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Review
. 2012 Dec;76(4):740-72.
doi: 10.1128/MMBR.00035-12.

Type IV pilin proteins: versatile molecular modules

Carmen L Giltner  1 , Ylan Nguyen, Lori L Burrows
Affiliations
Review

Type IV pilin proteins: versatile molecular modules

Carmen L Giltner et al. Microbiol Mol Biol Rev. 2012 Dec.

Abstract

Type IV pili (T4P) are multifunctional protein fibers produced on the surfaces of a wide variety of bacteria and archaea. The major subunit of T4P is the type IV pilin, and structurally related proteins are found as components of the type II secretion (T2S) system, where they are called pseudopilins; of DNA uptake/competence systems in both Gram-negative and Gram-positive species; and of flagella, pili, and sugar-binding systems in the archaea. This broad distribution of a single protein family implies both a common evolutionary origin and a highly adaptable functional plan. The type IV pilin is a remarkably versatile architectural module that has been adopted widely for a variety of functions, including motility, attachment to chemically diverse surfaces, electrical conductance, acquisition of DNA, and secretion of a broad range of structurally distinct protein substrates. In this review, we consider recent advances in this research area, from structural revelations to insights into diversity, posttranslational modifications, regulation, and function.

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Figures

Fig 1
Fig 1
Alignment of N-terminal sequences of type IV pilin proteins. The leader peptide and first 40 N-terminal residues of representative mature type IV pilin protein sequences were aligned based on the prepilin peptidase cleavage site (marked by an arrow). Pilin-like proteins share the type III signal sequence, which is cleaved before the hydrophobic stretch between the Gly (−1) and Phe (+1) residues, although the +1 residue can vary. The consensus signal sequence used by the PilFind algorithm (195) to identify putative type IV pilin proteins is shown below the alignment. The hydrophobic N terminus of mature pilin proteins is situated in the inner membrane and contains the highly conserved Glu5 (+5) residue (shown in bold); in GspK orthologs, there is a hydrophobic residue at that position. The transmembrane segments, as predicted by Geneious Pro v5.0.3 (Biomatters Ltd.) using TMHMM, are highlighted in blue. Pa, P. aeruginosa; Vc, V. cholerae; Aa, Aggregatibacter (Actinobacillus) actinomycetemcomitans; Mm, Methanococcus maripaludis; Nm, N. meningitidis; ETEC, enterotoxigenic E. coli; Bs, Bacillis subtilis; Mv, Methanococcus voltae.
Fig 2
Fig 2
Structures of T4 pilins and minor pilins. Type IV pilins are characterized by conserved N-terminal α-helices (cyan) connected to a β-sheet (gray) by the αβ-loop (magenta). The N-terminal α-helix is divided into two subdomains, α1-N (amino acids ∼1 to 28) and α1-C (amino acids ∼29 to 52), as indicated on the N. gonorrhoeae PilE structure. In most pilins, two cysteine residues form a disulfide bond to form the variable D region (blue). Although the Dichelobacter nodosus FimA protein lacks these characteristic residues, its C terminus retains a similar architecture through alternative interactions (169). (A) T4a pilins, represented by Neisseria gonorrhoeae PilE (Protein Data Bank [PDB] accession no. 1AY2), Dichelobacter nodosus FimA (PDB accession no. 3SOK), Pseudomonas aeruginosa PAK PilA (PDB accession no. 1DZO), and P. aeruginosa Pa110594 PilA (PDB accession no. 3JZZ), share a shallow S-shaped N-terminal helix and a four-stranded continuous antiparallel β-sheet. The architectures of the αβ-loop and the D region of T4a pilins vary, with defined secondary structure in the GC pilin structure (top left) and the Pa110594 PilA structure (top right). Pa110594 PilA has an extended loop region, with an additional α-helix and β-strand between β3 and β4 (green) differentiating it from other T4a pilins. (B) T4b pilins, represented by Salmonella Typhi PilS (PDB accession no. 1Q5F), Vibrio cholerae TcpA (PDB accession no. 1OQV), and enteropathogenic E. coli BfpA (PBD accession no. 1ZWT), have a different protein fold, with discontinuous β-strands making up the β-sheet. The D region is embedded in the protein, with the C terminus forming the last strand of the β-sheet. The αβ-loop contains an α-helix oriented roughly 90° relative to α1. The structure of the noncore minor pilin PilX from N. meningitidis (PDB accession no. 2OPD) is similar to that of the major pilins but has two α-helices in the αβ-loop. The D region contains a short α-helix with a hook implicated in function (176). Figures were prepared using MacPymol (DeLano Scientific).
Fig 3
Fig 3
Structures of T2S pseudopilins and minor pseudopilins. T2S pseudopilins are structurally similar to the T4 pilins, with an N-terminal α-helix (cyan) connected to a β-sheet (gray) by a variable loop (magenta). (A) The major pseudopilins, represented by Pseudomonas aeruginosa XcpT (PDB accession no. 2KEP), Vibrio cholerae EpsG (PDB accession no. 3FU1), enterohemorrhagic E. coli GspG (PDB accession no. 3G20), and Klebsiella pneumoniae PulG (PDB accession no. 1T92), have variable loops with a helical character followed by a 3-stranded β-sheet. Near the C terminus is a calcium-binding motif (with a calcium ion shown in orange) in EpsG and GspG, although calcium binding is expected to occur in all major pseudopilins (235). (B) The minor pseudopilins, represented by GspH from V. cholerae (PDB accession no. 2QV8), GspI and GspJ from V. vulnificus (PBD accession no. 2RET), and GspK from enterotoxigenic E. coli (PDB accession no. 3CI0), vary in architecture, with a large α-domain insertion (green) in GspK. Figures were prepared using MacPymol (DeLano Scientific).
