FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W3001266181> ?p ?o ?g. }

• W3001266181 endingPage "2639" @default.
• W3001266181 startingPage "2629" @default.
• W3001266181 abstract "Gram-positive bacteria, including major clinical pathogens such as Staphylococcus aureus, are becoming increasingly drug-resistant. Their cell walls are composed of a thick layer of peptidoglycan (PG) modified by the attachment of wall teichoic acid (WTA), an anionic glycopolymer that is linked to pathogenicity and regulation of cell division and PG synthesis. The transfer of WTA from lipid carriers to PG, catalyzed by the LytR–CpsA–Psr (LCP) enzyme family, offers a unique extracellular target for the development of new anti-infective agents. Inhibitors of LCP enzymes have the potential to manage a wide range of bacterial infections because the target enzymes are implicated in the assembly of many other bacterial cell wall polymers, including capsular polysaccharide of streptococcal species and arabinogalactan of mycobacterial species. In this study, we present the first crystal structure of S. aureus LcpA with bound substrate at 1.9 Å resolution and those of Bacillus subtilis LCP enzymes, TagT, TagU, and TagV, in the apo form at 1.6–2.8 Å resolution. The structures of these WTA transferases provide new insight into the binding of lipid-linked WTA and enable assignment of the catalytic roles of conserved active-site residues. Furthermore, we identified potential subsites for binding the saccharide core of PG using computational docking experiments, and multiangle light-scattering experiments disclosed novel oligomeric states of the LCP enzymes. The crystal structures and modeled substrate-bound complexes of the LCP enzymes reported here provide insights into key features linked to substrate binding and catalysis and may aid the structure-guided design of specific LCP inhibitors. Gram-positive bacteria, including major clinical pathogens such as Staphylococcus aureus, are becoming increasingly drug-resistant. Their cell walls are composed of a thick layer of peptidoglycan (PG) modified by the attachment of wall teichoic acid (WTA), an anionic glycopolymer that is linked to pathogenicity and regulation of cell division and PG synthesis. The transfer of WTA from lipid carriers to PG, catalyzed by the LytR–CpsA–Psr (LCP) enzyme family, offers a unique extracellular target for the development of new anti-infective agents. Inhibitors of LCP enzymes have the potential to manage a wide range of bacterial infections because the target enzymes are implicated in the assembly of many other bacterial cell wall polymers, including capsular polysaccharide of streptococcal species and arabinogalactan of mycobacterial species. In this study, we present the first crystal structure of S. aureus LcpA with bound substrate at 1.9 Å resolution and those of Bacillus subtilis LCP enzymes, TagT, TagU, and TagV, in the apo form at 1.6–2.8 Å resolution. The structures of these WTA transferases provide new insight into the binding of lipid-linked WTA and enable assignment of the catalytic roles of conserved active-site residues. Furthermore, we identified potential subsites for binding the saccharide core of PG using computational docking experiments, and multiangle light-scattering experiments disclosed novel oligomeric states of the LCP enzymes. The crystal structures and modeled substrate-bound complexes of the LCP enzymes reported here provide insights into key features linked to substrate binding and catalysis and may aid the structure-guided design of specific LCP inhibitors. The discovery of penicillin nearly a century ago ushered in an era of targeting bacterial cell wall peptidoglycan (PG) 2The abbreviations used are: PGpeptidoglycanLCPLytR-CpsA-PsrWTAwall teichoic acidCPcapsular polysaccharidePBPpenicillin-binding proteinsC10-PPgeranyl-pyrophosphateC55-Pundecaprenyl phosphateMurNAcN-acetylmuramic acidManNAcN-acetylmannosaminetriGlcNAcN,N′,N″-triacetylchitotriosePDBProtein Data BankSeMetselenomethionineTMtransmembraneRMSDroot-mean-square deviation. biosynthesis as an effective approach to combat a wide variety of bacterial infections. To survive the onslaught of β-lactams and other classes of typically bactericidal antibiotics, bacteria have evolved a myriad of resistance countermeasures in parallel. As a result, new therapeutic