with metal salts, exploiting the innate metal ion geometric requirements. A single structurally exposed negative amino acid cluster was responsible for metal coordination, despite the abundance of negatively charged residues. Density functional theory calculations showed that while most of the crystals were the most thermodynamically stable assemblies, some were kinetically trapped states. Thus, crystalline porosity diversity is achieved and controlled with metal coordination, opening a new scope in the application of proteins as biocompatible protein‐metal‐organic frameworks (POFs). In addition, metal‐dependent polymorphic crystals allow direct comparison of metal coordination preferences.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":null,"pages":null},"PeriodicalIF":8.0,"publicationDate":"2024-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140572790","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}
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