Pub Date : 2024-02-01Epub Date: 2024-01-24DOI: 10.1107/S2053230X24000359
Jordan S Dialpuri, Haroldas Bagdonas, Lucy C Schofield, Phuong Thao Pham, Lou Holland, Paul S Bond, Filomeno Sánchez Rodríguez, Stuart J McNicholas, Jon Agirre
Owing to the difficulties associated with working with carbohydrates, validating glycan 3D structures prior to deposition into the Protein Data Bank has become a staple of the structure-solution pipeline. The Privateer software provides integrative methods for the validation, analysis, refinement and graphical representation of 3D atomic structures of glycans, both as ligands and as protein modifiers. While Privateer is free software, it requires users to install any of the structural biology software suites that support it or to build it from source code. Here, the Privateer web app is presented, which is always up to date and available to be used online (https://privateer.york.ac.uk) without installation. This self-updating tool, which runs locally on the user's machine, will allow structural biologists to simply and quickly analyse carbohydrate ligands and protein glycosylation from a web browser whilst retaining all confidential information on their devices.
{"title":"Online carbohydrate 3D structure validation with the Privateer web app.","authors":"Jordan S Dialpuri, Haroldas Bagdonas, Lucy C Schofield, Phuong Thao Pham, Lou Holland, Paul S Bond, Filomeno Sánchez Rodríguez, Stuart J McNicholas, Jon Agirre","doi":"10.1107/S2053230X24000359","DOIUrl":"10.1107/S2053230X24000359","url":null,"abstract":"<p><p>Owing to the difficulties associated with working with carbohydrates, validating glycan 3D structures prior to deposition into the Protein Data Bank has become a staple of the structure-solution pipeline. The Privateer software provides integrative methods for the validation, analysis, refinement and graphical representation of 3D atomic structures of glycans, both as ligands and as protein modifiers. While Privateer is free software, it requires users to install any of the structural biology software suites that support it or to build it from source code. Here, the Privateer web app is presented, which is always up to date and available to be used online (https://privateer.york.ac.uk) without installation. This self-updating tool, which runs locally on the user's machine, will allow structural biologists to simply and quickly analyse carbohydrate ligands and protein glycosylation from a web browser whilst retaining all confidential information on their devices.</p>","PeriodicalId":7029,"journal":{"name":"Acta crystallographica. Section F, Structural biology communications","volume":" ","pages":"30-35"},"PeriodicalIF":0.9,"publicationDate":"2024-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10836424/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139541238","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}
Nocardia are Gram-positive bacteria from the Actinobacteria phylum. Some Nocardia species can infect humans and are usually considered to be opportunist pathogens, as they often infect immunocompromised patients. Although their clinical incidence is low, many Nocardia species are now considered to be emerging pathogens. Primary sites of infection by Nocardia are the skin or the lungs, but dissemination to other body parts is very frequent. These disseminated infections are very difficult to treat and thus are tackled with multiple classes of antibiotics, in addition to the traditional treatment targeting the folate pathway. β-Lactams are often included in the regimen, but many Nocardia species present moderate or strong resistance to some members of this drug class. Genomic, microbiological and biochemical studies have reported the presence of class A β-lactamases (ABLs) in a handful of Nocardia species, but no structural investigation of Nocardia β-lactamases has yet been performed. In this study, the expression, purification and preliminary biochemical characterization of an ABL from an N. cyriacigeorgica (NCY-1) clinical strain are reported. The crystallization and the very high resolution crystal structure of NCY-1 are also described. The sequence and structural analysis of the protein demonstrate that NCY-1 belongs to the class A1 β-lactamases and show its very high conservation with ABLs from other human-pathogenic Nocardia. In addition, the presence of one molecule of citrate tightly bound in the catalytic site of the enzyme is described. This structure may provide a solid basis for future drug development to specifically target Nocardia spp. β-lactamases.
