Open-access and free articles in Acta Crystallographica Section F: Structural Biology Communications
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Acta Crystallographica Section F: Structural Biology Communications is a rapid all-electronic journal, which provides a home for short communications on the crystallization and structure of biological macromolecules. Structures determined through structural genomics initiatives or from iterative studies such as those used in the pharmaceutical industry are particularly welcomed. Articles are available online when ready, making publication as fast as possible, and include unlimited free colour illustrations, movies and other enhancements. The editorial process is completely electronic with respect to deposition, submission, refereeing and publication.en-gbCopyright (c) 2024 International Union of CrystallographyInternational Union of CrystallographyInternational Union of Crystallographytexthttps://journals.iucr.orgOpen-access and free articles in Acta Crystallographica Section F Structural Biology CommunicationsActa Crystallographica Section F: Structural Biology Communications is a rapid all-electronic journal, which provides a home for short communications on the crystallization and structure of biological macromolecules. Structures determined through structural genomics initiatives or from iterative studies such as those used in the pharmaceutical industry are particularly welcomed. Articles are available online when ready, making publication as fast as possible, and include unlimited free colour illustrations, movies and other enhancements. The editorial process is completely electronic with respect to deposition, submission, refereeing and publication.urn:issn:1744-3091text/html1monthly2002-01-01T00:00+00:00Copyright (c) 2024 International Union of Crystallographymed@iucr.orgActa Crystallographica Section F Structural Biology Communicationsurn:issn:1744-3091Open-access and free articles in Acta Crystallographica Section F: Structural Biology Communicationshttp://journals.iucr.org/logos/rss10f.gif
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Still imageStructure of the GDP-bound state of the SRP GTPase FlhF
http://scripts.iucr.org/cgi-bin/paper?nd5006
The GTPase FlhF, a signal recognition particle (SRP)-type enzyme, is pivotal for spatial–numerical control and bacterial flagella assembly across diverse species, including pathogens. This study presents the X-ray structure of FlhF in its GDP-bound state at a resolution of 2.28 Å. The structure exhibits the classical N- and G-domain fold, consistent with related SRP GTPases such as Ffh and FtsY. Comparative analysis with GTP-loaded FlhF elucidates the conformational changes associated with GTP hydrolysis. These topological reconfigurations are similarly evident in Ffh and FtsY, and play a pivotal role in regulating the functions of these hydrolases.International Union of Crystallographytexthttps://creativecommons.org/licenses/by/4.0/urn:issn:2053-230Xen2024-02-20Dornes, A.Mais, C.-N.Bange, G.FLAGELLAR ASSEMBLY; SRP GTPASES; GTPASES; FLHF; NUCLEOTIDESStructure of the GDP-bound state of the SRP GTPase FlhFThe GTPase FlhF, a signal recognition particle (SRP)-type enzyme, is pivotal for spatial–numerical control and bacterial flagella assembly across diverse species, including pathogens. This study presents the X-ray structure of FlhF in its GDP-bound state at a resolution of 2.28 Å. The structure exhibits the classical N- and G-domain fold, consistent with related SRP GTPases such as Ffh and FtsY. Comparative analysis with GTP-loaded FlhF elucidates the conformational changes associated with GTP hydrolysis. These topological reconfigurations are similarly evident in Ffh and FtsY, and play a pivotal role in regulating the functions of these hydrolases.doi:10.1107/S2053230X24000979text/htmlThis study presents the X-ray structure of FlhF in its GDP-bound state at a resolution of 2.28 Å, exhibiting the classical N- and G-domain fold. Comparative analysis with GTP-loaded FlhF elucidates the conformational changes associated with GTP hydrolysis.2053-230XActa Crystallographica Section F: Structural Biology Communications58research communications53802053-230X2024-02-20med@iucr.orghttps://creativecommons.org/licenses/by/4.0/3March 2024Expression, purification and crystallization of the photosensory module of phytochrome B (phyB) from Sorghum bicolor
http://scripts.iucr.org/cgi-bin/paper?nj5324
Sorghum, a short-day tropical plant, has been adapted for temperate grain production, in particular through the selection of variants at the MATURITY loci (Ma1–Ma6) that reduce photoperiod sensitivity. Ma3 encodes phytochrome B (phyB), a red/far-red photochromic biliprotein photoreceptor. The multi-domain gene product, comprising 1178 amino acids, autocatalytically binds the phytochromobilin chromophore to form the photoactive holophytochrome (Sb.phyB). This study describes the development of an efficient heterologous overproduction system which allows the production of large quantities of various holoprotein constructs, along with purification and crystallization procedures. Crystals of the Pr (red-light-absorbing) forms of NPGP, PGP and PG (residues 1–655, 114–655 and 114–458, respectively), each C-terminally tagged with His6, were successfully produced. While NPGP crystals did not diffract, those of PGP and PG diffracted to 6 and 2.1 Å resolution, respectively. Moving the tag to the N-terminus and replacing phytochromobilin with phycocyanobilin as the ligand produced PG crystals that diffracted to 1.8 Å resolution. These results demonstrate that the diffraction quality of challenging protein crystals can be improved by removing flexible regions, shifting fusion tags and altering small-molecule ligands.International Union of Crystallographytexthttps://creativecommons.org/licenses/by/4.0/urn:issn:2053-230Xen2024-02-20Shenkutie, S.M.Nagano, S.Hughes, J.SORGHUM BICOLOR; PHYTOCHROME B; MATURITY; CRYSTALLIZATION; DIFFRACTION QUALITYExpression, purification and crystallization of the photosensory module of phytochrome B (phyB) from Sorghum bicolorSorghum, a short-day tropical plant, has been adapted for temperate grain production, in particular through the selection of variants at the MATURITY loci (Ma1–Ma6) that reduce photoperiod sensitivity. Ma3 encodes phytochrome B (phyB), a red/far-red photochromic biliprotein photoreceptor. The multi-domain gene product, comprising 1178 amino acids, autocatalytically binds the phytochromobilin chromophore to form the photoactive holophytochrome (Sb.phyB). This study describes the development of an efficient heterologous overproduction system which allows