agents are now urgently needed, and their development will rely on extensive research efforts on additional bacterial targets. Wall teichoic acid (WTA) is a Gram-positive bacterial cell wall polymer that is covalently attached to the N-acetylmuramic acid (MurNAc) C6-hydroxyl group of PG (Fig. S1). The inhibition of WTA biosynthesis is an attractive therapeutic approach because methicillin-resistant Staphylococcus aureus deficient in the production of WTAs are known to be resensitized to certain classes of β-lactam antibiotics (1Campbell J. Singh A.K. Santa Maria Jr., J.P. Kim Y. Brown S. Swoboda J.G. Mylonakis E. Wilkinson B.J. Walker S. Synthetic lethal compound combinations reveal a fundamental connection between wall teichoic acid and peptidoglycan biosyntheses in Staphylococcus aureus.ACS Chem. Biol. 2011; 6 (20961110): 106-11610.1021/cb100269fCrossref PubMed Scopus (206) Google Scholar, 2Farha M.A. Leung A. Sewell E.W. D'Elia M.A. Allison S.E. Ejim L. Pereira P.M. Pinho M.G. Wright G.D. Brown E.D. Inhibition of WTA synthesis blocks the cooperative action of PBPs and sensitizes MRSA to β-lactams.ACS Chem. Biol. 2013; 8 (23062620): 226-23310.1021/cb300413mCrossref PubMed Scopus (153) Google Scholar). This intriguing phenomenon is believed to stem from the role of WTA in guiding PG cross-linking through spatiotemporal localization of certain penicillin-binding proteins (PBPs) (1Campbell J. Singh A.K. Santa Maria Jr., J.P. Kim Y. Brown S. Swoboda J.G. Mylonakis E. Wilkinson B.J. Walker S. Synthetic lethal compound combinations reveal a fundamental connection between wall teichoic acid and peptidoglycan biosyntheses in Staphylococcus aureus.ACS Chem. Biol. 2011; 6 (20961110): 106-11610.1021/cb100269fCrossref PubMed Scopus (206) Google Scholar, 2Farha M.A. Leung A. Sewell E.W. D'Elia M.A. Allison S.E. Ejim L. Pereira P.M. Pinho M.G. Wright G.D. Brown E.D. Inhibition of WTA synthesis blocks the cooperative action of PBPs and sensitizes MRSA to β-lactams.ACS Chem. Biol. 2013; 8 (23062620): 226-23310.1021/cb300413mCrossref PubMed Scopus (153) Google Scholar, 3Atilano M.L. Pereira P.M. Yates J. Reed P. Veiga H. Pinho M.G. Filipe S.R. Teichoic acids are temporal and spatial regulators of peptidoglycan cross-linking in Staphylococcus aureus.Proc. Natl. Acad. Sci. U.S.A. 2010; 107 (20944066): 18991-1899610.1073/pnas.1004304107Crossref PubMed Scopus (186) Google Scholar). The absence of WTA disables these select classes of PBPs through mislocalization and sensitizes the organism to β-lactams that target unaffected PBPs. Another physiological role of WTA is the regulation of cell division through localization of autolysins to the division septum for PG breakdown (4Schlag M. Biswas R. Krismer B. Kohler T. Zoll S. Yu W. Schwarz H. Peschel A. Götz F. Role of staphylococcal wall teichoic acid in targeting the major autolysin Atl.Mol. Microbiol. 2010; 75 (20105277): 864-87310.1111/j.1365-2958.2009.07007.xCrossref PubMed Scopus (194) Google Scholar). In addition, this polymer is linked to pathogenicity because WTA-deficient mutants are defective in biofilm formation, host cell adherence, and colonization (5Holland L.M. Conlon B. O'Gara J.P. Mutation of tagO reveals an essential role for wall teichoic acids in Staphylococcus epidermidis biofilm development.Microbiology. 2011; 157 (21051486): 408-41810.1099/mic.0.042234-0Crossref PubMed Scopus (63) Google Scholar, 6Weidenmaier C. Peschel A. Xiong Y.-Q. Kristian S.A. Dietz K. Yeaman M.R. Bayer A.S. Lack of wall teichoic acids in Staphylococcus aureus leads to reduced interactions with endothelial cells and to attenuated virulence in a rabbit model of endocarditis.J. Infect. Dis. 2005; 191 (15838806): 1771-177710.1086/429692Crossref PubMed Scopus (178) Google Scholar, 7Misawa Y. Kelley K.A. Wang X. Wang L. Park W.B. Birtel J. Saslowsky D. Lee J.C. Staphylococcus aureus colonization of the mouse gastrointestinal tract is modulated by wall teichoic acid, capsule, and surface proteins.PLoS Pathog. 