诺卡氏菌是放线菌门的革兰氏阳性细菌。一些诺卡氏菌可感染人类,通常被认为是机会性病原体,因为它们经常感染免疫力低下的患者。虽然它们的临床发病率很低,但许多诺卡氏菌现在被认为是新出现的病原体。诺卡氏菌的主要感染部位是皮肤或肺部,但传播到身体其他部位的情况也很常见。这些播散性感染非常难以治疗,因此除了针对叶酸途径的传统治疗方法外,还需要使用多种抗生素。β-内酰胺类药物通常被纳入治疗方案,但许多诺卡氏菌对该类药物的某些成分具有中度或较强的耐药性。基因组学、微生物学和生物化学研究报告称,在少数诺卡氏菌中存在 A 类 β-内酰胺酶(ABLs),但尚未对诺卡氏菌 β-内酰胺酶进行结构研究。本研究报告了一种来自 N. cyriacigeorgica(NCY-1)临床菌株的 ABL 的表达、纯化和初步生化鉴定。研究还描述了 NCY-1 的结晶和高分辨率晶体结构。对该蛋白的序列和结构分析表明,NCY-1 属于 A1 类 β-内酰胺酶,并表明它与其他人类致病性诺卡氏菌的 ABL 有很高的一致性。此外,还描述了在该酶的催化位点存在一分子紧密结合的柠檬酸盐。该结构可为今后开发专门针对诺卡氏菌属β-内酰胺酶的药物奠定坚实的基础。
{"title":"Biochemical and structural characterization of a class A β-lactamase from Nocardia cyriacigeorgica","authors":"Jérôme Feuillard, Julie Couston, Yvonne Benito, Elisabeth Hodille, Oana Dumitrescu, Mickaël Blaise","doi":"10.1107/S2053230X23010671","DOIUrl":"10.1107/S2053230X23010671","url":null,"abstract":"<p><i>Nocardia</i> are Gram-positive bacteria from the Actinobacteria phylum. Some <i>Nocardia</i> species can infect humans and are usually considered to be opportunist pathogens, as they often infect immunocompromised patients. Although their clinical incidence is low, many <i>Nocardia</i> species are now considered to be emerging pathogens. Primary sites of infection by <i>Nocardia</i> are the skin or the lungs, but dissemination to other body parts is very frequent. These disseminated infections are very difficult to treat and thus are tackled with multiple classes of antibiotics, in addition to the traditional treatment targeting the folate pathway. β-Lactams are often included in the regimen, but many <i>Nocardia</i> species present moderate or strong resistance to some members of this drug class. Genomic, microbiological and biochemical studies have reported the presence of class A β-lactamases (ABLs) in a handful of <i>Nocardia</i> species, but no structural investigation of <i>Nocardia</i> β-lactamases has yet been performed. In this study, the expression, purification and preliminary biochemical characterization of an ABL from an <i>N. cyriacigeorgica</i> (NCY-1) clinical strain are reported. The crystallization and the very high resolution crystal structure of NCY-1 are also described. The sequence and structural analysis of the protein demonstrate that NCY-1 belongs to the class A1 β-lactamases and show its very high conservation with ABLs from other human-pathogenic <i>Nocardia</i>. In addition, the presence of one molecule of citrate tightly bound in the catalytic site of the enzyme is described. This structure may provide a solid basis for future drug development to specifically target <i>Nocardia</i> spp. β-lactamases.</p>","PeriodicalId":7029,"journal":{"name":"Acta crystallographica. Section F, Structural biology communications","volume":"80 1","pages":"13-21"},"PeriodicalIF":0.9,"publicationDate":"2024-01-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139085405","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 : 2024-01-01DOI: 10.1107/S2053230X23010749
Shivani Sharma, Tamar Skaist Mehlman, Reddy Sudheer Sagabala, Benoit Boivin, Daniel A Keedy
Protein tyrosine phosphatase 1B (PTP1B) plays important roles in cellular homeostasis and is a highly validated therapeutic target for multiple human ailments, including diabetes, obesity and breast cancer. However, much remains to be learned about how conformational changes may convey information through the structure of PTP1B to enable allosteric regulation by ligands or functional responses to mutations. High-resolution X-ray crystallography can offer unique windows into protein conformational ensembles, but comparison of even high-resolution structures is often complicated by differences between data sets, including non-isomorphism. Here, the highest resolution crystal structure of apo wild-type (WT) PTP1B to date is presented out of a total of ∼350 PTP1B structures in the PDB. This structure is in a crystal form that is rare for PTP1B, with two unique copies of the protein that exhibit distinct patterns of conformational heterogeneity, allowing a controlled comparison of local disorder across the two chains within the same asymmetric unit. The conformational differences between these chains are interrogated in the apo structure and between several recently reported high-resolution ligand-bound structures. Electron-density maps in a high-resolution structure of a recently reported activating double mutant are also examined, and unmodeled alternate conformations in the mutant structure are discovered that coincide with regions of enhanced conformational heterogeneity in the new WT structure. These results validate the notion that these mutations operate by enhancing local dynamics, and suggest a latent susceptibility to such changes in the WT enzyme. Together, these new data and analysis provide a detailed view of the conformational ensemble of PTP1B and highlight the utility of high-resolution crystallography for elucidating conformational heterogeneity with potential relevance for function.