Fig 4
Fig 4
Comparison of the T4 pilin D region and the T2S pseudopilin calcium-binding domain. One of the defining differences between T4 pilins (left) (P. aeruginosa PAK PilA [PBD accession no. 1DZO]) and T2S pseudopilins (right) (V. cholerae EspG [PBD accession no. 3FU1]) is found at the C terminus, where the majority of T4 pilins have a disulfide-bonded loop (D region) and the pseudopilins have a calcium-binding motif (with a calcium ion shown in orange) (235). The disulfide bond creates a loop with two β-turns (left inset), while calcium binding by both main chain and side chain residues stabilizes the corresponding region in the pseudopilins (green, right inset). Both structural motifs are implicated in function. Figures were prepared using MacPymol (DeLano Scientific).
Fig 5
Fig 5
Models of pilus and pseudopilus fibers. Using structural subunit information, low-resolution EM data, and biochemical data, fiber models of the T4aP (left), T4bP (center), and pseudopilus (right) were generated. The T4aP model can be viewed as a one-start or four-start right-handed helix or a three-start left-handed helix (one strand is pictured, with two subunits shown as a green cartoon) with a diameter of approximately 60 Å. Tight packing of the N-terminal helices holds the structure together, with additional polar interactions between the C-terminal head groups. The three-start left-handed helix T4bP model has a larger diameter, at approximately 90 Å, due to the larger subunit size along with the more loose packing of the subunits, exposing portions of the N-terminal helix and producing deep grooves and bulges along the fiber (model kindly provided by Lisa Craig). The right-handed one-start helix T2S pseudopilus model is slightly larger than the T4aP model, at 65 Å, with hydrophobic and electrostatic interactions stabilizing subunit interactions (model kindly provided by Olivera Francetic). Figures were prepared using MacPymol (DeLano Scientific).
Fig 6
Fig 6
Force-dependent conformational changes in T4aP fibers. Biais et al. (46) showed that applying pulling forces just below those that cause Neisseria pili to break can lead to stretching of the pili in their long dimension, making them ∼40% narrower, with only 2/3 the mass per unit length of normal fibers (right). This conformational distortion was reversible and exposed epitopes (left) for the SM1 monoclonal antibody (green) that were hidden in normal fibers (red). (Left panel reprinted from reference with permission of the publisher; right panel courtesy of Nicolas Biais.)
Fig 7
Fig 7
Proposed model of T4a pilin autoregulation by intramembrane interactions with the PilS sensor kinase. The PilR-PilS two-component system regulates major pilin biosynthesis in many T4aP-producing bacteria. Because PilS lacks a typical periplasmic sensor domain and controls the expression of pilins with diverse C-terminal sequences, it may instead detect the highly conserved N-terminal domain of PilA orthologs via intramembrane interactions (11). High intracellular levels of PilA, as in pilus assembly mutants, may inhibit pilA transcription in a manner that depends on sequences in the pilin's N terminus, possibly through modulation of PilS dimerization or stimulation of phosphatase activity of PilS on the response regulator, PilR (37, 338). When pilin levels are low—as in retraction-deficient mutants, where the pilins are trapped outside the cell—PilS kinase activity is stimulated, phosphorylating PilR and upregulating PilA transcription. The organization of the pilABCD genes in P. aeruginosa is shown. pilA is transcribed by σ54, RNAP, and phospho-PilR from a divergent promoter that also controls expression of PilB (the pilin polymerase), PilC (the platform protein), and PilD (the prepilin peptidase).
Fig 8
Fig 8
Type IV pilin glycosylation systems. Three different type IV pilin O-glycosylation systems have been characterized to date, one from Neisseria and two from Pseudomonas aeruginosa. The Pgl system (purple) from Neisseria generates undecaprenol-linked di- or trisaccharides via the action of cytoplasmic enzymes PglA, PglB, and PglE and translocates them via the activity of the flippase PglF (purple) to the periplasmic face of the membrane, where they are attached to Ser63 of PilE by one of two oligosaccharyltransferases, PglL or PglO (tan) (199, 256, 257). In P. aeruginosa, group I pilins are modified at the C-terminal Ser148 by TfpO (PilO) (tan) with an undecaprenol-linked O-antigen subunit generated by the Wbp enzymes of the lipopolysaccharide pathway (orange) and are flipped to the periplasmic face of the membrane by the O-unit flippase, Wzx (orange) (67, 87, 150). Group IV pilins are modified at multiple Ser and Thr residues by monomers, dimers, and longer polymers of d-arabinofuranose synthesized by cytoplasmic enzymes on a lipid carrier to form decaprenol-arabinofuranose (DPA) (blue), polymerized into α1,5-linked homopolymers, translocated to the periplasm by an unknown enzyme(s), and attached to the pilin by TfpW (tan) (241, 242, 405). TfpW is significantly larger than TfpO (694 versus 461 amino acids) and may be responsible for both the flippase and α1,5-arabinosyltransferase (AT) functions (yellow) in the group IV system.
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