2015; 11 (26201029): e100506110.1371/journal.ppat.1005061Crossref PubMed Scopus (39) Google Scholar). peptidoglycan LytR-CpsA-Psr wall teichoic acid capsular polysaccharide penicillin-binding proteins geranyl-pyrophosphate undecaprenyl phosphate N-acetylmuramic acid N-acetylmannosamine N,N′,N″-triacetylchitotriose Protein Data Bank selenomethionine transmembrane root-mean-square deviation. WTA is synthesized in the cytoplasm on undecaprenyl-phosphate (C55-P) for translocation across the lipid bilayer and subsequent attachment to PG (8Caveney N.A. Li F.K. Strynadka N.C. Enzyme structures of the bacterial peptidoglycan and wall teichoic acid biogenesis pathways.Curr. Opin. Struct. Biol. 2018; 53 (29885610): 45-5810.1016/j.sbi.2018.05.002Crossref PubMed Scopus (30) Google Scholar). The ribitol-phosphate polymer of S. aureus WTA is anchored to PG through a sugar-based linkage unit comprised of phosphate–GlcNAc–N-acetylmannosamine–[glycerol-phosphate]2 (Fig. S1). The transfer of WTA to PG is catalyzed on the outer leaflet of the cytosolic membrane by members of the LytR–CpsA–Psr (LCP) protein family that are unique to predominantly Gram-positive bacteria (9Kawai Y. Marles-Wright J. Cleverley R.M. Emmins R. Ishikawa S. Kuwano M. Heinz N. Bui N.K. Hoyland C.N. Ogasawara N. Lewis R.J. Vollmer W. Daniel R.A. Errington J. A widespread family of bacterial cell wall assembly proteins.EMBO J. 2011; 30 (21964069): 4931-494110.1038/emboj.2011.358Crossref PubMed Scopus (181) Google Scholar). Deletion of Bacillus subtilis LCP enzymes (TagTBS, TagUBS, and TagVBS) has been shown to be lethal presumably because of the accumulation of nonfunctional lipid-bound WTA intermediates and depletion of the pool of lipid carriers required for PG synthesis (9Kawai Y. Marles-Wright J. Cleverley R.M. Emmins R. Ishikawa S. Kuwano M. Heinz N. Bui N.K. Hoyland C.N. Ogasawara N. Lewis R.J. Vollmer W. Daniel R.A. Errington J. A widespread family of bacterial cell wall assembly proteins.EMBO J. 2011; 30 (21964069): 4931-494110.1038/emboj.2011.358Crossref PubMed Scopus (181) Google Scholar, 10D'Elia M.A. Millar K.E. Bhavsar A.P. Tomljenovic A.M. Hutter B. Schaab C. Moreno-Hagelsieb G. Brown E.D. Probing teichoic acid genetics with bioactive molecules reveals new interactions among diverse processes in bacterial cell wall biogenesis.Chem. Biol. 2009; 16 (19477419): 548-55610.1016/j.chembiol.2009.04.009Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). In S. aureus, deletion of LCP enzymes (LcpASA, LcpBSA, and LcpCSA) resulted in WTA-deficient mutants with physiological and pathogenic defects (11Chan Y.G. Frankel M.B. Dengler V. Schneewind O. Missiakas D. Staphylococcus aureus mutants lacking the LytR–CpsA–Psr family of enzymes release cell wall teichoic acids into the extracellular medium.J. Bacteriol. 2013; 195 (23935043): 4650-465910.1128/JB.00544-13Crossref PubMed Scopus (78) Google Scholar, 12Over B. Heusser R. McCallum N. Schulthess B. Kupferschmied P. Gaiani J.M. Sifri C.D. Berger-Bächi B. Stutzmann Meier P. LytR–CpsA–Psr proteins in Staphylococcus aureus display partial functional redundancy and the deletion of all three severely impairs septum placement and cell separation.FEMS Microbiol. Lett. 2011; 320 (21554381): 142-15110.1111/j.1574-6968.2011.02303.xCrossref PubMed Scopus (42) Google Scholar). The LCP family represents an attractive class of drug targets in that the soluble catalytic region is on the extracellular face of the cytosolic membrane, and there are no mammalian orthologs (13Hübscher J. Lüthy L. Berger-Bächi B. Stutzmann Meier P. Phylogenetic distribution and membrane topology of the LytR–CpsA–Psr protein family.BMC Genomics. 2008; 9 (19099556): 61710.1186/1471-2164-9-617Crossref PubMed Scopus (58) Google Scholar). Furthermore, LCP enzymes are involved in the PG attachment of not only WTA but other secondary polymers of therapeutic interest, including Streptococcus pneumonia capsular polysaccharide (CP) and Mycobacterium tuberculosis arabinogalactan (14Eberhardt A. Hoyland C.N. Vollmer D. Bisle S. Cleverley R.M. Johnsborg O. Håvarstein L.S. Lewis R.J. Vollmer W. Attachment of capsular polysaccharide to the cell wall in Streptococcus pneumoniae.Microb. Drug Resist. 