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. 2008 Mar 14;377(1):91-103.
doi: 10.1016/j.jmb.2007.08.041. Epub 2007 Aug 23.

Structure of the minor pseudopilin EpsH from the Type 2 secretion system of Vibrio cholerae

Marissa E Yanez  1 , Konstantin V Korotkov, Jan Abendroth, Wim G J Hol
Affiliations

Structure of the minor pseudopilin EpsH from the Type 2 secretion system of Vibrio cholerae

Marissa E Yanez et al. J Mol Biol. .

Abstract

Many Gram-negative bacteria use the multi-protein type II secretion system (T2SS) to selectively translocate virulence factors from the periplasmic space into the extracellular environment. In Vibrio cholerae the T2SS is called the extracellular protein secretion (Eps) system,which translocates cholera toxin and several enzymes in their folded state across the outer membrane. Five proteins of the T2SS, the pseudopilins, are thought to assemble into a pseudopilus, which may control the outer membrane pore EpsD, and participate in the active export of proteins in a "piston-like" manner. We report here the 2.0 A resolution crystal structure of an N-terminally truncated variant of EpsH, a minor pseudopilin from Vibrio cholerae. While EpsH maintains an N-terminal alpha-helix and C-terminal beta-sheet consistent with the type 4a pilin fold, structural comparisons reveal major differences between the minor pseudopilin EpsH and the major pseudopilin GspG from Klebsiella oxytoca: EpsH contains a large beta-sheet in the variable domain, where GspG contains an alpha-helix. Most importantly, EpsH contains at its surface a hydrophobic crevice between its variable and conserved beta-sheets, wherein a majority of the conserved residues within the EpsH family are clustered. In a tentative model of a T2SS pseudopilus with EpsH at its tip, the conserved crevice faces away from the helix axis. This conserved surface region may be critical for interacting with other proteins from the T2SS machinery.

