Three-dimensional Structure of Wza, the Protein Required for Translocation of Group 1 Capsular Polysaccharide across the Outer Membrane of Escherichia coli

Wza is a highly conserved multimeric outer membrane protein complex required for the surface expression of the serotype K30 group 1 capsular polysaccharide in Escherichia coli. Here we present the first three-dimensional structure of this type of polysaccharide exporter at a 15.5-Å resolution obtained using single particle averaging on a dataset of cryo-negatively stained protein. Previous structural studies on purified Wza have revealed a homo-oligomeric ring structure that is most probably composed of eight subunits. Symmetry analysis of the three-dimensional structure combined with biochemical two- and three-dimensional crystallographic data strongly suggest that Wza is an octameric complex with a C4 quasi-rotational symmetry and is organized as a tetramer of dimeric subunits. Wza is best described as a stack of two 4-Å high rings with differing diameters providing a mushroom-like aspect from the side. The larger ring has a distinctive square shape with a diameter of 115 Å, whereas the smaller is almost circular with a diameter of 90 Å. In the center of the complex and enclosed by the four symmetrical arms is a small elliptical cagelike cavity of ∼40 Å in diameter. The central cavity is effectively sealed at the top and bottom of the complex but has small inter-arm holes when viewed from the side. We discuss the structure of this complex and implications in the surface translocation of cell-surface polysaccharide. Wza is a highly conserved multimeric outer membrane protein complex required for the surface expression of the serotype K30 group 1 capsular polysaccharide in Escherichia coli. Here we present the first three-dimensional structure of this type of polysaccharide exporter at a 15.5-Å resolution obtained using single particle averaging on a dataset of cryo-negatively stained protein. Previous structural studies on purified Wza have revealed a homo-oligomeric ring structure that is most probably composed of eight subunits. Symmetry analysis of the three-dimensional structure combined with biochemical two- and three-dimensional crystallographic data strongly suggest that Wza is an octameric complex with a C4 quasi-rotational symmetry and is organized as a tetramer of dimeric subunits. Wza is best described as a stack of two 4-Å high rings with differing diameters providing a mushroom-like aspect from the side. The larger ring has a distinctive square shape with a diameter of 115 Å, whereas the smaller is almost circular with a diameter of 90 Å. In the center of the complex and enclosed by the four symmetrical arms is a small elliptical cagelike cavity of ∼40 Å in diameter. The central cavity is effectively sealed at the top and bottom of the complex but has small inter-arm holes when viewed from the side. We discuss the structure of this complex and implications in the surface translocation of cell-surface polysaccharide. Escherichia coli produces >80 structurally and immunochemically distinct capsular polysaccharides termed K antigens (1Jann K. Jann B. Sussman M. Escherichia coli: Mechanisms of Virulence. Cambridge University Press, Cambridge, United Kingdom1997: 113-143Google Scholar). These polymers vary in composition, linkage specificity, and substitution. Capsules are important virulence determinants that enable pathogenic bacteria to evade or counteract the unspecific host defense during the early (preimmune) phase of infection. They also interfere with the action of complement and phagocytes, although this effect is generally transient and overcome by capsule-specific antibodies in the immune phase of the host defense. In some cases, capsules are not immunogenic (or are poorly immunogenic) as a result of a structural mimicry or identity with host material, and these capsule types are correlated with highly virulent isolates. Examples include the group 2 of K1 and K5 serotypes (2Jann K. Jann B. Can. J. Microbiol. 