the production of large quantities of various holoprotein constructs, along with purification and crystallization procedures. Crystals of the Pr (red-light-absorbing) forms of NPGP, PGP and PG (residues 1–655, 114–655 and 114–458, respectively), each C-terminally tagged with His6, were successfully produced. While NPGP crystals did not diffract, those of PGP and PG diffracted to 6 and 2.1 Å resolution, respectively. Moving the tag to the N-terminus and replacing phytochromobilin with phycocyanobilin as the ligand produced PG crystals that diffracted to 1.8 Å resolution. These results demonstrate that the diffraction quality of challenging protein crystals can be improved by removing flexible regions, shifting fusion tags and altering small-molecule ligands.doi:10.1107/S2053230X24000827text/htmlA heterologous holophytochrome overproduction system has been developed to produce large quantities of three holoprotein constructs of phytochrome B from S. bicolor for crystallization. The results showed that the diffraction quality of the crystals could be improved by removing flexible regions, shifting the fusion tag and changing the type of ligand.research communications5980Acta Crystallographica Section F: Structural Biology Communications2053-230X662024-02-20https://creativecommons.org/licenses/by/4.0/med@iucr.org3March 20242053-230XCrystal structure of the RNA-recognition motif of Drosophila melanogaster tRNA (uracil-5-)-methyltransferase homolog A
http://scripts.iucr.org/cgi-bin/paper?ek5035
Human tRNA (uracil-5-)-methyltransferase 2 homolog A (TRMT2A) is the dedicated enzyme for the methylation of uridine 54 in transfer RNA (tRNA). Human TRMT2A has also been described as a modifier of polyglutamine (polyQ)-derived neuronal toxicity. The corresponding human polyQ pathologies include Huntington's disease and constitute a family of devastating neurodegenerative diseases. A polyQ tract in the corresponding disease-linked protein causes neuronal death and symptoms such as impaired motor function, as well as cognitive impairment. In polyQ disease models, silencing of TRMT2A reduced polyQ-associated cell death and polyQ protein aggregation, suggesting this protein as a valid drug target against this class of disorders. In this paper, the 1.6 Å resolution crystal structure of the RNA-recognition motif (RRM) from Drosophila melanogaster, which is a homolog of human TRMT2A, is described and analysed.International Union of Crystallographytexthttps://creativecommons.org/licenses/by/4.0/urn:issn:2053-230Xen2024-01-25Witzenberger, M.Janowski, R.Niessing, D.RRMS; TRMT2A; METHYLTRANSFERASES; X-RAY CRYSTALLOGRAPHY; DROSOPHILA MELANOGASTER; NEURODEGENERATIVE DISEASECrystal structure of the RNA-recognition motif of Drosophila melanogaster tRNA (uracil-5-)-methyltransferase homolog AHuman tRNA (uracil-5-)-methyltransferase 2 homolog A (TRMT2A) is the dedicated enzyme for the methylation of uridine 54 in transfer RNA (tRNA). Human TRMT2A has also been described as a modifier of polyglutamine (polyQ)-derived neuronal toxicity. The corresponding human polyQ pathologies include Huntington's disease and constitute a family of devastating neurodegenerative diseases. A polyQ tract in the corresponding disease-linked protein causes neuronal death and symptoms such as impaired motor function, as well as cognitive impairment. In polyQ disease models, silencing of TRMT2A reduced polyQ-associated cell death and polyQ protein aggregation, suggesting this protein as a valid drug target against this class of disorders. In this paper, the 1.6 Å resolution crystal structure of the RNA-recognition motif (RRM) from Drosophila melanogaster, which is a homolog of human TRMT2A, is described and analysed.doi:10.1107/S2053230X24000645text/htmlThe 1.6 Å resolution crystal structure of the RNA-recognition motif of D. melanogaster tRNA (uracil-5-)-methyltransferase homolog A is reported.80research communications36422053-230XActa Crystallographica Section F: Structural Biology Communications2February 20242024-01-25https://creativecommons.org/licenses/by/4.0/med@iucr.org2053-230XOnline carbohydrate 3D structure validation with the Privateer web app
http://scripts.iucr.org/cgi-bin/paper?va5056
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.International Union of Crystallographytexthttps://creativecommons.org/licenses/by/4.0/urn:issn:2053-230Xen2024-01-24Dialpuri, J.S.Bagdonas, H.Schofield, L.C.Pham, P.T.Holland, L.Bond, P.S.Sánchez Rodríguez, F.McNicholas, S.J.Agirre, J.PRIVATEER; VALIDATION; POLYSACCHARIDES; CARBOHYDRATES; N-GLYCOSYLATION; N-GLYCANS; WEB APPSOnline carbohydrate 3D structure validation with the Privateer web appOwing 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.doi:10.1107/S2053230X24000359text/htmlThe Privateer carbohydrate 3D structure-validation software is now freely available as a web app. Here, its use is described, including a practical example.2053-230X2February 20242024-01-24https://creativecommons.org/licenses/by/4.0/med@iucr.org352053-230XActa Crystallographica Section F: Structural Biology Communications80methods communications30High-resolution double vision of the allosteric phosphatase PTP1B
http://scripts.iucr.org/cgi-bin/paper?jt5072
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.International Union of Crystallographytexthttps://creativecommons.org/licenses/by/4.0/urn:issn:2053-230Xen2024-01-01Sharma, S.Skaist Mehlman, T.Sagabala, R.S.Boivin, B.Keedy, D.A.PROTEIN TYROSINE PHOSPHATASE 1B; ALLOSTERIC REGULATION; CONFORMATIONAL HETEROGENEITY; HIGH-RESOLUTION CRYSTALLOGRAPHYHigh-resolution double vision of the allosteric phosphatase PTP1BProtein 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.doi:10.1107/S2053230X23010749text/htmlHigh-resolution X-ray crystallography reveals new details of conformational heterogeneity for the dynamic enzyme PTP1B.Acta Crystallographica Section F: Structural Biology Communications2053-230X12research communications1802053-230X2024-01-01med@iucr.orghttps://creativecommons.org/licenses/by/4.0/1January 2024Structure of the outer membrane porin OmpW from the pervasive pathogen Klebsiella pneumoniae
http://scripts.iucr.org/cgi-bin/paper?pg5094
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.International Union of Crystallographytexthttps://creativecommons.org/licenses/by/4.0/urn:issn:2053-230Xen2024-01-01Seddon, C.Frankel, G.Beis, K.KLEBSIELLA PNEUMONIAE; OUTER MEMBRANE PORIN; OMPW; BACTERIAL CONJUGATION; [BETA]-BARRELStructure of the outer membrane porin OmpW from the pervasive pathogen Klebsiella pneumoniaeConjugation 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.doi:10.1107/S2053230X23010579text/htmlThe crystal structure of the outer membrane protein OmpW from Klebsiella pneumoniae is reported.research communications22802053-230XActa Crystallographica Section F: Structural Biology Communications272024-01-01med@iucr.orghttps://creativecommons.org/licenses/by/4.0/1January 20242053-230XThe crystal structure of the human smacovirus 1 Rep domain