{"title":"High-resolution double vision of the allosteric phosphatase PTP1B.","authors":"Shivani Sharma, Tamar Skaist Mehlman, Reddy Sudheer Sagabala, Benoit Boivin, Daniel A Keedy","doi":"10.1107/S2053230X23010749","DOIUrl":"10.1107/S2053230X23010749","url":null,"abstract":"<p><p>Protein tyrosine phosphatase 1B (PTP1B) plays important roles in cellular homeostasis and is a highly validated therapeutic target for multiple human ailments, including diabetes, obesity and breast cancer. However, much remains to be learned about how conformational changes may convey information through the structure of PTP1B to enable allosteric regulation by ligands or functional responses to mutations. High-resolution X-ray crystallography can offer unique windows into protein conformational ensembles, but comparison of even high-resolution structures is often complicated by differences between data sets, including non-isomorphism. Here, the highest resolution crystal structure of apo wild-type (WT) PTP1B to date is presented out of a total of ∼350 PTP1B structures in the PDB. This structure is in a crystal form that is rare for PTP1B, with two unique copies of the protein that exhibit distinct patterns of conformational heterogeneity, allowing a controlled comparison of local disorder across the two chains within the same asymmetric unit. The conformational differences between these chains are interrogated in the apo structure and between several recently reported high-resolution ligand-bound structures. Electron-density maps in a high-resolution structure of a recently reported activating double mutant are also examined, and unmodeled alternate conformations in the mutant structure are discovered that coincide with regions of enhanced conformational heterogeneity in the new WT structure. These results validate the notion that these mutations operate by enhancing local dynamics, and suggest a latent susceptibility to such changes in the WT enzyme. Together, these new data and analysis provide a detailed view of the conformational ensemble of PTP1B and highlight the utility of high-resolution crystallography for elucidating conformational heterogeneity with potential relevance for function.</p>","PeriodicalId":7029,"journal":{"name":"Acta crystallographica. Section F, Structural biology communications","volume":" ","pages":"1-12"},"PeriodicalIF":1.1,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10833341/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138827635","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 : 2024-01-01DOI: 10.1107/S2053230X23010579
Chloe Seddon, Gad Frankel, Konstantinos Beis
Conjugation is the process by which plasmids, including those that carry antibiotic-resistance genes, are mobilized from one bacterium (the donor) to another (the recipient). The conjugation efficiency of IncF-like plasmids relies on the formation of mating-pair stabilization via intimate interactions between outer membrane proteins on the donor (a plasmid-encoded TraN isoform) and recipient bacteria. Conjugation of the R100-1 plasmid into Escherichia coli and Klebsiella pneumoniae (KP) recipients relies on pairing between the plasmid-encoded TraNα in the donor and OmpW in the recipient. Here, the crystal structure of K. pneumoniae OmpW (OmpWKP) is reported at 3.2 Å resolution. OmpWKP forms an eight-stranded β-barrel flanked by extracellular loops. The structures of E. coli OmpW (OmpWEC) and OmpWKP show high conservation despite sequence variability in the extracellular loops.
