Klebsiella pneumoniae (Kp) poses an escalating threat to public health, particularly given its association with nosocomial infections and its emergence as a leading cause of neonatal sepsis, particularly in low- and middle-income countries (LMICs). Host cell adherence and biofilm formation of Kp is mediated by type 1 and type 3 fimbriae whose major fimbrial subunits are encoded by the fimA and mrkA genes, respectively. In this study, we focus on MrkA subunit, which is a 20 KDa protein whose 3D molecular structure remains elusive. We applied solution NMR to characterize a recombinant version of MrkA in which the donor strand segment situated at the protein’s N-terminus is relocated to the C-terminus, preceded by a hexaglycine linker. This construct yields a self-complemented variant of MrkA. Remarkably, the self-complemented MrkA monomer loses its capacity to interact with other monomers and to extend into fimbriae structures. Here, we report the nearly complete assignment of the 13C,15N labelled self-complemented MrkA monomer. Furthermore, an examination of its internal mobility unveiled that relaxation parameters are predominantly uniform across the polypeptide sequence, except for the glycine-rich region within loop 176–181. These data pave the way to a comprehensive structural elucidation of the MrkA monomer and to structurally map the molecular interaction regions between MrkA and antigen-induced antibodies.
{"title":"1H, 13C and 15N assignment of self-complemented MrkA protein antigen from Klebsiella pneumoniae","authors":"Valentina Monaci, Gianmarco Gasperini, Lucia Banci, Francesca Micoli, Francesca Cantini","doi":"10.1007/s12104-024-10185-3","DOIUrl":"10.1007/s12104-024-10185-3","url":null,"abstract":"<div><p>Klebsiella pneumoniae (Kp) poses an escalating threat to public health, particularly given its association with nosocomial infections and its emergence as a leading cause of neonatal sepsis, particularly in low- and middle-income countries (LMICs). Host cell adherence and biofilm formation of Kp is mediated by type 1 and type 3 fimbriae whose major fimbrial subunits are encoded by the <i>fimA</i> and <i>mrkA</i> genes, respectively. In this study, we focus on MrkA subunit, which is a 20 KDa protein whose 3D molecular structure remains elusive. We applied solution NMR to characterize a recombinant version of MrkA in which the donor strand segment situated at the protein’s N-terminus is relocated to the C-terminus, preceded by a hexaglycine linker. This construct yields a self-complemented variant of MrkA. Remarkably, the self-complemented MrkA monomer loses its capacity to interact with other monomers and to extend into fimbriae structures. Here, we report the nearly complete assignment of the <sup>13</sup>C,<sup>15</sup>N labelled self-complemented MrkA monomer. Furthermore, an examination of its internal mobility unveiled that relaxation parameters are predominantly uniform across the polypeptide sequence, except for the glycine-rich region within loop 176–181. These data pave the way to a comprehensive structural elucidation of the MrkA monomer and to structurally map the molecular interaction regions between MrkA and antigen-induced antibodies.</p></div>","PeriodicalId":492,"journal":{"name":"Biomolecular NMR Assignments","volume":"18 2","pages":"171 - 179"},"PeriodicalIF":0.8,"publicationDate":"2024-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11511707/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141625602","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-07-04DOI: 10.1007/s12104-024-10188-0
Sahil Ahlawat, Surabhi Mehra, Chandrakala M. Gowda, Samir K Maji, Vipin Agarwal
Synucleinopathies are neurodegenerative diseases characterized by the accumulation of α-synuclein protein aggregates in the neurons and glial cells. Both ex vivo and in vitro α-synuclein fibrils tend to show polymorphism. Polymorphism results in structure variations among fibrils originating from a single polypeptide/protein. The polymorphs usually have different biophysical, biochemical and pathogenic properties. The various pathologies of a single disease might be associated with distinct polymorphs. Similarly, in the case of different synucleinopathies, each condition might be associated with a different polymorph. Fibril formation is a nucleation-dependent process involving the formation of transient and heterogeneous intermediates from monomers. Polymorphs are believed to arise from heterogeneous oligomer populations because of distinct selection mechanisms in different conditions. To test this hypothesis, we isolated and incubated different intermediates during in vitro fibrillization of α-synuclein to form different polymorphs. Here, we report 13C and 15N chemical shifts and the secondary structure of fibrils prepared from the helical intermediate using solid-state nuclear magnetic spectroscopy.