http://scripts.iucr.org/cgi-bin/paper?ek5034
Replication initiator proteins (Reps) from the HUH endonuclease family process specific single-stranded DNA sequences to initiate rolling-circle replication in viruses. Here, the first crystal structure of the apo state of a Rep domain from the smacovirus family is reported. The structure of the human smacovirus 1 Rep domain was obtained at 1.33 Å resolution and represents an expansion of the HUH endonuclease superfamily, allowing greater diversity in bioconjugation-tag applications.International Union of Crystallographytexthttps://creativecommons.org/licenses/by/4.0/urn:issn:2053-230Xen2023-12-05Limón, L.K.Shi, K.Dao, A.Rugloski, J.Tompkins, K.J.Aihara, H.Gordon, W.R.Evans, R.L.HUH ENDONUCLEASES; REP DOMAINS; HUH-TAGS; SSDNA; BIOCONJUGATION; SMACOVIRUSES; CRYSTAL STRUCTUREThe crystal structure of the human smacovirus 1 Rep domainReplication initiator proteins (Reps) from the HUH endonuclease family process specific single-stranded DNA sequences to initiate rolling-circle replication in viruses. Here, the first crystal structure of the apo state of a Rep domain from the smacovirus family is reported. The structure of the human smacovirus 1 Rep domain was obtained at 1.33 Å resolution and represents an expansion of the HUH endonuclease superfamily, allowing greater diversity in bioconjugation-tag applications.doi:10.1107/S2053230X23009536text/htmlThe structure of the human smacovirus 1 Rep domain was obtained at 1.33 Å resolution. This new HUH endonuclease offers a new ssDNA-binding sequence specificity that will be exploited to increase orthogonality among Rep families.79295research communications300Acta Crystallographica Section F: Structural Biology Communications2053-230XDecember 202312med@iucr.orghttps://creativecommons.org/licenses/by/4.0/2023-12-052053-230XMaking your raw data available to the macromolecular crystallography community
http://scripts.iucr.org/cgi-bin/paper?va5053
A recent editorial in the IUCr macromolecular crystallography journals [Helliwell et al. (2019), Acta Cryst. D75, 455–457] called for the implementation of the FAIR data principles. This implies that the authors of a paper that describes research on a macromolecular structure should make their raw diffraction data available. Authors are already used to submitting the derived data (coordinates) and the processed data (structure factors, merged or unmerged) to the PDB, but may still be uncomfortable with making the raw diffraction images available. In this paper, some guidelines and instructions on depositing raw data to Zenodo are given.International Union of Crystallographytexthttps://creativecommons.org/licenses/by/4.0/urn:issn:2053-230Xen2023-09-29Kroon-Batenburg, L.M.J.RAW DATA DEPOSITION; ZENODO; FAIR PRINCIPLESMaking your raw data available to the macromolecular crystallography communityA recent editorial in the IUCr macromolecular crystallography journals [Helliwell et al. (2019), Acta Cryst. D75, 455–457] called for the implementation of the FAIR data principles. This implies that the authors of a paper that describes research on a macromolecular structure should make their raw diffraction data available. Authors are already used to submitting the derived data (coordinates) and the processed data (structure factors, merged or unmerged) to the PDB, but may still be uncomfortable with making the raw diffraction images available. In this paper, some guidelines and instructions on depositing raw data to Zenodo are given.doi:10.1107/S2053230X23007987text/htmlGuidance is provided on depositing raw diffraction data to support the results described in a macromolecular crystallography paper.2053-230X2023-09-29med@iucr.orghttps://creativecommons.org/licenses/by/4.0/10October 2023Acta Crystallographica Section F: Structural Biology Communications2053-230X273methods communications26779Biochemical and X-ray analyses of the players involved in the faRel2/aTfaRel2 toxin–antitoxin operon
http://scripts.iucr.org/cgi-bin/paper?ek5033
The aTfaRel2/faRel2 operon from Coprobacillus sp. D7 encodes a bicistronic type II toxin–antitoxin (TA) module. The FaRel2 toxin is a toxic small alarmone synthetase (toxSAS) that inhibits translation through the pyrophosphorylation of uncharged tRNAs at the 3′-CCA end. The toxin is neutralized by the antitoxin ATfaRel2 through the formation of an inactive TA complex. Here, the production, biophysical analysis and crystallization of ATfaRel2 and FaRel2 as well as of the ATfaRel2–FaRel2 complex are reported. ATfaRel2 is monomeric in solution. The antitoxin crystallized in space group P21212 with unit-cell parameters a = 53.3, b = 34.2, c = 37.6 Å, and the best crystal diffracted to a resolution of 1.24 Å. Crystals of FaRel2 in complex with APCPP, a nonhydrolysable ATP analogue, belonged to space group P21, with unit-cell parameters a = 31.5, b = 60.6, c = 177.2 Å, β = 90.6°, and diffracted to 2.6 Å resolution. The ATfaRel2–FaRel2Y128F complex forms a heterotetramer in solution composed of two toxins and two antitoxins. This complex crystallized in two space groups: F4132, with unit-cell parameters a = b = c = 227.1 Å, and P212121, with unit-cell parameters a = 51.7, b = 106.2, c = 135.1 Å. The crystals diffracted to 1.98 and 2.1 Å resolution, respectively.International Union of Crystallographytexthttps://creativecommons.org/licenses/by/4.0/urn:issn:2053-230Xen2023-09-20Dominguez-Molina, L.Talavera, A.Cepauskas, A.Kurata, T.Echemendia-Blanco, D.Hauryliuk, V.Garcia-Pino, A.ATFAREL2-FAREL2 COMPLEX; COPROBACILLUS; TOXIN-ANTITOXIN MODULES; ALARMONES; TOXSAS; RSH PROTEINS; TRNA MODIFICATIONBiochemical and X-ray analyses of the players involved in the faRel2/aTfaRel2 toxin–antitoxin operonThe aTfaRel2/faRel2 operon from Coprobacillus sp. D7 encodes a bicistronic type II toxin–antitoxin (TA) module. The FaRel2 toxin is a toxic small alarmone synthetase (toxSAS) that inhibits translation through the pyrophosphorylation of uncharged tRNAs at the 3′-CCA end. The toxin is neutralized by the antitoxin ATfaRel2 through the formation of an inactive TA complex. Here, the production, biophysical analysis and crystallization of ATfaRel2 and FaRel2 as well as of the ATfaRel2–FaRel2 complex are reported. ATfaRel2 is monomeric in solution. The antitoxin crystallized in space group P21212 with unit-cell parameters a = 53.3, b = 34.2, c = 37.6 