2012; 18 (22432711): 240-25510.1089/mdr.2011.0232Crossref PubMed Scopus (80) Google Scholar, 15Harrison J. Lloyd G. Joe M. Lowary T.L. Reynolds E. Walters-Morgan H. Bhatt A. Lovering A. Besra G.S. Alderwick L.J. Lcp1 is a phosphotransferase responsible for ligating arabinogalactan to peptidoglycan in Mycobacterium tuberculosis.MBio. 2016; 7 (27486192): e00972-16Crossref PubMed Scopus (31) Google Scholar). Multiple copies of the lcp gene are often found in Gram-positive bacteria, and the gene products display catalytic and functional differences. B. subtilis TagU was found to have higher catalytic activity than TagT and TagV variants in that species, whereas M. tuberculosis Rv3484 was shown to be the only essential LCP enzyme in vivo (16Gale R.T. Li F.K.K. Sun T. Strynadka N.C.J. Brown E.D. B. subtilis LytR–CpsA–Psr enzymes transfer wall teichoic acids from authentic lipid-linked substrates to mature peptidoglycan in vitro.Cell Chem. Biol. 2017; 24 (29107701): 1537-1546.e410.1016/j.chembiol.2017.09.006Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 17Malm S. Maaß S. Schaible U.E. Ehlers S. Niemann S. In vivo virulence of Mycobacterium tuberculosis depends on a single homologue of the LytR–CpsA–Psr proteins.Sci. Rep. 2018; 8 (29500450): 393610.1038/s41598-018-22012-6Crossref PubMed Scopus (7) Google Scholar). In S. aureus, LcpA was discovered to be the primary WTA transferase, whereas LcpC acts as the primary CP transferase (18Schaefer K. Matano L.M. Qiao Y. Kahne D. Walker S. In vitro reconstitution demonstrates the cell wall ligase activity of LCP proteins.Nat. Chem. Biol. 2017; 13 (28166208): 396-40110.1038/nchembio.2302Crossref PubMed Scopus (49) Google Scholar, 19Rausch M. Deisinger J.P. Ulm H. Müller A. Li W. Hardt P. Wang X. Li X. Sylvester M. Engeser M. Vollmer W. Müller C.E. Sahl H.G. Lee J.C. Schneider T. Coordination of capsule assembly and cell wall biosynthesis in Staphylococcus aureus.Nat. Commun. 2019; 10 (30926919): 140410.1038/s41467-019-09356-xCrossref PubMed Scopus (40) Google Scholar). The natural PG acceptor substrate of LCP enzymes also appears variable. The disaccharide lipid-linked PG precursor, lipid II, was demonstrated to be a substrate of S. aureus LcpC but not for LcpA (18Schaefer K. Matano L.M. Qiao Y. Kahne D. Walker S. In vitro reconstitution demonstrates the cell wall ligase activity of LCP proteins.Nat. Chem. Biol. 2017; 13 (28166208): 396-40110.1038/nchembio.2302Crossref PubMed Scopus (49) Google Scholar, 19Rausch M. Deisinger J.P. Ulm H. Müller A. Li W. Hardt P. Wang X. Li X. Sylvester M. Engeser M. Vollmer W. Müller C.E. Sahl H.G. Lee J.C. Schneider T. Coordination of capsule assembly and cell wall biosynthesis in Staphylococcus aureus.Nat. Commun. 2019; 10 (30926919): 140410.1038/s41467-019-09356-xCrossref PubMed Scopus (40) Google Scholar). In addition, S. aureus LcpB was found to be incapable of utilizing cross-linked S. aureus PG as the acceptor substrate, suggesting that the attachment of WTA occurs prior to cross-linking of PG strands (20Schaefer K. Owens T.W. Kahne D. Walker S. Substrate preferences establish the order of cell wall assembly in Staphylococcus aureus.J. Am. Chem. Soc. 2018; 140 (29402087): 2442-244510.1021/jacs.7b13551Crossref PubMed Scopus (18) Google Scholar). In contrast, a study conducted on B. subtilis LCP enzymes demonstrated WTA ligation to mature cross-linked B. subtilis PG in vitro (16Gale R.T. Li F.K.K. Sun T. Strynadka N.C.J. Brown E.D. B. subtilis LytR–CpsA–Psr enzymes transfer wall teichoic acids from authentic lipid-linked substrates to mature peptidoglycan in vitro.Cell Chem. Biol. 2017; 24 (29107701): 1537-1546.e410.1016/j.chembiol.2017.09.006Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Given the observed functional variations among species, understanding the underlying molecular details becomes important. Prior crystallographic studies have been conducted on LCP enzymes with S. pneumoniae Cps2A and B. subtilis TagT characterized in complex with analogs of the polyprenyl substrate (Table S1). In the former, the bound lipids lack the saccharide headgroup that differentiates CP/WTA precursors from lipid II (9Kawai Y. Marles-Wright J. Cleverley R.M. Emmins R. Ishikawa S. Kuwano M. Heinz N. Bui N.K. Hoyland C.N. Ogasawara N. Lewis R.J. Vollmer W. Daniel R.A. Errington J. A widespread family of bacterial cell wall assembly proteins.EMBO J. 2011; 30 (21964069): 4931-494110.1038/emboj.2011.358Crossref PubMed Scopus (181) Google Scholar, 14Eberhardt A. Hoyland C.N. Vollmer D. Bisle S. Cleverley R.M. Johnsborg O. Håvarstein L.S. Lewis R.J. Vollmer W. Attachment of capsular polysaccharide to the cell wall in Streptococcus pneumoniae.Microb. Drug Resist. 