Three-Dimensional Structure of the
            <i>Neisseria meningitidis</i>
            Secretin PilQ Determined from Negative-Stain Transmission Electron Microscopy

Collins, Richard F.; Ford, Robert C.; Kitmitto, Ashraf; Olsen, Ranveig Ottoey; Tønjum, Tone; Derrick, Jeremy P.

doi:10.1128/jb.185.8.2611-2617.2003

Cited by 72 publications

(80 citation statements)

References 55 publications

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“…Secretins are multimeric OM proteins in which a conserved C-terminal ␤-barrel rich domain is implicated in forming the multimeric OM channel (39 -42). Typically, 6 -14 identical subunits form the ringlike structure of the secretin (39,(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52). Despite similarities in overall architecture of the secretin and Wza multimers, the respective monomers share no primary sequence similarity.…”

Section: Discussionmentioning

confidence: 99%

“…The channels formed by protein export secretins are gated (48,55), and low resolution three-dimensional structures of three secretins suggest an open state for these channel proteins in the OM and a closed state in the periplasm entrance (42,44,49). Gated channels are also formed by TolC, an outer membrane protein involved in type I protein secretion and multidrug efflux (56).…”

Section: Discussionmentioning

confidence: 99%

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Translocation of Group 1 Capsular Polysaccharide in Escherichia coli Serotype K30

Nesper

Hill

Paiment

et al. 2003

Journal of Biological Chemistry

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The late steps in assembly of capsular polysaccharides (CPS) and their translocation to the bacterial cell surface are not well understood. The Wza protein was shown previously to be required for the formation of the prototype group 1 capsule structure on the surface of Escherichia coli serotype K30 (Drummelsmith, J., and Whitfield, C. (2000) EMBO J. 19, 57-66). Wza is a conserved outer membrane lipoprotein that forms multimers adopting a ringlike structure, and collective evidence suggests a role for these structures in the export of capsular polymer across the outer membrane. Wza was purified in the native form and with a C-terminal hexahistidine tag. Wza His 6 was acylated and functional in capsule assembly, although its efficiency was slightly reduced in comparison to the native Wza protein. Ordered two-dimensional crystals of Wza His 6 were obtained after reconstitution of purified multimers into lipids. Electron microscopy of negatively stained crystals and Fourier filtering revealed ringlike multimers with an average outer diameter of 8.84 nm and an average central cavity diameter of 2.28 nm. Single particle analysis yielded projection structures at an estimated resolution of 3 nm, favoring a structure for the Wza His 6 containing eight identical subunits. A derivative of Wza (Wza*) in which the original signal sequence was replaced with that from OmpF showed that the native acylated N terminus of Wza is critical for formation of normal multimeric structures and for their competence for CPS assembly, but not for targeting Wza to the outer membrane. In the presence of Wza*, CPS accumulated in the periplasm but was not detected on the cell surface. Chemical cross-linking of intact cells suggested formation of a transmembrane complex minimally containing Wza and the inner membrane tyrosine autokinase Wzc.In Gram-negative bacteria, macromolecules destined for the cell surface or the extracellular environment must cross both the inner (IM) 1 and the outer membrane (OM). In protein export, where the processes are arguably best understood, secretion systems with varying complexity accomplish the translocation steps; all involve multi-enzyme complexes where an outer membrane protein (channel) is linked directly, or via helper proteins, to IM components.Cell-associated capsular polysaccharides (CPS) and their secreted (cell-free) counterparts, exopolysaccharides (EPS), represent another type of macromolecule that must be transported across the cell envelope. The early steps in assembly of these polymers are reasonably well established. However, there is little understanding of the terminal steps, including the mechanism by which they cross the bacterial cell envelope and the machinery involved.In Escherichia coli, more than 80 antigenically distinct capsular (K) polysaccharide structures are recognized. They are divided into four groups based on the organization of their genetic loci, polymerization mechanisms, and regulation (1). Group 1 and 2 capsules have received the most attention, and they involve biosynthet...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Translocation of Group 1 Capsular Polysaccharide in Escherichia coli Serotype K30

Nesper

Hill

Paiment

et al. 2003

Journal of Biological Chemistry

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“…Purification of the PilQ complex from meningococcal outer membranes was performed as previously described (Collins et al, 2001(Collins et al, , 2003(Collins et al, , 2004.…”