Å, and the best crystal diffracted to a resolution of 1.24 Å. Crystals of FaRel2 in complex with APCPP, a nonhydrolysable ATP analogue, belonged to space group P21, with unit-cell parameters a = 31.5, b = 60.6, c = 177.2 Å, β = 90.6°, and diffracted to 2.6 Å resolution. The ATfaRel2–FaRel2Y128F complex forms a heterotetramer in solution composed of two toxins and two antitoxins. This complex crystallized in two space groups: F4132, with unit-cell parameters a = b = c = 227.1 Å, and P212121, with unit-cell parameters a = 51.7, b = 106.2, c = 135.1 Å. The crystals diffracted to 1.98 and 2.1 Å resolution, respectively.doi:10.1107/S2053230X23007288text/htmlCrystallization strategies for and structural analyses of all of the players in the faRel2/aTfaRel2 toxin–antitoxin system are reported.2023-09-20med@iucr.orghttps://creativecommons.org/licenses/by/4.0/10October 20232053-230Xresearch communications247792053-230XActa Crystallographica Section F: Structural Biology Communications256High-resolution crystal structure of the Mu8.1 conotoxin from Conus mucronatus
http://scripts.iucr.org/cgi-bin/paper?ow5036
Marine cone snails produce a wealth of peptide toxins (conotoxins) that bind their molecular targets with high selectivity and potency. Therefore, conotoxins constitute valuable biomolecular tools with a variety of biomedical purposes. The Mu8.1 conotoxin from Conus mucronatus is the founding member of the newly identified saposin-like conotoxin class of conotoxins and has been shown to target Cav2.3, a voltage-gated calcium channel. Two crystal structures have recently been determined of Mu8.1 at 2.3 and 2.1 Å resolution. Here, a high-resolution crystal structure of Mu8.1 was determined at 1.67 Å resolution in the high-symmetry space group I4122. The asymmetric unit contained one molecule, with a symmetry-related molecule generating a dimer equivalent to that observed in the two previously determined structures. The high resolution allows a detailed atomic analysis of a water-filled cavity buried at the dimer interface, revealing a tightly coordinated network of waters that shield a lysine residue (Lys55) with a predicted unusually low side-chain pKa value. These findings are discussed in terms of a potential functional role of Lys55 in target interaction.International Union of Crystallographytexthttps://creativecommons.org/licenses/by/4.0/urn:issn:2053-230Xen2023-08-29Müller, E.Hackney, C.M.Ellgaard, L.Morth, J.P.CONOTOXINS; ZINC BINDING; TOXINS; HYDROGEN BONDING; MU8.1High-resolution crystal structure of the Mu8.1 conotoxin from Conus mucronatusMarine cone snails produce a wealth of peptide toxins (conotoxins) that bind their molecular targets with high selectivity and potency. Therefore, conotoxins constitute valuable biomolecular tools with a variety of biomedical purposes. The Mu8.1 conotoxin from Conus mucronatus is the founding member of the newly identified saposin-like conotoxin class of conotoxins and has been shown to target Cav2.3, a voltage-gated calcium channel. Two crystal structures have recently been determined of Mu8.1 at 2.3 and 2.1 Å resolution. Here, a high-resolution crystal structure of Mu8.1 was determined at 1.67 Å resolution in the high-symmetry space group I4122. The asymmetric unit contained one molecule, with a symmetry-related molecule generating a dimer equivalent to that observed in the two previously determined structures. The high resolution allows a detailed atomic analysis of a water-filled cavity buried at the dimer interface, revealing a tightly coordinated network of waters that shield a lysine residue (Lys55) with a predicted unusually low side-chain pKa value. These findings are discussed in terms of a potential functional role of Lys55 in target interaction.doi:10.1107/S2053230X23007070text/htmlThe crystal structure of the Mu8.1 conotoxin was determined in the high-symmetry space group I4122 at 1.67 Å resolution. This crystal structure reveals a surface-exposed Zn2+-binding site and establishes a hydrogen-bonding network around Lys55 buried at the dimer interface.med@iucr.orghttps://creativecommons.org/licenses/by/4.0/2023-08-29September 202392053-230X240research communications79Acta Crystallographica Section F: Structural Biology Communications2053-230X246Drastic alterations in the loop structure around colchicine upon complex formation with an engineered lipocalin indicate a conformational selection mechanism
http://scripts.iucr.org/cgi-bin/paper?ji5030
Using Anticalin technology, a lipocalin protein dubbed Colchicalin, with the ability to bind the toxic plant alkaloid colchicine with picomolar affinity, has previously been engineered, thus offering a potential antidote in vivo and also allowing its sensitive detection in biological samples. To further analyze the mode of ligand recognition, the crystal structure of Colchicalin is now reported in its unliganded form and is compared with the colchicine complex. A superposition of the protein structures revealed major rearrangements in the four structurally variable loops of the engineered lipocalin. Notably, the binding pocket in the unbound protein is largely occupied by the inward-bent loop #3, in particular Ile97, as well as by the phenylalanine side chain at position 71 in loop #2. Upon binding of colchicine, a dramatic shift of loop #3 by up to 11.1 Å occurs, in combination with a side-chain flip of Phe71, thus liberating the necessary space within the ligand pocket. Interestingly, the proline residue at the neighboring position 72, which arose during the combinatorial engineering of Colchicalin, remained in a cis configuration in both structures. These findings provide a striking example of a conformational adaptation mechanism, which is a long-known phenomenon for antibodies in immunochemistry, during the recognition of a small ligand by an engineered lipocalin, thus illustrating the general similarity between the mode of antigen/ligand binding by immunoglobulins and lipocalins.International Union of Crystallographytexthttps://creativecommons.org/licenses/by/4.0/urn:issn:2053-230Xen2023-08-16Jerschke, E.Eichinger, A.Skerra, A.ANTICALINS; HAPTEN RECOGNITION; INDUCED FIT; PROTEIN DESIGNDrastic alterations in the loop structure around colchicine upon complex formation with an engineered lipocalin indicate a conformational selection mechanismUsing Anticalin technology, a lipocalin protein dubbed Colchicalin, with the ability to bind the toxic plant alkaloid colchicine with picomolar affinity, has previously been engineered, thus offering a potential antidote in vivo and also allowing its sensitive detection in biological samples. To further analyze the mode of ligand recognition, the