2012; 18 (22432711): 240-25510.1089/mdr.2011.0232Crossref PubMed Scopus (80) Google Scholar). More recently, C30-PP–GlcNAc and C30-PP–GlcNAc–ManNAc were successfully co-crystallized with B. subtilis TagT; however, the saccharide headgroups were found in different orientations, and the expected specificity determining polar contacts between the saccharide substrate moieties and the enzyme active site were not observed (20Schaefer K. Owens T.W. Kahne D. Walker S. Substrate preferences establish the order of cell wall assembly in Staphylococcus aureus.J. Am. Chem. Soc. 2018; 140 (29402087): 2442-244510.1021/jacs.7b13551Crossref PubMed Scopus (18) Google Scholar). A notable structural variant of LCP enzymes is that of Actinomyces oris LcpA, which possesses unique structural features around the active site presumably associated with binding target proteins rather than PG for glycosylation (21Siegel S.D. Amer B.R. Wu C. Sawaya M.R. Gosschalk J.E. Clubb R.T. Ton-That H. Structure and mechanism of LcpA, a phosphotransferase that mediates glycosylation of a Gram-positive bacterial cell wall–anchored protein.MBio. 2019; 10 (30782654): e01580-18PubMed Google Scholar). Further structural characterization of LCP enzymes is required to investigate PG binding and to clarify the structural relationship between the donor lipid headgroup and the enzyme. In this study, we present four crystal structures of S. aureus and B. subtilis LCP enzymes for a comparative analysis that provides the molecular basis of residues assessed in published mutagenesis studies. Importantly, our structure of S. aureus LcpA complexed to C40-PP–GlcNAc provides clarity on the orientation of the saccharide headgroup and reveals an interface reliant on van der Waals and hydrophobic contacts rather than direct polar interactions. Furthermore, the structure of S. aureus LcpA provides a clinically relevant target for structure-guided design of inhibitors. We complement our crystallographic work by modeling PG-bound complexes, and we report hitherto unknown oligomeric states of various LCP enzymes in solution. Our crystallographic study yielded the first structure of the primary S. aureus WTA transferase, LcpASA (Fig. 1 and Table 1). The crystallized enzyme captured C40-PP–GlcNAc, an endogenous lipid with central features in keeping with the natural lipid donor substrate. The construct (residues 80–327; ΔTM) used for crystallization encompasses the extracellular catalytic region, known as the LCP domain, and lacks the single N-terminal transmembrane anchor (Fig. S2a). Our structure of LcpASA was solved by molecular replacement to 1.9 Å resolution, providing an excellent template for future structure-based drug design work.Table 1X-ray data collection and refinement statisticsS. aureus LcpAB. subtilis TagTB. subtilis TagU (SeMet)B. subtilis TagVResidues80–32746–32262–30672–332Protein Data Bank code6UEX6UF56UF66UF3Data collectionWavelength (Å)0.979490.979640.979490.97949SpacegroupC2221P41212P3221P212121Cell dimensionsa, b, c (Å)89.25, 90.54, 94.7565.66, 65.66, 143.0748.79, 48.79, 234.2740.99, 66.10, 81.72a, b, c (°)90, 90, 9090, 90, 9090, 90, 12090, 90, 90Resolution (Å)aThe values in parentheses represent the highest-resolution shell.47.38–1.90 (1.97–1.90)48.37–2.80 (2.90–2.80)41.58–2.20 (2.28–2.20)36.64–1.6 (1.66–1.60)CC1/20.998 (0.696)0.997 (0.655)0.999 (0.862)1 (0.709)Rpim0.02126 (0.6351)0.05905 (0.5196)0.02982 (0.523)0.02434 (0.5561)Rmeas0.05442 (1.643)0.2232 (1.989)0.09296 (1.665)0.0674 (1.561)I/σI16.31 (1.36)13.10 (1.55)14.69 (1.46)19.00 (1.50)Completeness (%)99.84 (99.80)99.84 (99.87)99.59 (99.11)99.82 (99.87)Redundancy6.6 (6.6)13.9 (14.4)9.7 (10.0)7.6 (7.7)RefinementResolution (Å)47.38–1.9048.37–2.8041.58–2.2036.64–1.60No. of reflections30,547 (3015)8230 (794)17,404 (1671)29,994 (2959)Rwork/Rfreeb5% of reflections were excluded from refinement and used to calculate Rfree.0.1922/0.22580.2307/0.27660.2445/0.25880.2004/0.2288No. of atoms2063188118112133Protein1896187917701966Ligand/ion1100360Water5725167B factors (Å2)67.3263.1293.2536.23Protein65.9863.1592.5735.69Ligand/ion91.91–129.37–Water64.6243.6673.0642.53RMSDBond lengths (Å)0.0080.0050.0060.006Bond angles (°)1.191.061.081.04Ramachandran (%)Favored97.9694.9896.4198.79Allowed2.045.023.591.21Outliers0000MolProbityClashscore3.355.898.093.85a The values in parentheses represent the highest-resolution shell.b 5% of reflections were excluded from refinement and used to calculate Rfree. Open table in a new tab The LCP domain of LcpASA is comprised of a six-stranded