{"title":"Structure of the outer membrane porin OmpW from the pervasive pathogen Klebsiella pneumoniae.","authors":"Chloe Seddon, Gad Frankel, Konstantinos Beis","doi":"10.1107/S2053230X23010579","DOIUrl":"10.1107/S2053230X23010579","url":null,"abstract":"<p><p>Conjugation is the process by which plasmids, including those that carry antibiotic-resistance genes, are mobilized from one bacterium (the donor) to another (the recipient). The conjugation efficiency of IncF-like plasmids relies on the formation of mating-pair stabilization via intimate interactions between outer membrane proteins on the donor (a plasmid-encoded TraN isoform) and recipient bacteria. Conjugation of the R100-1 plasmid into Escherichia coli and Klebsiella pneumoniae (KP) recipients relies on pairing between the plasmid-encoded TraNα in the donor and OmpW in the recipient. Here, the crystal structure of K. pneumoniae OmpW (OmpW<sub>KP</sub>) is reported at 3.2 Å resolution. OmpW<sub>KP</sub> forms an eight-stranded β-barrel flanked by extracellular loops. The structures of E. coli OmpW (OmpW<sub>EC</sub>) and OmpW<sub>KP</sub> show high conservation despite sequence variability in the extracellular loops.</p>","PeriodicalId":7029,"journal":{"name":"Acta crystallographica. Section F, Structural biology communications","volume":"80 Pt 1","pages":"22-27"},"PeriodicalIF":0.9,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10833342/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139416013","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 : 2023-12-18DOI: 10.1107/S2053230X23010646
Ratna A. Utami, Hiromi Yoshida, Lydia H. Kartadinata, Virgi A. Abdillah, Cut R. Faratilla, Debbie S. Retnoningrum, Wangsa T. Ismaya
The copper–zinc superoxide dismutase (CuZnSOD) from lemon (SOD_CL) is active in an acidic environment and resists proteolytic degradation. The enzyme occurs as a dimer, which has an indirect effect on the enzyme activity as the monomer retains only ∼35% of the activity. Here, the crystal structure of SOD_CL at 1.86 Å resolution is reported that may explain this peculiarity. The crystal belonged to space group P21, with unit-cell parameters a = 61.11, b = 74.55, c = 61.69 Å, β = 106.86°, and contained four molecules in the asymmetric unit. The overall structure of SOD_CL resembles that of CuZnSOD from plants. The structure of SOD_CL shows a unique arrangement of surface loop IV that connects the dimer interface and the active site, which is located away from the dimer-interface region. This arrangement allows direct interaction between the residues residing in the dimer interface and those in the active site. The arrangement also includes Leu62 and Gln164, which are conserved in cytoplasmic CuZnSOD. This supports the classification of SOD_CL as a cytoplasmic CuZnSOD despite sharing the highest amino-acid sequence homology with CuZnSODs from spinach and tomato, which are chloroplastic.
{"title":"Direct relationship between dimeric form and activity in the acidic copper–zinc superoxide dismutase from lemon","authors":"Ratna A. Utami, Hiromi Yoshida, Lydia H. Kartadinata, Virgi A. Abdillah, Cut R. Faratilla, Debbie S. Retnoningrum, Wangsa T. Ismaya","doi":"10.1107/S2053230X23010646","DOIUrl":"10.1107/S2053230X23010646","url":null,"abstract":"<p>The copper–zinc superoxide dismutase (CuZnSOD) from lemon (SOD_CL) is active in an acidic environment and resists proteolytic degradation. The enzyme occurs as a dimer, which has an indirect effect on the enzyme activity as the monomer retains only ∼35% of the activity. Here, the crystal structure of SOD_CL at 1.86 Å resolution is reported that may explain this peculiarity. The crystal belonged to space group <i>P</i>2<sub>1</sub>, with unit-cell parameters <i>a</i> = 61.11, <i>b</i> = 74.55, <i>c</i> = 61.69 Å, β = 106.86°, and contained four molecules in the asymmetric unit. The overall structure of SOD_CL resembles that of CuZnSOD from plants. The structure of SOD_CL shows a unique arrangement of surface loop IV that connects the dimer interface and the active site, which is located away from the dimer-interface region. This arrangement allows direct interaction between the residues residing in the dimer interface and those in the active site. The arrangement also includes Leu62 and Gln164, which are conserved in cytoplasmic CuZnSOD. This supports the classification of SOD_CL as a cytoplasmic CuZnSOD despite sharing the highest amino-acid sequence homology with CuZnSODs from spinach and tomato, which are chloroplastic.</p>","PeriodicalId":7029,"journal":{"name":"Acta crystallographica. Section F, Structural biology communications","volume":"79 12","pages":"301-307"},"PeriodicalIF":0.9,"publicationDate":"2023-12-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138717613","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}