crystal structure of Colchicalin is now reported in its unliganded form and is compared with the colchicine complex. A superposition of the protein structures revealed major rearrangements in the four structurally variable loops of the engineered lipocalin. Notably, the binding pocket in the unbound protein is largely occupied by the inward-bent loop #3, in particular Ile97, as well as by the phenylalanine side chain at position 71 in loop #2. Upon binding of colchicine, a dramatic shift of loop #3 by up to 11.1 Å occurs, in combination with a side-chain flip of Phe71, thus liberating the necessary space within the ligand pocket. Interestingly, the proline residue at the neighboring position 72, which arose during the combinatorial engineering of Colchicalin, remained in a cis configuration in both structures. These findings provide a striking example of a conformational adaptation mechanism, which is a long-known phenomenon for antibodies in immunochemistry, during the recognition of a small ligand by an engineered lipocalin, thus illustrating the general similarity between the mode of antigen/ligand binding by immunoglobulins and lipocalins.doi:10.1107/S2053230X23006817text/htmlColchicalin is an engineered binding protein derived from the lipocalin scaffold, which provides a binding site comprised of four loops exhibiting structural plasticity. Structural analysis of this lipocalin in the absence and the presence of its ligand colchicine reveals drastic structural changes which indicate a conformational selection mechanism.Acta Crystallographica Section F: Structural Biology Communications2053-230X239231research communications792053-230Xmed@iucr.orghttps://creativecommons.org/licenses/by/4.0/2023-08-16September 20239Structure of the imine reductase from Ajellomyces dermatitidis in three crystal forms
http://scripts.iucr.org/cgi-bin/paper?no5202
The NADPH-dependent imine reductase from Ajellomyces dermatitidis (AdRedAm) catalyzes the reductive amination of certain ketones with amine donors supplied in an equimolar ratio. The structure of AdRedAm has been determined in three forms. The first form, which belongs to space group P3121 and was refined to 2.01 Å resolution, features two molecules (one dimer) in the asymmetric unit in complex with the redox-inactive cofactor NADPH4. The second form, which belongs to space group C21 and was refined to 1.73 Å resolution, has nine molecules (four and a half dimers) in the asymmetric unit, each complexed with NADP+. The third form, which belongs to space group P3121 and was refined to 1.52 Å resolution, has one molecule (one half-dimer) in the asymmetric unit. This structure was again complexed with NADP+ and also with the substrate 2,2-difluoroacetophenone. The different data sets permit the analysis of AdRedAm in different conformational states and also reveal the molecular basis of stereoselectivity in the transformation of fluorinated acetophenone substrates by the enzyme.International Union of Crystallographytexthttps://creativecommons.org/licenses/by/4.0/urn:issn:2053-230Xen2023-08-15Sharma, M.Cuetos, A.Willliams, A.González-Martínez, D.Grogan, G.IMINE REDUCTASES; BIOCATALYSIS; AJELLOMYCES DERMATITIDISStructure of the imine reductase from Ajellomyces dermatitidis in three crystal formsThe NADPH-dependent imine reductase from Ajellomyces dermatitidis (AdRedAm) catalyzes the reductive amination of certain ketones with amine donors supplied in an equimolar ratio. The structure of AdRedAm has been determined in three forms. The first form, which belongs to space group P3121 and was refined to 2.01 Å resolution, features two molecules (one dimer) in the asymmetric unit in complex with the redox-inactive cofactor NADPH4. The second form, which belongs to space group C21 and was refined to 1.73 Å resolution, has nine molecules (four and a half dimers) in the asymmetric unit, each complexed with NADP+. The third form, which belongs to space group P3121 and was refined to 1.52 Å resolution, has one molecule (one half-dimer) in the asymmetric unit. This structure was again complexed with NADP+ and also with the substrate 2,2-difluoroacetophenone. The different data sets permit the analysis of AdRedAm in different conformational states and also reveal the molecular basis of stereoselectivity in the transformation of fluorinated acetophenone substrates by the enzyme.doi:10.1107/S2053230X23006672text/htmlThe structure of the imine reductase from A. dermatitidis is presented in three crystal forms, each of which provides information on conformational dynamics and cofactor and substrate binding within the enzyme.2053-230XActa Crystallographica Section F: Structural Biology Communications230research communications224792053-230X2023-08-15med@iucr.orghttps://creativecommons.org/licenses/by/4.0/9September 2023Crystal structure of a GCN5-related N-acetyltransferase from Lactobacillus curiae
http://scripts.iucr.org/cgi-bin/paper?us5147
Members of the GCN5-related N-acetyltransferase (GNAT) family are found in all domains of life and are involved in processes ranging from protein synthesis and gene expression to detoxification and virulence. Due to the variety of their macromolecular targets, GNATs are a highly diverse family of proteins. Currently, 3D structures of only a small number of GNAT representatives are available and thus the family remains poorly characterized. Here, the crystal structure of the guanidine riboswitch-associated GNAT from Lactobacillus curiae (LcGNAT) that acetylates canavanine, a structural analogue of arginine with antimetabolite properties, is reported. LcGNAT shares the conserved fold of the members of the GNAT superfamily, but does not contain an N-terminal β0 strand and instead contains a C-terminal β7 strand. Its P-loop, which coordinates the pyrophosphate moiety of the acetyl-coenzyme A cosubstrate, is degenerated. These features are shared with its closest homologues in the polyamine acetyltransferase subclass. Site-directed mutagenesis revealed a central role of the conserved residue Tyr142 in catalysis, as well as the semi-conserved Tyr97 and Glu92, suggesting that despite its individual substrate specificity LcGNAT performs the classical reaction mechanism of this family.International Union of Crystallographytexthttps://creativecommons.org/licenses/by/4.0/urn:issn:2053-230Xen2023-08-09Fleming, J.R.Hauth, F.Hartig, J.S.Mayans, O.GCN5-RELATED N-ACETYLTRANSFERASES; LACTOBACILLUS CURIAE; CANAVANINE; GUANIDINE RIBOSWITCH-ASSOCIATED GENE FUNCTIONS; X-RAY CRYSTALLOGRAPHY; ACETYLATIONCrystal structure of a GCN5-related N-acetyltransferase from Lactobacillus curiaeMembers of the GCN5-related N-acetyltransferase (GNAT) family are