1992; 38: 705-710Crossref PubMed Scopus (89) Google Scholar). The capsular K antigens of E. coli are classified into four categories (denoted as groups 1–4) based on their surface organization, their assembly mechanism, the organization of their biosynthetic gene loci, and the regulation of their expression (3Whitfield C. Roberts I.S. Mol. Microbiol. 1999; 31: 1307-1319Crossref PubMed Scopus (412) Google Scholar). However, in terms of their assembly, there are only two pathways used in E. coli, and the capsules of groups 1 and 2 provide the prototypes. Essentially identical pathways are found in other bacterial species (for review see Ref. 3Whitfield C. Roberts I.S. Mol. Microbiol. 1999; 31: 1307-1319Crossref PubMed Scopus (412) Google Scholar). In all of the cases, capsular polysaccharides are synthesized at the inner membrane and must be translocated across the outer membrane for final assembly on the cell surface. Group 1 capsules have a relatively low charge density and contain hexuronic acids as acidic components (3Whitfield C. Roberts I.S. Mol. Microbiol. 1999; 31: 1307-1319Crossref PubMed Scopus (412) Google Scholar). Group 1 K antigens are found in two forms on the cell surface. Short K-antigenic oligosaccharides are attached to lipid A in a form resembling lipopolysaccharides (5Maclachlan P.R. Keenleyside W.J. Dodgson C. Whitfield C. J. Bacteriol. 1993; 175: 7515-7522Crossref PubMed Google Scholar). In contrast, high molecular weight polymers forming the capsular structure are not attached to lipid A and are assembled on the cell surface in a translocation pathway independent from lipopolysaccharides (6Drummelsmith J. Whitfield C. Mol. Microbiol. 1999; 31: 1321-1332Crossref PubMed Scopus (123) Google Scholar). Group 2 capsules have a high charge density and may contain hexuronic acids, N-acetylneuraminic acid, or 2-keto-2-deoxymanno-octulosonic acid as acidic components (2Jann K. Jann B. Can. J. Microbiol. 1992; 38: 705-710Crossref PubMed Scopus (89) Google Scholar, 7Jann K. Jann B. Rev. Infect. Dis. 1987; 9: S517-S526Crossref PubMed Google Scholar). Group 2 polysaccharides have phosphatidic acid at their reducing termini, and this is thought to act as the surface anchor (8Gotschlich E.C. Fraser B.A. Nishimura O. Robbins J.B. Liu T.Y. J. Biol. Chem. 1981; 256: 8915-8921Abstract Full Text PDF PubMed Google Scholar, 9Schmidt M.A. Jann K. FEMS Microbiol. Lett. 1982; 14: 69-74Crossref Scopus (58) Google Scholar). The oligosaccharide repeat units of the group 1 K30 antigen from E. coli (O9a:K30) are assembled on an undecaprenylphosphate carrier, and the glycosyltransferases have been identified (6Drummelsmith J. Whitfield C. Mol. Microbiol. 1999; 31: 1321-1332Crossref PubMed Scopus (123) Google Scholar). They are then transferred across the inner membrane by an unknown process involving Wzx and polymerized at the periplasmic face of the membrane by the polymerase Wzy. These proteins are the characteristic components of a Wzy-dependent assembly pathway and are best studied in the assembly of lipopolysaccharide O antigens (reviewed in Ref. 10Raetz C.R.H. Whitfield C. Ann. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3356) Google Scholar). High level polymerization of group 1 capsular polysaccharide requires phosphorylation of an inner membrane tyrosine autokinase, Wzc (11Wugeditsch T. Paiment A. Hocking J. Drummelsmith J. Forrester C. Whitfield C. J. Biol. Chem. 2001; 276: 2361-2371Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Wzc is dephosphorylated by its cognate phosphatase, Wzb, and this protein is also essential for capsule assembly (11Wugeditsch T. Paiment A. Hocking J. Drummelsmith J. Forrester C. Whitfield C. J. Biol. Chem. 2001; 276: 2361-2371Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 12Paiment A. Hocking J. Whitfield C. J. Bacteriol. 