{"title":"Solid-state NMR assignment of α-synuclein polymorph prepared from helical intermediate","authors":"Sahil Ahlawat, Surabhi Mehra, Chandrakala M. Gowda, Samir K Maji, Vipin Agarwal","doi":"10.1007/s12104-024-10188-0","DOIUrl":"10.1007/s12104-024-10188-0","url":null,"abstract":"<div><p>Synucleinopathies are neurodegenerative diseases characterized by the accumulation of α-synuclein protein aggregates in the neurons and glial cells. Both ex vivo and in vitro α-synuclein fibrils tend to show polymorphism. Polymorphism results in structure variations among fibrils originating from a single polypeptide/protein. The polymorphs usually have different biophysical, biochemical and pathogenic properties. The various pathologies of a single disease might be associated with distinct polymorphs. Similarly, in the case of different synucleinopathies, each condition might be associated with a different polymorph. Fibril formation is a nucleation-dependent process involving the formation of transient and heterogeneous intermediates from monomers. Polymorphs are believed to arise from heterogeneous oligomer populations because of distinct selection mechanisms in different conditions. To test this hypothesis, we isolated and incubated different intermediates during in vitro fibrillization of α-synuclein to form different polymorphs. Here, we report <sup>13</sup>C and <sup>15</sup>N chemical shifts and the secondary structure of fibrils prepared from the helical intermediate using solid-state nuclear magnetic spectroscopy.</p></div>","PeriodicalId":492,"journal":{"name":"Biomolecular NMR Assignments","volume":"18 2","pages":"193 - 200"},"PeriodicalIF":0.8,"publicationDate":"2024-07-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11511750/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141496713","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-29DOI: 10.1007/s12104-024-10186-2
Zigmantas Toleikis, Piotr Paluch, Ewelina Kuc, Jana Petkus, Darius Sulskis, Mai-Liis Org-Tago, Ago Samoson, Vytautas Smirnovas, Jan Stanek, Alons Lends
The α-synuclein (α-syn) amyloid fibrils are involved in various neurogenerative diseases. Solid-state NMR (ssNMR) has been showed as a powerful tool to study α-syn aggregates. Here, we report the 1H, 13C and 15N back-bone chemical shifts of a new α-syn polymorph obtained using proton-detected ssNMR spectroscopy under fast (95 kHz) magic-angle spinning conditions. The manual chemical shift assignments were cross-validated using FLYA algorithm. The secondary structural elements of α-syn fibrils were calculated using 13C chemical shift differences and TALOS software.
{"title":"Solid-state NMR backbone chemical shift assignments of α-synuclein amyloid fibrils at fast MAS regime","authors":"Zigmantas Toleikis, Piotr Paluch, Ewelina Kuc, Jana Petkus, Darius Sulskis, Mai-Liis Org-Tago, Ago Samoson, Vytautas Smirnovas, Jan Stanek, Alons Lends","doi":"10.1007/s12104-024-10186-2","DOIUrl":"10.1007/s12104-024-10186-2","url":null,"abstract":"<div><p>The α-synuclein (α-syn) amyloid fibrils are involved in various neurogenerative diseases. Solid-state NMR (ssNMR) has been showed as a powerful tool to study α-syn aggregates. Here, we report the <sup>1</sup>H, <sup>13</sup>C and <sup>15</sup>N back-bone chemical shifts of a new α-syn polymorph obtained using proton-detected ssNMR spectroscopy under fast (95 kHz) magic-angle spinning conditions. The manual chemical shift assignments were cross-validated using FLYA algorithm. The secondary structural elements of α-syn fibrils were calculated using <sup>13</sup>C chemical shift differences and TALOS software.</p></div>","PeriodicalId":492,"journal":{"name":"Biomolecular NMR Assignments","volume":"18 2","pages":"181 - 186"},"PeriodicalIF":0.8,"publicationDate":"2024-06-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141475633","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-26DOI: 10.1007/s12104-024-10187-1
Muzaddid Sarker, Yves Aubin
Adalimumab is a therapeutic monoclonal antibody developed to target human TNF an important mediator of immune-mediated inflammatory diseases such as rheumatoid arthritis, amongst others. The 48 kDa Fab fragment of adalimumab was produced in Escherichia coli using a single chain approach to allow complete isotopic incorporation of deuterium, carbon-13 and nitrogen-15 along with the protonated isoleucine-d, valine and leucine methyl groups. Here we report the near complete resonance assignment of the polypeptide backbone and the methyl groups of isoleucine, leucine and valine residues.