β-sheet sandwiched between multiple α-helices and several double-stranded β-sheets (Fig. 1a and Fig. S2b). A large hydrophobic lipid-binding pocket with a narrow opening and a wide base is formed by the central β-sheet and helices 3–7 (Fig. 1b). An electropositive region for binding the pyrophosphate moiety of the lipid donor substrate is found at the entrance of the hydrophobic pocket highlighting the location of the active site. LcpASA shows the highest overall structural similarity to Enterococcus faecalis EF0465 with an RMSD of 2.5 Å for 227 Cα pairs, indicating regions of significant difference (Table S1). However, structural comparison of just the highly conserved active-site residues shows that LcpASA is most similar to apo and C30-PP–GlcNAc–ManNAc–bound TagTBS with a closely matched RMSD of 0.85 Å for 10 Cα pairs in both instances and 1.3–1.6 Å for 44 common side chain heavy atoms therein. The active site of LcpASA is surrounded by four regions, designated here as regions A (residues 92–100), B (residues 188–201), C (residues 217–224), and D (residues 296–312), that display structural variability when compared with the LCP enzymes of other species (Fig. 1a). To this end, we have expanded our structural understanding of LCP enzymes by solving three additional structures from the prototypical Gram-positive bacterium, B. subtilis (Fig. 2 and Table 1). The first is the novel structure of TagUBS (residues 62–306; selenomethionine (SeMet)-substituted) phased by single isomorphous replacement and refined to 2.2 Å resolution. Additional insights into the architecture of the active site are provided by the structure of TagVBS (residues 72–332; 1.6 Å resolution) at a higher resolution than previously reported (2.6 Å resolution) and the structure of TagTBS (residues 46–322; 2.8 Å resolution) with additional electron density for a previously disordered and unmodeled region in the apo structure. The electrostatic surfaces of the LCP enzymes are highly variable in addition to the electropositive region formed by conserved arginine residues at the active site. This electropositive region becomes more difficult to observe when the guanidinium side chains are not localized by a pyrophosphate group (Fig. 2c). Between our four structures, the most significant difference in secondary structure is found in region B, where LcpASA has a large loop, TagTBS has an α-helix, and both TagUBS and TagVBS have a double-stranded β-sheet (Figs. 1a and 2a). Regions A and C encompass flexible loops, and region D adopts a two-stranded β-sheet with an enrichment of aromatic residues that we predict bind to the carbohydrate groups of PG. In the structure of TagUBS, the rearrangements of helices 3–7 on one side of the central β-sheet resulted in the collapse of the lipid-binding site (Fig. 2b). These structural differences are facilitated by the association of two protein molecules forming a modest interfacial surface area of 760 Å2 across a crystallographic 2-fold symmetry axis at one end of the lipid-binding site (helices 6 and 7 and β-strand 9) (Fig. S2c). The association is stabilized by hydrogen bonds and hydrophobic interactions mainly consisting of aromatic residues from the interior of the hydrophobic pocket. Notably, several crystal structures of LCP enzymes, including TagTBS here, display disorder of helix 6 (Fig. 2a). The exposure of the hydrophobic core to bulk solvent may be the driving force behind the dimerization of TagUBS. The hydrophobic and aromatic nature of this exposed region also suggests a possible surface for association with membrane. In the structure of S. aureus LcpA, we have fortuitously captured a lipid substrate produced by the overexpression strain BL21(DE3) Escherichia coli for the synthesis of O7-specific lipopolysaccharide (22Kim H. Kim S. Yoon S.H. Metabolic network reconstruction and phenome analysis of the industrial microbe, Escherichia coli BL21(DE3).PLoS One. 2018; 13 (30240424): e020437510.1371/journal.pone.0204375PubMed Google Scholar). The clear density at the extended hydrophobic lipid-binding pocket (surface area ≈ 680 Å2; volume ≈ 550 Å3) allowed unambiguous modeling of a lipid tail and a monosaccharide-pyrophosphate headgroup (Fig. S2d). The electron density of the monosaccharide is in keeping with a GlcNAc moiety commonly appended to polyprenyl groups through the action of WecA in E. coli (23Alexander D.C. Valvano M.A. Role of the rfe gene in the biosynthesis of the Escherichia coli O7-specific lipopolysaccharide and other O-specific polysaccharides containing N-acetylglucosamine.J. Bacteriol. 