2002; 184: 6437-6447Crossref PubMed Scopus (97) Google Scholar). Two outer membrane proteins are involved in K30 assembly. The Wzi β-barrel protein acts late in the assembly process and appears to be involved in surface association (but not synthesis) of capsular polymer, although its exact role is unknown (13Nesper J. Hill C.M.D. Paiment A. Harauz G. Beis K. Naismith J.H. Whitfield C. J. Biol. Chem. 2003; 278: 49763-49772Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Wza proteins are lipoproteins that form multimeric outer membrane complexes that are involved in translocation of the capsular polymer across the outer membrane (6Drummelsmith J. Whitfield C. Mol. Microbiol. 1999; 31: 1321-1332Crossref PubMed Scopus (123) Google Scholar, 13Nesper J. Hill C.M.D. Paiment A. Harauz G. Beis K. Naismith J.H. Whitfield C. J. Biol. Chem. 2003; 278: 49763-49772Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Group 2 capsules are synthesized by a hetero-oligomeric membrane-bound biosynthetic complex on the inner face of the cytoplasmic membrane by the sequential action of glycosyltransferases that elongate the polysaccharide at its non-reducing end (14Finke A. Bronner D. Nikolaev A.V. Jann B. Jann K. J. Bacteriol. 1991; 173: 4088-4094Crossref PubMed Google Scholar). Translocation across the inner membrane characteristically requires an ABC-2 (ATP-binding cassette) transporter (3Whitfield C. Roberts I.S. Mol. Microbiol. 1999; 31: 1307-1319Crossref PubMed Scopus (412) Google Scholar, 15Bliss J.M. Silver R.P. J. Bacteriol. 1997; 179: 1400-1403Crossref PubMed Google Scholar, 16Silver R.P. Prior K. Nsahlai C. Wright L.F. Res. Microbiol. 2001; 152: 357-364Crossref PubMed Scopus (43) Google Scholar). Although some group 2 genetic loci encode homologs of Wza (6Drummelsmith J. Whitfield C. Mol. Microbiol. 1999; 31: 1321-1332Crossref PubMed Scopus (123) Google Scholar) and models for export have been proposed (16Silver R.P. Prior K. Nsahlai C. Wright L.F. Res. Microbiol. 2001; 152: 357-364Crossref PubMed Scopus (43) Google Scholar), little is known regarding their structure function. The outer membrane component involved in the translocation of group 2 capsular polysaccharides in E. coli is unknown. The Wza protein assembles into a stable oligomeric complex, which can form two- (13Nesper J. Hill C.M.D. Paiment A. Harauz G. Beis K. Naismith J.H. Whitfield C. J. Biol. Chem. 2003; 278: 49763-49772Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) and three-dimensional crystals (17Beis K. Nesper J. Whitfield C. Naismith J.H. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 60: 558-560Crossref Scopus (10) Google Scholar). Negatively stained two-dimensional crystals revealed ringlike multimers with an average outer diameter of ∼9–10 nm and central stain-excluding region of ∼2–3 nm (13Nesper J. Hill C.M.D. Paiment A. Harauz G. Beis K. Naismith J.H. Whitfield C. J. Biol. Chem. 2003; 278: 49763-49772Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Single particle analysis of the same detergent-solubilized material identified a putative C8 rotational symmetry, suggesting that the Wza·His6 complex is octameric (13Nesper J. Hill C.M.D. Paiment A. Harauz G. Beis K. Naismith J.H. Whitfield C. J. Biol. Chem. 2003; 278: 49763-49772Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). At a gross structural level, Wza multimers resemble the secretins such as PilQ (18Collins R.F. Ford R.C. Kitmitto A. Olsen R. Tonjum T. Derrick J.P. J. Bacteriol. 2003; 183: 3825-3832Crossref Scopus (108) Google Scholar), pIV (19Alm R.A. Mattick J.S. Gene (Amst.). 1997; 192: 89-98Crossref PubMed Scopus (134) Google Scholar), PulD (20Nouwen N. Stahleberg H. Pugsley A.P. Engel A. EMBO J. 2000; 19: 2229-2236Crossref PubMed Scopus (108) Google Scholar), XcpQ (21Bitter W. Koster M. Latjinhouwers M. de Cock H. Tommassen J. Mol. Microbiol. 1998; 27: 209-219Crossref PubMed Scopus (193) Google Scholar), and YscC (22Cornelis G.R. Wolf-Watz H. Mol. Microbiol. 1997; 23: 861-867Crossref PubMed Scopus (478) Google Scholar), which export proteins in Neisseria sp., Pseudomonas aeruginosa, Klebsiella oxytoca, P. aeruginosa, and Yersinia sp., respectively. However, these similarities do not extend to the primary sequence features (13Nesper J. Hill C.M.D. Paiment A. Harauz G. Beis K. Naismith J.H. Whitfield C. J. Biol. Chem. 2003; 278: 49763-49772Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Nevertheless, the higher order structural similarities suggest that detailed studies of one protein will provide information on the biology of the other systems. Electron microscopy (EM) 1The abbreviations used are: EM, electron microscopy; FSC, Fourier shell correlation. 