阿达木单抗是一种治疗性单克隆抗体,针对人类 TNF 而开发,TNF 是类风湿性关节炎等免疫介导炎症性疾病的重要介质。阿达木单抗的 48 kDa Fab 片段是在大肠杆菌中使用单链方法生产的,这种方法可以将氘、碳-13 和氮-15 以及质子化的异亮氨酸-d、缬氨酸和亮氨酸甲基完全同位素化。在此,我们报告了多肽骨架以及异亮氨酸、亮氨酸和缬氨酸残基甲基的近乎完整的共振分配。
{"title":"Backbone and methyl side-chain resonance assignments of the Fab fragment of adalimumab","authors":"Muzaddid Sarker, Yves Aubin","doi":"10.1007/s12104-024-10187-1","DOIUrl":"10.1007/s12104-024-10187-1","url":null,"abstract":"<div><p>Adalimumab is a therapeutic monoclonal antibody developed to target human TNF an important mediator of immune-mediated inflammatory diseases such as rheumatoid arthritis, amongst others. The 48 kDa Fab fragment of adalimumab was produced in <i>Escherichia coli</i> using a single chain approach to allow complete isotopic incorporation of deuterium, carbon-13 and nitrogen-15 along with the protonated isoleucine-d, valine and leucine methyl groups. Here we report the near complete resonance assignment of the polypeptide backbone and the methyl groups of isoleucine, leucine and valine residues.</p></div>","PeriodicalId":492,"journal":{"name":"Biomolecular NMR Assignments","volume":"18 2","pages":"187 - 192"},"PeriodicalIF":0.8,"publicationDate":"2024-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11511761/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141454426","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-25DOI: 10.1007/s12104-024-10184-4
Patrick M. Keating, Jinwoo Lee
Lassa virus (LASV) is the most prevalent member of the arenavirus family and the causative agent of Lassa fever, a viral hemorrhagic fever. Although there are annual outbreaks in West Africa, and recently isolated cases worldwide, there are no current therapeutics or vaccines. As such, LASV poses a significant global public health threat. One of the key steps in LASV infection is delivering its genetic material by fusing its viral membrane with the host cell membrane. This process is facilitated by significant conformational changes within glycoprotein 2 (GP2), yielding distinct prefusion and postfusion structural states. However, structural information is missing to understand the changes that occur in the transmembrane domain (TM) during the fusion process. Previously, we showed that the TM undergoes pH-dependent structural changes that result in a helical extension. Here, we provide the 1H, 15N, and 13C assignment of the LASV TM backbone in the prefusion and postfusion states. We also provide the 1H, 15N, and 13C assignment of two mutants, G429P and D432P, which prevent this helical extension. These results will help understand the role the TM plays in membrane fusion and can lead to the design of therapeutics against LASV infection.