found in all domains of life and are involved in processes ranging from protein synthesis and gene expression to detoxification and virulence. Due to the variety of their macromolecular targets, GNATs are a highly diverse family of proteins. Currently, 3D structures of only a small number of GNAT representatives are available and thus the family remains poorly characterized. Here, the crystal structure of the guanidine riboswitch-associated GNAT from Lactobacillus curiae (LcGNAT) that acetylates canavanine, a structural analogue of arginine with antimetabolite properties, is reported. LcGNAT shares the conserved fold of the members of the GNAT superfamily, but does not contain an N-terminal β0 strand and instead contains a C-terminal β7 strand. Its P-loop, which coordinates the pyrophosphate moiety of the acetyl-coenzyme A cosubstrate, is degenerated. These features are shared with its closest homologues in the polyamine acetyltransferase subclass. Site-directed mutagenesis revealed a central role of the conserved residue Tyr142 in catalysis, as well as the semi-conserved Tyr97 and Glu92, suggesting that despite its individual substrate specificity LcGNAT performs the classical reaction mechanism of this family.doi:10.1107/S2053230X2300571Xtext/htmlThe 3D structure of a GCN5-related N-acetyltransferase enzyme that is selective for canavanine has been elucidated and shown to share the fold and catalytic mechanism of the polyamine acetyltransferase subclass.med@iucr.orghttps://creativecommons.org/licenses/by/4.0/2023-08-09August 202382053-230X217research communications792053-230XActa Crystallographica Section F: Structural Biology Communications223Crystal structure of Prp16 in complex with ADP
http://scripts.iucr.org/cgi-bin/paper?ek5032
DEAH-box helicases play a crucial role in pre-mRNA splicing as they are responsible for major rearrangements of the spliceosome and are involved in various quality-ensuring steps. Prp16 is the driving force during spliceosomal catalysis, remodeling the C state into the C* state. Here, the first crystal structure of Prp16 from Chaetomium thermophilum in complex with ADP is reported at 1.9 Å resolution. Comparison with the other spliceosomal DEAH-box helicases Prp2, Prp22 and Prp43 reveals an overall identical domain architecture. The β-hairpin, which is a structural element of the RecA2 domain, exhibits a unique position, punctuating its flexibility. Analysis of cryo-EM models of spliceosomal complexes containing Prp16 reveals that these models show Prp16 in its nucleotide-free state, rendering the model presented here the first structure of Prp16 in complex with a nucleotide.International Union of Crystallographytexthttps://creativecommons.org/licenses/by/4.0/urn:issn:2053-230Xen2023-07-25Garbers, T.B.Enders, M.Neumann, P.Ficner, R.DEAH-BOX HELICASES; PRP16; CHAETOMIUM THERMOPHILUM; SPLICEOSOMECrystal structure of Prp16 in complex with ADPDEAH-box helicases play a crucial role in pre-mRNA splicing as they are responsible for major rearrangements of the spliceosome and are involved in various quality-ensuring steps. Prp16 is the driving force during spliceosomal catalysis, remodeling the C state into the C* state. Here, the first crystal structure of Prp16 from Chaetomium thermophilum in complex with ADP is reported at 1.9 Å resolution. Comparison with the other spliceosomal DEAH-box helicases Prp2, Prp22 and Prp43 reveals an overall identical domain architecture. The β-hairpin, which is a structural element of the RecA2 domain, exhibits a unique position, punctuating its flexibility. Analysis of cryo-EM models of spliceosomal complexes containing Prp16 reveals that these models show Prp16 in its nucleotide-free state, rendering the model presented here the first structure of Prp16 in complex with a nucleotide.doi:10.1107/S2053230X23005721text/htmlPrp16 is a DEAH-box ATPase required for the splicing of pre-mRNA. The X-ray crystal structure of the Prp16-ADP complex was determined at a resolution of 1.9 Å.2053-230X8August 20232023-07-25med@iucr.orghttps://creativecommons.org/licenses/by/4.0/207Acta Crystallographica Section F: Structural Biology Communications2053-230X79research communications200Tetracycline-modifying enzyme SmTetX from Stenotrophomonas maltophilia
http://scripts.iucr.org/cgi-bin/paper?va5051
The resistance of the emerging human pathogen Stenotrophomonas maltophilia to tetracycline antibiotics mainly depends on multidrug efflux pumps and ribosomal protection enzymes. However, the genomes of several strains of this Gram-negative bacterium code for a FAD-dependent monooxygenase (SmTetX) homologous to tetracycline destructases. This protein was recombinantly produced and its structure and function were investigated. Activity assays using SmTetX showed its ability to modify oxytetracycline with a catalytic rate comparable to those of other destructases. SmTetX shares its fold with the tetracycline destructase TetX from Bacteroides thetaiotaomicron; however, its active site possesses an aromatic region that is unique in this enzyme family. A docking study confirmed tetracycline and its analogues to be the preferred binders amongst various classes of antibiotics.International Union of Crystallographytexthttps://creativecommons.org/licenses/by/4.0/urn:issn:2053-230Xen2023-07-05Malý, M.Kolenko, P.Stránský, J.Švecová, L.Dušková, J.Koval', T.Skálová, T.Trundová, M.Adámková, K.Černý, J.Božíková, P.Dohnálek, J.FAD-DEPENDENT MONOOXYGENASES; TETRACYCLINE; ANTIBIOTIC RESISTANCETetracycline-modifying enzyme SmTetX from Stenotrophomonas maltophiliaThe resistance of the emerging human pathogen Stenotrophomonas maltophilia to tetracycline antibiotics mainly depends on multidrug efflux pumps and ribosomal protection enzymes. However, the genomes of several strains of this Gram-negative bacterium code for a FAD-dependent monooxygenase (SmTetX) homologous to tetracycline destructases. This protein was recombinantly produced and its structure and function were investigated. Activity assays using SmTetX showed its ability to modify oxytetracycline with a catalytic rate comparable to those of other destructases. SmTetX shares its fold with the tetracycline destructase TetX from Bacteroides thetaiotaomicron; however, its active site possesses an aromatic region that is unique in this enzyme family. A docking study confirmed tetracycline and its analogues to be the preferred binders amongst various classes of antibiotics.doi:10.1107/S2053230X23005381text/htmlStenotrophomonas maltophilia codes for a tetracycline-modifying FAD-dependent monooxygenase that shares an overall fold with the tetracycline destructase TetX and possesses a unique active site within this enzyme family.180research communications792053-230XActa Crystallographica Section F: Structural Biology Communications192https://creativecommons.org/licenses/by/4.0/med@iucr.org2023-07-05July 202372053-230XThe catalytic domains of Streptococcus mutans glucosyltransferases: a structural analysis