1The abbreviations used are: EM, electron microscopy; FSC, Fourier shell correlation. has a particular value for examining large-scale conformation changes within oligomeric protein complexes (23Frank J. Three-dimensional Electron Microscopy of Macromolecular Assemblies. Plenum Press, San Diego1996Crossref Google Scholar). We aim to use EM to investigate how the components interact during polysaccharide export. As the necessary first step, we have determined the three-dimensional structure of native Wza multimers using cryo-negative staining combined with single particle analysis (22Cornelis G.R. Wolf-Watz H. Mol. Microbiol. 1997; 23: 861-867Crossref PubMed Scopus (478) Google Scholar, 23Frank J. Three-dimensional Electron Microscopy of Macromolecular Assemblies. Plenum Press, San Diego1996Crossref Google Scholar) to 15.5 Å. This technique imparts a higher contrast (for a relatively small complex) than is possible for particles observed by cryo-EM yet retains the advantages of sample preservation (24Adrian M. Dubochet J. Fuller S.D. Harris J.R. Micron. 1998; 2–3: 145-160Crossref Scopus (138) Google Scholar). Our analysis is the most detailed structural information currently for this of exporter protein. and of expression and that used to Wza have been described K. Nesper J. Whitfield C. Naismith J.H. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 60: 558-560Crossref Scopus Google Scholar). on the surface of a of Wza in for and then for on a then on a of or for and to in an all of the information to electron microscopy low and microscopy and analysis information for the three-dimensional low at to from resolution of at in a and three-dimensional structure of Wza using in The dataset of Wza particles using the of J. M. P. J. M. A. J. Biol. PubMed Scopus Google Scholar) or P.R. W. J. Biol. 1999; PubMed Scopus Google Scholar). The contrast for particle in the dataset determined using the 1992; Scopus Google Scholar, Lett. 2003; PubMed Scopus Google Scholar) and for and using then in a for P.R. W. J. Biol. 1999; PubMed Scopus Google Scholar), and the contrast The particles low to Å and in their to a A of then to Wza in particle in the a three-dimensional determined from that distinct of the Wza complex with a C4 symmetry The of the characteristic determined using a Fourier and the combined to the three-dimensional The three-dimensional structure using eight of with by examining the by of the Fourier shell of the three-dimensional models from The final three-dimensional eight of Symmetry of symmetry in in using particle the rotational with a for features and C4 symmetry, The of C4 symmetry then in (23Frank J. Three-dimensional Electron Microscopy of Macromolecular Assemblies. Plenum Press, San Diego1996Crossref Google Scholar) using a on rotational and analysis (18Collins R.F. Ford R.C. Kitmitto A. Olsen R. Tonjum T. Derrick J.P. J. Bacteriol. 2003; 183: 3825-3832Crossref Scopus (108) Google Scholar). and of determined by analysis by the in of two from of the final the same a for the final three-dimensional structure to the of the structure with a high of W. M. PubMed Scopus Google Scholar). of Wza and crystals of Wza using a as described G. Ford R.C. C. G. R. A. C. EMBO J. 2001; PubMed Scopus Google Scholar) and stained with Electron microscopy low on a electron at on and on a at at the and contrast as described G. Ford R.C. C. G. R. A. C. EMBO J. 2001; PubMed Scopus Google Scholar) using the of R. Mol. Biol. 1982; PubMed Scopus Google Scholar, R.A. R. J.M. J. Biol. PubMed Scopus Google Scholar). using the Rev. Google Scholar). Microscopy of of cryo-negatively stained Wza multimers within the staining complexes in with of stained Wza single particles J. Whitfield C. EMBO J. 2000; 19: PubMed Scopus Google Scholar), a of with a contrast with the