{"title":"Assignment of the Lassa virus transmembrane domain in the prefusion and postfusion states in detergent micelles","authors":"Patrick M. Keating, Jinwoo Lee","doi":"10.1007/s12104-024-10184-4","DOIUrl":"10.1007/s12104-024-10184-4","url":null,"abstract":"<div><p>Lassa virus (LASV) is the most prevalent member of the arenavirus family and the causative agent of Lassa fever, a viral hemorrhagic fever. Although there are annual outbreaks in West Africa, and recently isolated cases worldwide, there are no current therapeutics or vaccines. As such, LASV poses a significant global public health threat. One of the key steps in LASV infection is delivering its genetic material by fusing its viral membrane with the host cell membrane. This process is facilitated by significant conformational changes within glycoprotein 2 (GP2), yielding distinct prefusion and postfusion structural states. However, structural information is missing to understand the changes that occur in the transmembrane domain (TM) during the fusion process. Previously, we showed that the TM undergoes pH-dependent structural changes that result in a helical extension. Here, we provide the <sup>1</sup>H, <sup>15</sup>N, and <sup>13</sup>C assignment of the LASV TM backbone in the prefusion and postfusion states. We also provide the <sup>1</sup>H, <sup>15</sup>N, and <sup>13</sup>C assignment of two mutants, G429P and D432P, which prevent this helical extension. These results will help understand the role the TM plays in membrane fusion and can lead to the design of therapeutics against LASV infection.</p></div>","PeriodicalId":492,"journal":{"name":"Biomolecular NMR Assignments","volume":"18 2","pages":"165 - 169"},"PeriodicalIF":0.8,"publicationDate":"2024-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141445096","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-22DOI: 10.1007/s12104-024-10183-5
Loic Delcourte, Corinne Sanchez, Estelle Morvan, Mélanie Berbon, Axelle Grélard, Claire Saragaglia, Thierry Dakhli, Stéphane Thore, Benjamin Bardiaux, Birgit Habenstein, Brice Kauffmann, Sven J. Saupe, Antoine Loquet
Signalosomes are high-order protein machineries involved in complex mechanisms controlling regulated immune defense and cell death execution. The immune response is initiated by the recognition of exogeneous or endogenous signals, triggering the signalosome assembly process. The final step of signalosome fate often involves membrane-targeting and activation of pore-forming execution domains, leading to membrane disruption and ultimately cell death. Such cell death-inducing domains have been thoroughly characterized in plants, mammals and fungi, notably for the fungal cell death execution protein domain HeLo. However, little is known on the mechanisms of signalosome-based immune response in bacteria, and the conformation of cell death executors in bacterial signalosomes is still poorly characterized. We recently uncovered the existence of NLR signalosomes in various multicellular bacteria and used genome mining approaches to identify putative cell death executors in Streptomyces olivochromogenes. These proteins contain a C-terminal amyloid domain involved in signal transmission and a N-terminal domain, termed BELL for Bacteria analogous to fungal HeLL (HeLo-like), presumably responsible for membrane-targeting, pore-forming and cell death execution. In the present study, we report the high yield expression of S. olivochromogenes BELL2 and its characterization by solution NMR spectroscopy. BELL is folded in solution and we report backbone and sidechain assignments. We identified five α-helical secondary structure elements and a folded core much smaller than its fungal homolog HeLo. This study constitutes the first step toward the NMR investigation of the full-length protein assembly and its membrane targeting.
信号体是一种高阶蛋白质机制,参与控制调节免疫防御和细胞死亡执行的复杂机制。免疫反应是通过识别外源或内源信号,触发信号体组装过程而启动的。信号体命运的最后一步往往涉及膜靶向和激活孔形成执行域,导致膜破坏,最终导致细胞死亡。在植物、哺乳动物和真菌中,这种诱导细胞死亡的结构域已被彻底表征,特别是真菌细胞死亡执行蛋白结构域 HeLo。然而,人们对细菌中基于信号体的免疫反应机制知之甚少,而且对细菌信号体中细胞死亡执行蛋白的构象仍然知之甚少。我们最近发现了多种多细胞细菌中存在的 NLR 信号体,并利用基因组挖掘方法鉴定了橄榄色链霉菌中的推定细胞死亡执行体。这些蛋白质包含一个参与信号传递的 C 端淀粉样结构域和一个 N 端结构域(BELL,表示细菌类似于真菌的 HeLL(HeLo-like)),可能负责膜靶向、孔形成和细胞死亡执行。在本研究中,我们报告了 S. olivochromogenes BELL2 的高产率表达及其溶液核磁共振光谱特性。BELL 在溶液中折叠,我们报告了骨架和侧链的分配。我们发现了五个α螺旋二级结构元素和一个折叠核心,其体积远小于其真菌同源物 HeLo。这项研究为全长蛋白质的组装及其膜靶向的核磁共振研究迈出了第一步。