1994; 176 (7525537): 7079-708410.1128/JB.176.22.7079-7084.1994Crossref PubMed Google Scholar). The weak additional density extending from the C3-hydroxyl group of GlcNAc may indicate the presence of galactose presumably appended to GlcNAc by the enzyme WbbD in E. coli (24Riley J.G. Menggad M. Montoya-Peleaz P.J. Szarek W.A. Marolda C.L. Valvano M.A. Schutzbach J.S. Brockhausen I. The wbbD gene of E. coli strain VW187 (O7:K1) encodes a UDP-Gal:GlcNAcα-pyrophosphate-Rβ1,3-galactosyltransferase involved in the biosynthesis of O7-specific lipopolysaccharide.Glycobiology. 2005; 15 (15625181): 605-61310.1093/glycob/cwi038Crossref PubMed Scopus (29) Google Scholar). The interactions between LcpASA and C40-PP–GlcNAc are shown in Fig. 1c. Residues surrounding the lipid tail display low sequence conservation but are largely hydrophobic. Notably, the bulky aromatic side chain of Phe-171 introduces a kink at the third prenyl moiety, whereas the wide base of the pocket induces a twist in the lipid backbone to accommodate the remainder of the lipid tail. Invariant arginine residues form a positively charged entrance to the hydrophobic pocket, where their guanidinyl groups form salt bridges with the pyrophosphate moiety of the polyprenyl. Arg-99 and Arg-216 are in contact with the α-phosphate group and Arg-122, Arg-218, and Arg-227 are in contact with the β-phosphate group in keeping with earlier mutagenesis studies showing their importance in growth and activity in various species (summarized in Table S2) (9Kawai Y. Marles-Wright J. Cleverley R.M. Emmins R. Ishikawa S. Kuwano M. Heinz N. Bui N.K. Hoyland C.N. Ogasawara N. Lewis R.J. Vollmer W. Daniel R.A. Errington J. A widespread family of bacterial cell wall assembly proteins.EMBO J. 2011; 30 (21964069): 4931-494110.1038/emboj.2011.358Crossref PubMed Scopus (181) Google Scholar, 20Schaefer K. Owens T.W. Kahne D. Walker S. Substrate preferences establish the order of cell wall assembly in Staphylococcus aureus.J. Am. Chem. Soc. 2018; 140 (29402087): 2442-244510.1021/jacs.7b13551Crossref PubMed Scopus (18) Google Scholar, 25Baumgart M. Schubert K. Bramkamp M. Frunzke J. Impact of LytR–CpsA–Psr proteins on cell wall biosynthesis in Corynebacterium glutamicum.J. Bacteriol. 2016; 198 (27551018): 3045-305910.1128/JB.00406-16Crossref PubMed Scopus (22) Google Scholar). The orientation of the lipid-bound GlcNAc moiety is stabilized by an intramolecular hydrogen bond (3.0 Å) between the nitrogen atom of the N-acetyl group and a phosphoryl oxygen of the α-phosphate group (Fig. 1c). In addition, the position of the GlcNAc moiety is stabilized by hydrophobic and van der Waals interactions between the N-acetyl group and a shallow pocket outlined by residues Asn-194, Ile-195, Arg-216, Phe-217, Arg-218, and His-219 of regions B and C (Fig. 1d). These observations are also supported by the structure of TagTBS bound to C30-PP–GlcNAc–ManNAc (Fig. S3). The lack of specific interactions between the enzyme and the N-acetyl group likely allows for the significant observed differences in the secondary structure of region B as mentioned above. Region C possesses two highly conserved residues, a pyrophosphate-binding arginine (Arg-218) and an aspartate (Asp-224). The loop of region C is commonly held away from the active site by a resident acidic residue that forms a salt bridge with a nearby basic residue (Fig. S4). Region C is fully modeled in our structures of LcpASA, TagTBS, and TagUBS. A comparison between all available LCP enzyme structures reveals that ordering of the loop in region C depends heavily on the extension of helix 5 beyond" @default.
• W3001266181 created "2020-01-30" @default.
• W3001266181 creator A5004347046 @default.
• W3001266181 creator A5022292484 @default.
• W3001266181 creator A5028883134 @default.
• W3001266181 creator A5046230856 @default.
• W3001266181 creator A5067661409 @default.
• W3001266181 creator A5076663151 @default.
• W3001266181 date "2020-02-01" @default.
• W3001266181 modified "2023-09-30" @default.
• W3001266181 title "Crystallographic analysis of Staphylococcus aureus LcpA, the primary wall teichoic acid ligase" @default.