The most observed the distinctive high contrast with an of rotational symmetry and of these A also of the particles to of the Wza complex with to the electron as in This sample of also a of the three-dimensional required for Symmetry of the Wza rotational symmetry used for of Wza based on the of the data in that the particle top the of rotational has a distinctive square and suggest that the symmetrical of Wza is EM data have that Wza has an octameric the of a three-dimensional structure using C8 symmetry than C4 that to the data and a resolution three-dimensional which not not with other only for and C4 some of the Wza two-dimensional and the from the three-dimensional structure and a of and of the the two-dimensional with the from the final three-dimensional structure that there a structural this not in the data with C8 the of the final Wza at the density to be M. C. Full Text Full Text PDF PubMed Scopus Google Scholar). This a protein of and is with an eight Wza from three-dimensional data (17Beis K. Nesper J. Whitfield C. Naismith J.H. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 60: 558-560Crossref Scopus (10) Google Scholar) using the Acta Crystallogr. Sect. D Biol. Crystallogr. PubMed Scopus Google Scholar). The data C4 and C8 rotational as in These data are all with a complex with a C4 rotational In these data strongly suggest that the octameric Wza is a tetramer composed of Wza subunits. Three-dimensional of of to Wza particles to the final three-dimensional of the three-dimensional structure of Wza viewed from the and top are in The Wza complex can be into two A larger is at the top with a smaller at the are by four with of a or at the top and bottom The bottom is to the top an to in the Although the top is relatively on its the into a of the four subunits. The that these two are by four the rotational the two The complex has an of Å in this this is the of an average lipid In the (or are four circular holes of Å in diameter that to a central This cavity is elliptical in shape and is Å in and at the The of the central cavity and its features within the complex also are in At this we are to units within the and with the Three-dimensional the resolution by for the final two three-dimensional from or structure only of the The of structural then is in Fourier by analysis M. J. Electron 9: PubMed Scopus Google Scholar), and the data are Fourier in three-dimensional to a of the for have that a of M. J. Electron 9: PubMed Scopus Google Scholar) be although this may be a R. J. Mol. Biol. 2003; PubMed Scopus Google Scholar). of a resolution of 15.5 Å for the Wza structure with the three-dimensional As in some small of high at the are the of the This is probably of a of in At the bottom of the the top and bottom there is of high within the protein density and this may of the in this The of high the of rotational symmetry are and are of that be necessary to a polymer translocation in the In this the top and bottom with the top a high at the center of the whereas the bottom a low in the of structural analysis and using an electron crystallographic crystals of the detergent-solubilized Wza complex obtained using a G. Ford R.C. C. G. R. A. C. EMBO J. 2001; PubMed Scopus Google Scholar), and an of crystals The crystals are composed of single of Wza but with two or can be observed in In small in the observed Wza particles in into the a Fourier of a of the Wza to of The the of a the crystallographic a The crystallographic symmetry two Wza in the cell for the of a that the crystals form in the group with the two Wza in the cell and there is some staining of the two of the there is a from a This one as to one of the Wza appears to be smaller in this has its smaller by and this to its The and shape of in the cell is with the single particle averaging described is with a central of Å in diameter. is from this crystallographic of an symmetry in with the of C4 symmetry for the single particle averaging D and the two in the cell the of symmetry Three-dimensional of mushroom-like complex is into two and appears to be by eight with