{"title":"NMR resonance assignment of the cell death execution domain BELL2 from multicellular bacterial signalosomes","authors":"Loic Delcourte, Corinne Sanchez, Estelle Morvan, Mélanie Berbon, Axelle Grélard, Claire Saragaglia, Thierry Dakhli, Stéphane Thore, Benjamin Bardiaux, Birgit Habenstein, Brice Kauffmann, Sven J. Saupe, Antoine Loquet","doi":"10.1007/s12104-024-10183-5","DOIUrl":"10.1007/s12104-024-10183-5","url":null,"abstract":"<div><p>Signalosomes are high-order protein machineries involved in complex mechanisms controlling regulated immune defense and cell death execution. The immune response is initiated by the recognition of exogeneous or endogenous signals, triggering the signalosome assembly process. The final step of signalosome fate often involves membrane-targeting and activation of pore-forming execution domains, leading to membrane disruption and ultimately cell death. Such cell death-inducing domains have been thoroughly characterized in plants, mammals and fungi, notably for the fungal cell death execution protein domain HeLo. However, little is known on the mechanisms of signalosome-based immune response in bacteria, and the conformation of cell death executors in bacterial signalosomes is still poorly characterized. We recently uncovered the existence of NLR signalosomes in various multicellular bacteria and used genome mining approaches to identify putative cell death executors in <i>Streptomyces olivochromogenes</i>. These proteins contain a C-terminal amyloid domain involved in signal transmission and a N-terminal domain, termed BELL for Bacteria analogous to fungal HeLL (HeLo-like), presumably responsible for membrane-targeting, pore-forming and cell death execution. In the present study, we report the high yield expression of <i>S. olivochromogenes</i> BELL2 and its characterization by solution NMR spectroscopy. BELL is folded in solution and we report backbone and sidechain assignments. We identified five α-helical secondary structure elements and a folded core much smaller than its fungal homolog HeLo. This study constitutes the first step toward the NMR investigation of the full-length protein assembly and its membrane targeting.</p></div>","PeriodicalId":492,"journal":{"name":"Biomolecular NMR Assignments","volume":"18 2","pages":"159 - 164"},"PeriodicalIF":0.8,"publicationDate":"2024-06-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141439906","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-21DOI: 10.1007/s12104-024-10181-7
Maria Luiza Caldas Nogueira, Renuk Lakshmanan, Gwladys Rivière, Mario Mietzsch, Antonette Bennett, Robert McKenna, Joanna R. Long
Parvovirus B19 (B19V) is a human pathogen that is the causative agent of several diseases in infants and adults. Due to a lack of antivirals against this virus, treatment options are limited. The minor capsid protein of B19V has a unique N terminus, named VP1u, which is essential for infection. The VP1u encodes a receptor binding domain (RBD), necessary for host cell entry, and a phospholipase A2 (PLA2) domain, crucial for endosomal escape during cellular trafficking. Both domains are indispensable for infection, making the RBD a plausible drug target for inhibitors against B19V, as it is located on the exterior surface of the virus. To date, no experimental structural information has been available for the VP1u component for any Parvovirus. Here we report the backbone NMR resonance assignments for the RBD of B19V and demonstrate it forms a stable structure. The backbone chemical shifts are in good agreement with a structure predicted by AlphaFold, validating that the RBD contains three helices connected by tight turns. This RBD construct can now be used for further NMR studies, including assignment of full-length VP1u, determination of protein-protein interaction interfaces, and development of B19 antivirals specific to the RBD domain.