Section: Construction Of Meningococcal In-frame Pilq-his Fusionsmentioning

confidence: 99%

“…In addition, we assessed the potential of meningococcal PilQ to influence the integrity of the outer membrane, as viewed by its ability to allow passage of vancomycin. This new information is important for interpretation of 3D structural data, and further modelling of the PilQ complex (Collins et al, 2003(Collins et al, , 2004, as well as for studies of PilQ antigenicity and immunogenicity (Corbett et al, 1988;Hansen & Wilde, 1984). The findings may also be relevant for homologous secretins from the Gram-negative type II and III secretion systems.…”

Section: Introductionmentioning

confidence: 99%

Topology of the outer-membrane secretin PilQ from Neisseria meningitidis

et al. 2006

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Neisseria meningitidis is the causative agent of epidemic meningococcal meningitis and septicaemia. Type IV pili are surface organelles that mediate a variety of functions, including adhesion, twitching motility, and competence for DNA binding and uptake in transformation. The secretin PilQ is required for type IV pilus expression at the cell surface, and forms a dodecameric cage-like macromolecular complex in the meningococcal outer membrane. PilQ-null mutants are devoid of surface pili, and prevailing evidence suggests that the PilQ complex facilitates extrusion and retraction of type IV pili across the outer membrane. Defining the orientation of the meningococcal PilQ complex in the membrane is a prerequisite for understanding the structure-function relationships of this important protein in pilus biology. In order to begin to define the topology of the PilQ complex in the outer membrane, polyhistidine insertions in N-and C-terminal regions of PilQ were constructed, and their subcellular locations examined. Notably, the insertion epitopes at residues 205 and 678 were located within the periplasm, whereas residue 656 was exposed at the outer surface of the outer membrane. Using electron microscopy with Ni-NTA gold labelling, it was demonstrated that the insertion at residue 205 within the N-terminus mapped to a site on the arm-like features of the 3D structure of the PilQ multimer. Interestingly, mutation of the same region gave rise to an increase in vancomycin permeability through the PilQ complex. The results yield novel information on the PilQ N-terminal location and function in the periplasm, and reveal a complex organization of the membrane-spanning secretin in vivo.

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“…Further analysis by single particle averaging methods suggested that the complex has C12 symmetry, and a three-dimensional reconstruction from a sample in negative stain at 25 Å resolution shows a bowl-like structure (11). To obtain more detailed structural information on the PilQ complex, we have applied cryo-negative staining in conjunction with single particle averaging methods to extend the resolution of the structure considerably to 12 Å.…”

mentioning

confidence: 99%

Structure of the Neisseria meningitidis Outer Membrane PilQ Secretin Complex at 12 Å Resolution

Collins

Frye

Kitmitto

et al. 2004

Journal of Biological Chemistry

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118

123

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The bacterial pathogen Neisseria meningitidis expresses long, thin, retractile fibers (called type IV pili) from its cell surface and uses these adhesive structures to mediate primary attachment to epithelial cells during host colonization and invasion. PilQ is an outer membrane protein complex that is essential for the translocation of these pili across the outer membrane. Here, we present the structure of the PilQ complex determined by cryoelectron microscopy to 12 Å resolution. The dominant feature of the structure is a large central cavity, formed by four arm features that spiral upwards from a squared ring base and meet to form a prominent cap region. The cavity, running through the center of the complex, is continuous and is effectively sealed at both the top and bottom. Analysis of the complex using selforientation and by examination of two-dimensional crystals indicates a strong C4 rotational symmetry, with a much weaker C12 rotational symmetry, consistent with PilQ possessing true C4 symmetry with C12 quasisymmetry. We therefore suggest that the complex is a homododecamer, formed by association of 12 PilQ polypeptide chains into a tetramer of trimers. The structure of the PilQ complex, with its large and well defined central chamber, suggests that it may not function solely as a passive portal in the outer membrane, but could be actively involved in mediating pilus assembly or modification.

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Three-Dimensional Structure of the Neisseria meningitidis Secretin PilQ Determined from Negative-Stain Transmission Electron Microscopy

Cited by 72 publications

References 55 publications

Translocation of Group 1 Capsular Polysaccharide in Escherichia coli Serotype K30

Translocation of Group 1 Capsular Polysaccharide in Escherichia coli Serotype K30

Topology of the outer-membrane secretin PilQ from Neisseria meningitidis

Structure of the Neisseria meningitidis Outer Membrane PilQ Secretin Complex at 12 Å Resolution

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