• W3001266181 cites W1584292656 @default.
• W3001266181 cites W1726597626 @default.
• W3001266181 cites W1782933731 @default.
• W3001266181 cites W1821189467 @default.
• W3001266181 cites W1963863783 @default.
• W3001266181 cites W1973035681 @default.
• W3001266181 cites W1979880724 @default.
• W3001266181 cites W1991892385 @default.
• W3001266181 cites W1999475434 @default.
• W3001266181 cites W2002886801 @default.
• W3001266181 cites W2012192342 @default.
• W3001266181 cites W2013798053 @default.
• W3001266181 cites W2018479585 @default.
• W3001266181 cites W2025816743 @default.
• W3001266181 cites W2056405753 @default.
• W3001266181 cites W2074179331 @default.
• W3001266181 cites W2074473216 @default.
• W3001266181 cites W2109361578 @default.
• W3001266181 cites W2123661253 @default.
• W3001266181 cites W2124026197 @default.
• W3001266181 cites W2132629607 @default.
• W3001266181 cites W2150815982 @default.
• W3001266181 cites W2154019529 @default.
• W3001266181 cites W2154197653 @default.
• W3001266181 cites W2159779488 @default.
• W3001266181 cites W2160821408 @default.
• W3001266181 cites W2163306024 @default.
• W3001266181 cites W2163341755 @default.
• W3001266181 cites W2168974130 @default.
• W3001266181 cites W2476533984 @default.
• W3001266181 cites W2508199602 @default.
• W3001266181 cites W2533901947 @default.
• W3001266181 cites W2587265764 @default.
• W3001266181 cites W2761755625 @default.
• W3001266181 cites W2765748851 @default.
• W3001266181 cites W2787707083 @default.
• W3001266181 cites W2806087678 @default.
• W3001266181 cites W2807702980 @default.
• W3001266181 cites W2892285465 @default.
• W3001266181 cites W2925443009 @default.
• W3001266181 cites W4255677297 @default.
• W3001266181 cites W955952240 @default.
• W3001266181 doi "https://doi.org/10.1074/jbc.ra119.011469" @default.
• W3001266181 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/7049971" @default.
• W3001266181 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/31969390" @default.
• W3001266181 hasPublicationYear "2020" @default.
• W3001266181 type Work @default.
• W3001266181 sameAs 3001266181 @default.
• W3001266181 citedByCount "20" @default.
• W3001266181 countsByYear W30012661812020 @default.
• W3001266181 countsByYear W30012661812021 @default.
• W3001266181 countsByYear W30012661812022 @default.
• W3001266181 countsByYear W30012661812023 @default.
• W3001266181 crossrefType "journal-article" @default.
• W3001266181 hasAuthorship W3001266181A5004347046 @default.
• W3001266181 hasAuthorship W3001266181A5022292484 @default.
• W3001266181 hasAuthorship W3001266181A5028883134 @default.
• W3001266181 hasAuthorship W3001266181A5046230856 @default.
• W3001266181 hasAuthorship W3001266181A5067661409 @default.
• W3001266181 hasAuthorship W3001266181A5076663151 @default.
• W3001266181 hasBestOaLocation W30012661811 @default.
• W3001266181 hasConcept C175114707 @default.
• W3001266181 hasConcept C185592680 @default.
• W3001266181 hasConcept C2778253522 @default.
• W3001266181 hasConcept C2779489039 @default.
• W3001266181 hasConcept C523546767 @default.
• W3001266181 hasConcept C54355233 @default.
• W3001266181 hasConcept C552990157 @default.
• W3001266181 hasConcept C55493867 @default.
• W3001266181 hasConcept C86803240 @default.
• W3001266181 hasConcept C89423630 @default.
• W3001266181 hasConceptScore W3001266181C175114707 @default.
• W3001266181 hasConceptScore W3001266181C185592680 @default.
• W3001266181 hasConceptScore W3001266181C2778253522 @default.
• W3001266181 hasConceptScore W3001266181C2779489039 @default.
• W3001266181 hasConceptScore W3001266181C523546767 @default.
• W3001266181 hasConceptScore W3001266181C54355233 @default.
• W3001266181 hasConceptScore W3001266181C552990157 @default.
• W3001266181 hasConceptScore W3001266181C55493867 @default.
• W3001266181 hasConceptScore W3001266181C86803240 @default.
• W3001266181 hasConceptScore W3001266181C89423630 @default.
• W3001266181 hasFunder F4320306082 @default.
• W3001266181 hasFunder F4320319952 @default.
• W3001266181 hasFunder F4320320994 @default.
• W3001266181 hasFunder F4320334506 @default.
• W3001266181 hasFunder F4320334593 @default.
• W3001266181 hasFunder F4320335271 @default.

• next