{"title":"Backbone NMR resonance assignments for the VP1u N-terminal receptor-binding domain of the human parvovirus pathogen B19","authors":"Maria Luiza Caldas Nogueira, Renuk Lakshmanan, Gwladys Rivière, Mario Mietzsch, Antonette Bennett, Robert McKenna, Joanna R. Long","doi":"10.1007/s12104-024-10181-7","DOIUrl":"10.1007/s12104-024-10181-7","url":null,"abstract":"<div><p>Parvovirus B19 (B19V) is a human pathogen that is the causative agent of several diseases in infants and adults. Due to a lack of antivirals against this virus, treatment options are limited. The minor capsid protein of B19V has a unique N terminus, named VP1u, which is essential for infection. The VP1u encodes a receptor binding domain (RBD), necessary for host cell entry, and a phospholipase A2 (PLA<sub>2</sub>) domain, crucial for endosomal escape during cellular trafficking. Both domains are indispensable for infection, making the RBD a plausible drug target for inhibitors against B19V, as it is located on the exterior surface of the virus. To date, no experimental structural information has been available for the VP1u component for any Parvovirus. Here we report the backbone NMR resonance assignments for the RBD of B19V and demonstrate it forms a stable structure. The backbone chemical shifts are in good agreement with a structure predicted by AlphaFold, validating that the RBD contains three helices connected by tight turns. This RBD construct can now be used for further NMR studies, including assignment of full-length VP1u, determination of protein-protein interaction interfaces, and development of B19 antivirals specific to the RBD domain.</p></div>","PeriodicalId":492,"journal":{"name":"Biomolecular NMR Assignments","volume":"18 2","pages":"147 - 152"},"PeriodicalIF":0.8,"publicationDate":"2024-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141431063","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-18DOI: 10.1007/s12104-024-10182-6
Laura Okmane, Mats Sandgren, Jerry Ståhlberg, Gustav Nestor
A glycoside hydrolase family 45 (GH45) enzyme from the white-rot basidiomycete fungus Phanerochaete chrysosporium (PcCel45A) was expressed in Pichia pastoris with 13C and 15N labelling. A nearly complete assignment of 1H, 13C and 15N backbone resonances was obtained, as well as the secondary structure prediction based on the assigned chemical shifts using the TALOS-N software. The predicted secondary structure was almost identical to previously published crystal structures of the same enzyme, except for differences in the termini of the sequence. This is the first NMR study using an isotopically labelled GH45 enzyme.
{"title":"1H, 13C and 15N backbone resonance assignment of Cel45A from Phanerochaete chrysosporium","authors":"Laura Okmane, Mats Sandgren, Jerry Ståhlberg, Gustav Nestor","doi":"10.1007/s12104-024-10182-6","DOIUrl":"10.1007/s12104-024-10182-6","url":null,"abstract":"<div><p>A glycoside hydrolase family 45 (GH45) enzyme from the white-rot basidiomycete fungus <i>Phanerochaete chrysosporium</i> (<i>Pc</i>Cel45A) was expressed in <i>Pichia pastoris</i> with <sup>13</sup>C and <sup>15</sup>N labelling. A nearly complete assignment of <sup>1</sup>H, <sup>13</sup>C and <sup>15</sup>N backbone resonances was obtained, as well as the secondary structure prediction based on the assigned chemical shifts using the TALOS-N software. The predicted secondary structure was almost identical to previously published crystal structures of the same enzyme, except for differences in the termini of the sequence. This is the first NMR study using an isotopically labelled GH45 enzyme.</p></div>","PeriodicalId":492,"journal":{"name":"Biomolecular NMR Assignments","volume":"18 2","pages":"153 - 157"},"PeriodicalIF":0.8,"publicationDate":"2024-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11511684/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141417127","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-10DOI: 10.1007/s12104-024-10179-1
Sunirmala Sahoo, Nitin Dhaka, Sulakshana P. Mukherjee
NF-kappaB is a family of inducible transcription factors playing an important role in immune response in vertebrates. All the five members of the family function as dimers in various combinations. Though all the family members recognize and bind to similar DNA elements to regulate the transcription of its target genes, the dimer composition can lead to differential transcriptional outcomes. Here we report the backbone resonance assignment of the 24.2 kDa homodimer of p52 subunit of the NF-kB family. The p52 subunit of NF-kB is a crucial player in the non-canonical NF-kB pathway and its dysregulation has shown detrimental effects in immune response leading to various inflammatory diseases and cancers. While the β-strands predicted using the backbone chemical shifts in this study largely conform with the available crystal structure, the helical turns present in the crystal structure are not observed in our results.