rotational symmetry, suggesting a tetramer of A and C4 symmetry have been observed in other membrane the A. 2003; PubMed Scopus Google Scholar) and the O. N. 2001; 9: Full Text Full Text PDF PubMed Scopus Google Scholar), although in the the ring a surface and has a In these the structural data that the larger ring is observed in the of these we suggest that the larger ring of the Wza complex is in the The detailed structure by this also some structural Wza and the protein export secretins and some important PilQ from Neisseria forms a oligomeric outer membrane complex that is for the and of to the PilQ also is and PilQ and Wza complexes have four arms that from the bottom a central cavity and to form a on the top of the However, the and cavity of PilQ are larger than and the complex is probably from than viewed from the PilQ also has a The cavity of PilQ is also almost sealed on all of the whereas the cavity of Wza has The pIV which has in a distinct structure to However, the central cavity of pIV Å in is in two by the central ring of the complex N. R. N. M. J. Mol. Biol. 2003; PubMed Scopus Google Scholar). In contrast to Wza the structure a at the and The of the PulD revealed a structure with a and the of the protein Although the of the EM that the is N. N. H. B. Engel A. A. Pugsley A.P. A. 1999; PubMed Scopus Google Scholar). However, electron microscopy analysis of PulD that the three-dimensional structure may be to that of pIV (20Nouwen N. Stahleberg H. Pugsley A.P. Engel A. EMBO J. 2000; 19: 2229-2236Crossref PubMed Scopus (108) Google Scholar). of is for proteins but has been observed relatively for other oligomeric is a of membrane and protein complexes such as PilQ and R. C. The protein assembly forms a ring with the inner membrane M. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The molecular of the complex is and that of the rotational units is a The structural of these is but the symmetry has a the export will have symmetry or a symmetry with the exporter for K the for group 1 capsule the ring of the Wza is its almost the of the be is that the smaller ring is in the of the complex in the membrane the to be the lipid although this The structure is with a in which Wza with K antigen components at the inner biochemical with the inner membrane protein Wzc (13Nesper J. Hill C.M.D. Paiment A. Harauz G. Beis K. Naismith J.H. Whitfield C. J. Biol. Chem. 2003; 278: 49763-49772Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). This be to the of secretins as with the inner membrane components in type complex A. C. Mol. Microbiol. 1993; PubMed Scopus Google Scholar, A. N. E. P. C. P. A. Mol. Microbiol. 2001; PubMed Scopus Google Scholar). is an to the from E. coli H. J. Mol. Microbiol. 2001; Google Scholar). The can be to an transporter or an on the export M.A. J. Mol. Biol. 1999; PubMed Scopus Google Scholar, C. C. Biol. 2001; PubMed Scopus Google Scholar, C. C. EMBO 2000; PubMed Scopus Google Scholar, T. E. C. EMBO J. 1998; PubMed Scopus Google Scholar, A. E. B. C. 2000; PubMed Scopus Google Scholar). The Wza not an transporter for translocation of the K The Wza multimeric structure the complex to the but to the complex cavity may be the holes from the may be that this is the for the capsular polysaccharide with the the of one of the two We have proposed that is the ring that is in the this the in the the used for and electron the this However, Wza and secretins act as across the outer appears that we are the form of Wza In order for the to the have to that the Wza is this in the As the data that there some in the larger (but not with for and of the larger This may be by or with protein. is a protein that can be to Wza in a higher order complex (13Nesper J. Hill C.M.D. Paiment A. Harauz G. Beis K. Naismith J.H. Whitfield C. J. Biol. Chem. 2003; 278: 49763-49772Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). This structural information for Wza multimers a to investigate the of the Wza complex and the of on its function. We and for with We also and Fuller for

Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.