http://scripts.iucr.org/cgi-bin/paper?jc5056
Streptococcus mutans, found in the human oral cavity, is a significant contributor to the pathogenesis of dental caries. This bacterium expresses three genetically distinct types of glucosyltransferases named GtfB (GTF-I), GtfC (GTF-SI) and GtfD (GTF-S) that play critical roles in the development of dental plaque. The catalytic domains of GtfB, GtfC and GtfD contain conserved active-site residues for the overall enzymatic activity that relate to hydrolytic glycosidic cleavage of sucrose to glucose and fructose, release of fructose and generation of a glycosyl-enzyme intermediate in the reducing end. In a subsequent transglycosylation step, the glucosyl moiety is transferred to the nonreducing end of an acceptor to form a growing glucan polymer chain made up of glucose molecules. It has been proposed that both sucrose breakdown and glucan synthesis occur in the same active site of the catalytic domain, although the active site does not appear to be large enough to accommodate both functions. These three enzymes belong to glycoside hydrolase family 70 (GH70), which shows homology to glycoside hydrolase family 13 (GH13). GtfC synthesizes both soluble and insoluble glucans (α-1,3 and α-1,6 glycosidic linkages), while GtfB and GtfD synthesize only insoluble or soluble glucans, respectively. Here, crystal structures of the catalytic domains of GtfB and GtfD are reported. These structures are compared with previously determined structures of the catalytic domain of GtfC. With this work, apo structures and inhibitor-complex structures with acarbose are now available for the catalytic domains of GtfC and GtfB. The structure of GtfC with maltose allows further identification and comparison of active-site residues. A model of sucrose binding to GtfB is also included. The new structure of the catalytic domain of GtfD affords a structural comparison of the three S. mutans glycosyltransferases. Unfortunately, the catalytic domain of GtfD is not complete since crystallization resulted in the structure of a truncated protein lacking approximately 200 N-terminal residues of domain IV.International Union of Crystallographytexthttps://creativecommons.org/licenses/by/4.0/urn:issn:2053-230Xen2023-05-05Schormann, N.Patel, M.Thannickal, L.Purushotham, S.Wu, R.Mieher, J.L.Wu, H.Deivanayagam, C.GLUCANSUCRASES; GLYCOSYLTRANSFERASES; CATALYTIC DOMAINS; SOLUBLE AND INSOLUBLE GLUCANS; GTFB; GTFC; GTFD; DENTAL PLAQUE; STREPTOCOCCUS MUTANS; SRCR1 BINDINGThe catalytic domains of Streptococcus mutans glucosyltransferases: a structural analysisStreptococcus mutans, found in the human oral cavity, is a significant contributor to the pathogenesis of dental caries. This bacterium expresses three genetically distinct types of glucosyltransferases named GtfB (GTF-I), GtfC (GTF-SI) and GtfD (GTF-S) that play critical roles in the development of dental plaque. The catalytic domains of GtfB, GtfC and GtfD contain conserved active-site residues for the overall enzymatic activity that relate to hydrolytic glycosidic cleavage of sucrose to glucose and fructose, release of fructose and generation of a glycosyl-enzyme intermediate in the reducing end. In a subsequent transglycosylation step, the glucosyl moiety is transferred to the nonreducing end of an acceptor to form a growing glucan polymer chain made up of glucose molecules. It has been proposed that both sucrose breakdown and glucan synthesis occur in the same active site of the catalytic domain, although the active site does not appear to be large enough to accommodate both functions. These three enzymes belong to glycoside hydrolase family 70 (GH70), which shows homology to glycoside hydrolase family 13 (GH13). GtfC synthesizes both soluble and insoluble glucans (α-1,3 and α-1,6 glycosidic linkages), while GtfB and GtfD synthesize only insoluble or soluble glucans, respectively. Here, crystal structures of the catalytic domains of GtfB and GtfD are reported. These structures are compared with previously determined structures of the catalytic domain of GtfC. With this work, apo structures and inhibitor-complex structures with acarbose are now available for the catalytic domains of GtfC and GtfB. The structure of GtfC with maltose allows further identification and comparison of active-site residues. A model of sucrose binding to GtfB is also included. The new structure of the catalytic domain of GtfD affords a structural comparison of the three S. mutans glycosyltransferases. Unfortunately, the catalytic domain of GtfD is not complete since crystallization resulted in the structure of a truncated protein lacking approximately 200 N-terminal residues of domain IV.doi:10.1107/S2053230X23003199text/htmlApo and acarbose-complex structures of the catalytic domain of GtfB from Streptococcus mutans and the truncated structure of the catalytic domain of GtfD are described.2053-230XActa Crystallographica Section F: Structural Biology Communications127119research communications792053-230Xhttps://creativecommons.org/licenses/by/4.0/med@iucr.org2023-05-05May 20235Crystal structure of MbnF: an NADPH-dependent flavin monooxygenase from Methylocystis strain SB2
http://scripts.iucr.org/cgi-bin/paper?no5198
Methanobactins (MBs) are ribosomally produced and post-translationally modified peptides (RiPPs) that are used by methanotrophs for copper acquisition. The signature post-translational modification of MBs is the formation of two heterocyclic groups, either an oxazolone, pyrazinedione or imidazolone group, with an associated thioamide from an X-Cys dipeptide. The precursor peptide (MbnA) for MB formation is found in a gene cluster of MB-associated genes. The exact biosynthetic pathway of MB formation is not yet fully understood, and there are still uncharacterized proteins in some MB gene clusters, particularly those that produce pyrazinedione or imidazolone rings. One such protein is MbnF, which is proposed to be a flavin monooxygenase (FMO) based on homology. To help to elucidate its possible function, MbnF from Methylocystis sp. strain SB2 was recombinantly produced in Escherichia coli and its X-ray crystal structure was resolved to 2.6 Å resolution. Based on its structural features, MbnF appears to be a type A FMO, most of which catalyze hydroxylation reactions. Preliminary functional characterization shows that MbnF preferentially oxidizes