{"title":"Backbone triple resonance assignments of the dimerization domain of NF-kappaB p52 subunit","authors":"Sunirmala Sahoo, Nitin Dhaka, Sulakshana P. Mukherjee","doi":"10.1007/s12104-024-10179-1","DOIUrl":"10.1007/s12104-024-10179-1","url":null,"abstract":"<div><p>NF-kappaB is a family of inducible transcription factors playing an important role in immune response in vertebrates. All the five members of the family function as dimers in various combinations. Though all the family members recognize and bind to similar DNA elements to regulate the transcription of its target genes, the dimer composition can lead to differential transcriptional outcomes. Here we report the backbone resonance assignment of the 24.2 kDa homodimer of p52 subunit of the NF-kB family. The p52 subunit of NF-kB is a crucial player in the non-canonical NF-kB pathway and its dysregulation has shown detrimental effects in immune response leading to various inflammatory diseases and cancers. While the β-strands predicted using the backbone chemical shifts in this study largely conform with the available crystal structure, the helical turns present in the crystal structure are not observed in our results.</p></div>","PeriodicalId":492,"journal":{"name":"Biomolecular NMR Assignments","volume":"18 2","pages":"135 - 138"},"PeriodicalIF":0.8,"publicationDate":"2024-06-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141295328","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-07DOI: 10.1007/s12104-024-10180-8
Anaísa Coelho, José M. Silva, Francesca Cantini, Mario Piccioli, Ricardo O. Louro, Catarina M. Paquete
The contribution of Fe(II)-oxidizing bacteria to iron cycling in freshwater, groundwater, and marine environments has been widely recognized in recent years. These organisms perform extracellular electron transfer (EET), which constitutes the foundations of bioelectrochemical systems for the production of biofuels and bioenergy. It was proposed that the Gram-negative bacterium Sideroxydans lithotrophicus ES-1 oxidizes soluble ferrous Fe(II) at the surface of the cell and performs EET through the Mto redox pathway. This pathway is composed by the periplasmic monoheme cytochrome MtoD that is proposed to bridge electron transfer between the cell exterior and the cytoplasm. This makes its functional and structural characterization, as well as evaluating the interaction process with its physiological partners, essential for understanding the mechanisms underlying EET. Here, we report the complete assignment of the heme proton and carbon signals together with a near-complete assignment of 1H, 13C and 15N backbone and side chain resonances for the reduced, diamagnetic form of the protein. These data pave the way to identify and structurally map the molecular interaction regions between the cytochrome MtoD and its physiological redox partners, to explore the EET processes of S. lithotrophicus ES-1.
{"title":"Resonance assignments of cytochrome MtoD from the extracellular electron uptake pathway of sideroxydans lithotrophicus ES-1","authors":"Anaísa Coelho, José M. Silva, Francesca Cantini, Mario Piccioli, Ricardo O. Louro, Catarina M. Paquete","doi":"10.1007/s12104-024-10180-8","DOIUrl":"10.1007/s12104-024-10180-8","url":null,"abstract":"<div><p>The contribution of Fe(II)-oxidizing bacteria to iron cycling in freshwater, groundwater, and marine environments has been widely recognized in recent years. These organisms perform extracellular electron transfer (EET), which constitutes the foundations of bioelectrochemical systems for the production of biofuels and bioenergy. It was proposed that the Gram-negative bacterium <i>Sideroxydans lithotrophicus</i> ES-1 oxidizes soluble ferrous Fe(II) at the surface of the cell and performs EET through the Mto redox pathway. This pathway is composed by the periplasmic monoheme cytochrome MtoD that is proposed to bridge electron transfer between the cell exterior and the cytoplasm. This makes its functional and structural characterization, as well as evaluating the interaction process with its physiological partners, essential for understanding the mechanisms underlying EET. Here, we report the complete assignment of the heme proton and carbon signals together with a near-complete assignment of <sup>1</sup>H, <sup>13</sup>C and <sup>15</sup>N backbone and side chain resonances for the reduced, diamagnetic form of the protein. These data pave the way to identify and structurally map the molecular interaction regions between the cytochrome MtoD and its physiological redox partners, to explore the EET processes of <i>S. lithotrophicus</i> ES-1.</p></div>","PeriodicalId":492,"journal":{"name":"Biomolecular NMR Assignments","volume":"18 2","pages":"139 - 146"},"PeriodicalIF":0.8,"publicationDate":"2024-06-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11511738/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141282649","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}