NADPH over NADH, supporting NAD(P)H-mediated flavin reduction, which is the initial step in the reaction cycle of several type A FMO enzymes. It is also shown that MbnF binds the precursor peptide for MB, with subsequent loss of the leader peptide sequence as well as the last three C-terminal amino acids, suggesting that MbnF might be needed for this process to occur. Finally, molecular-dynamics simulations revealed a channel in MbnF that is capable of accommodating the core MbnA fragment minus the three C-terminal amino acids.International Union of Crystallographytexthttps://creativecommons.org/licenses/by/4.0/urn:issn:2053-230Xen2023-05-05Stewart, A.Dershwitz, P.Stewart, C.Sawaya, M.R.Yeates, T.O.Semrau, J.D.Zischka, H.DiSpirito, A.A.Bobik, T.A.METHANOBACTINS; MBNF; FLAVIN MONOOXYGENASES; METHYLOCYSTIS SP. STRAIN SB2; WILSON'S DISEASECrystal structure of MbnF: an NADPH-dependent flavin monooxygenase from Methylocystis strain SB2Methanobactins (MBs) are ribosomally produced and post-translationally modified peptides (RiPPs) that are used by methanotrophs for copper acquisition. The signature post-translational modification of MBs is the formation of two heterocyclic groups, either an oxazolone, pyrazinedione or imidazolone group, with an associated thioamide from an X-Cys dipeptide. The precursor peptide (MbnA) for MB formation is found in a gene cluster of MB-associated genes. The exact biosynthetic pathway of MB formation is not yet fully understood, and there are still uncharacterized proteins in some MB gene clusters, particularly those that produce pyrazinedione or imidazolone rings. One such protein is MbnF, which is proposed to be a flavin monooxygenase (FMO) based on homology. To help to elucidate its possible function, MbnF from Methylocystis sp. strain SB2 was recombinantly produced in Escherichia coli and its X-ray crystal structure was resolved to 2.6 Å resolution. Based on its structural features, MbnF appears to be a type A FMO, most of which catalyze hydroxylation reactions. Preliminary functional characterization shows that MbnF preferentially oxidizes NADPH over NADH, supporting NAD(P)H-mediated flavin reduction, which is the initial step in the reaction cycle of several type A FMO enzymes. It is also shown that MbnF binds the precursor peptide for MB, with subsequent loss of the leader peptide sequence as well as the last three C-terminal amino acids, suggesting that MbnF might be needed for this process to occur. Finally, molecular-dynamics simulations revealed a channel in MbnF that is capable of accommodating the core MbnA fragment minus the three C-terminal amino acids.doi:10.1107/S2053230X23003035text/htmlMethanobactins are post-translationally modified copper-binding peptides that have a number of potential environmental and biomedical applications. This report presents the crystal structure and preliminary biochemical characterization of the putative methanobactin biosynthesis protein MbnF.2053-230X5May 20232023-05-05med@iucr.orghttps://creativecommons.org/licenses/by/4.0/1182053-230XActa Crystallographica Section F: Structural Biology Communications79research communications111Monomeric crystal structure of the vaccine carrier protein CRM197 and implications for vaccine development
http://scripts.iucr.org/cgi-bin/paper?jg5007
CRM197 is a genetically detoxified mutant of diphtheria toxin (DT) that is widely used as a carrier protein in conjugate vaccines. Protective immune responses to several bacterial diseases are obtained by coupling CRM197 to glycans from these pathogens. Wild-type DT has been described in two oligomeric forms: a monomer and a domain-swapped dimer. Their proportions depend on the chemical conditions and especially the pH, with a large kinetic barrier to interconversion. A similar situation occurs in CRM197, where the monomer is preferred for vaccine synthesis. Despite 30 years of research and the increasing application of CRM197 in conjugate vaccines, until now all of its available crystal structures have been dimeric. Here, CRM197 was expressed as a soluble, intracellular protein in an Escherichia coli strain engineered to have an oxidative cytoplasm. The purified product, called EcoCRM, remained monomeric throughout crystallization. The structure of monomeric EcoCRM is reported at 2.0 Å resolution with the domain-swapping hinge loop (residues 379–387) in an extended, exposed conformation, similar to monomeric wild-type DT. The structure enables comparisons across expression systems and across oligomeric states, with implications for monomer–dimer interconversion and for the optimization of conjugation.International Union of Crystallographytexthttps://creativecommons.org/licenses/by/4.0/urn:issn:2053-230Xen2023-03-30Gallagher, D.T.Oganesyan, N.Lees, A.CARRIERS; CONJUGATE VACCINES; CRM197; DIPHTHERIA TOXIN; DOMAIN SWAPPING; TOXOIDSMonomeric crystal structure of the vaccine carrier protein CRM197 and implications for vaccine developmentCRM197 is a genetically detoxified mutant of diphtheria toxin (DT) that is widely used as a carrier protein in conjugate vaccines. Protective immune responses to several bacterial diseases are obtained by coupling CRM197 to glycans from these pathogens. Wild-type DT has been described in two oligomeric forms: a monomer and a domain-swapped dimer. Their proportions depend on the chemical conditions and especially the pH, with a large kinetic barrier to interconversion. A similar situation occurs in CRM197, where the monomer is preferred for vaccine synthesis. Despite 30 years of research and the increasing application of CRM197 in conjugate vaccines, until now all of its available crystal structures have been dimeric. Here, CRM197 was expressed as a soluble, intracellular protein in an Escherichia coli strain engineered to have an oxidative cytoplasm. The purified product, called EcoCRM, remained monomeric throughout crystallization. The structure of monomeric EcoCRM is reported at 2.0 Å resolution with the domain-swapping hinge loop (residues 379–387) in an extended, exposed conformation, similar to monomeric wild-type DT. The structure enables comparisons across expression systems and across oligomeric states, with implications for monomer–dimer interconversion and for the optimization of conjugation.doi:10.1107/S2053230X23002364text/htmlThe first crystal structure of the widely used vaccine carrier protein CRM197 in its monomeric form reveals features underlying its dimerization and vaccine-conjugation reaction.April 20234https://creativecommons.org/licenses/by/4.0/med@iucr.org2023-03-302053-230X7982research communications862053-230XActa Crystallographica Section F: Structural Biology Communications