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Figures

Figure 1
Figure 1. The structure, topology and crystal contacts of EpsH
a) The structure of monomer A of EpsH30-188 with the corresponding topology diagram. Both the structure and topology diagram are colored according to the Type 4a pilin fold, with the conserved N-terminal α-helix in blue, the variable region with β-sheet I in purple and the conserved β-sheet II in green. A stretch of residues from Phe122 to Lys134 is disordered, and could not be included in the model, is denoted by a dotted black line. Chain B is very similar to Chain A, but is missing a larger part of the flexible loop (Gly104-Lys134). b) Two molecules A and B of EpsH30-188 in one asymmetric unit (ASU) with neighboring ASU’s on either side. Monomer A of EpsH30-188, denoted in blue by the letter A, forms three substantial contacts (labeled A-B, A-B’ and A-B”) with three adjacent chains within the crystal lattice. Symmetry mates are labeled A’ and B’ for chains in the ASU to the left of monomer A, and A” and B” for chains to the right of Chain A.
Figure 2
Figure 2. Family sequence alignment of EpsH/GspH
A black arrow denotes the start of the construct for EpsH30-188. Secondary structure elements are shown on top and colored according to the Type 4a pilin fold, with the conserved N-terminal α-helix in blue, the variable region in purple and the conserved β-sheet labeled in green. Invariant residues are highlighted in red, while residues that are conserved in all but 1 or 2 homologues are highlighted in pink. A yellow background denotes medium sequence conservation and white denotes poor sequence conservation. Residues at positions that have a hydrophobic residue in all 10 homologues are indicated with red letters. The solvent accessibility as assigned by ESPRIPT on the basis of the EpsH30-188 structure is denoted by the bar acc, with dark blue meaning residues that are highly accessible, cyan meaning residues that are partially accessible and white denoting residues that are buried in the EpsH structure. Intermolecular contacts between A-B, A-B’ and A-B” are represented by triangles, which represent hydrophobic contacts, and by stars, which indicate residues that form H-bonds. Symbols for the contact residues in the A-B interface, (Gln37, Tyr40, Gln41, Leu44, Asn47, Glu48, Ile51, Leu52, Lyβ86, Asn174, Gly175, Thr176, Leu179) are colored in blue; in the A-B’ interface, (Asp30, Ala32, Gln33, Pro158, Gly160, Gln161, Glu162, Asp164, Glu165, Gln166, Trp167, Ala181, Pro182, Gly183, Glu184, Ser185) are colored in green; and in the A-B” interface, (Gly54, Gln55, Asp56, Leu73, Thr74, Ala75, Asp76, Gly78, Trp79, Phe142, Leu144, Ser145, Ser146, Glu148, Val149, Thr150, Pro151) are colored in pink. The black circles denote conserved hydrophobic residues that make up a conserved patch on the surface of EpsH.
Figure 3
Figure 3. Sequence conservation and electrostatics surface view of Vibrio cholerae EpsH
a) The sequence conservation as determined by Consurf plotted on the surface of EpsH. Blue indicates high, red indicates low sequence conservation. A black arrow points to a highly conserved groove on the surface of EpsH, which is positioned between the variable and conserved β-sheets. b) The electrostatic surface of EpsH30-188 as calculated by APBS tools, in the same orientation as (a). A black arrow, which points to the highly conserved groove indicated in (a), shows that highly conserved residues form a hydrophobic crevice on the surface of EpsH. c) A cluster of 17 conserved residues in stick representation that form the hydrophobic groove shown in figures 3a and 3b. These 17 residues, of which 15 are hydrophobic, may form a binding site for EpsH to interact with partner proteins or small molecules. Six residues that are invariant in all 10 homologues are labeled in red. Eight residues that conserve a hydrophobic residue are labeled in orange. Three residues that are highly conserved in 8-9 homologues are labeled in black. Four residues that are part of the interface 3 between monomers A and B” in the crystal lattice are highlighted with a yellow background.
Figure 4
Figure 4. Comparison of Vibrio cholerae EpsH with all known Type 4a-like Pilin structures to date
a) EpsH is shown schematically along side the structures of equivalent structures of GspG from K. oxytoca (PDB code 1T92), PilAPAK from P. aeruginosa (PDB code 1DZO), PilE from N. gonorrhoeae (PDB code 2HI2), and PilAK1224 from P. aeruginosa (PDB code 1RGO). To facilitate the comparison, all structures are shown in the same orientation of the conserved β-sheet colored in green. The fourth β-strand of K. oxytoca GspG is colored yellow to indicate a strand exchange between two equivalent β-strands of two chains observed in the asymmetric unit of K. oxytoca GspG. These authors propose that the 4th strand under physiological conditions is provided by the same subunit as the three other strands. b) Topology Diagrams of the three T4BP pilins and the two T2SS pseudopilins of known structure. c) A structure-based alignment of 67 residues of EpsH and PilAPAK that can be superimposed with an r.m.s.d. of 2.5 Å for 67 Cα. The region that is most conserved between PilAPAK and EpsH is indicated by a pink box. This corresponds to the same region that has the highest sequence conservation within the EpsH30-188 structure (Figure 2). Three residues that have their numbers highlighted in red (Pro139 in EpsH, Gly175 in EpsH and Gly125 in PilAPAK) are conserved within the EpsH and PilA families, respectively. The black arrows denote two cis prolines in PilA that are in the same location in EpsH but are in the trans conformation. d) N-terminal alignment of EpsH and PilAPAK. Shown are the 29 N-terminal residues in EpsH, and the 33 N-terminal residues in PilAPAK, that were deleted from these proteins to obtain the respective crystal structures.
Figure 4
Figure 4. Comparison of Vibrio cholerae EpsH with all known Type 4a-like Pilin structures to date
a) EpsH is shown schematically along side the structures of equivalent structures of GspG from K. oxytoca (PDB code 1T92), PilAPAK from P. aeruginosa (PDB code 1DZO), PilE from N. gonorrhoeae (PDB code 2HI2), and PilAK1224 from P. aeruginosa (PDB code 1RGO). To facilitate the comparison, all structures are shown in the same orientation of the conserved β-sheet colored in green. The fourth β-strand of K. oxytoca GspG is colored yellow to indicate a strand exchange between two equivalent β-strands of two chains observed in the asymmetric unit of K. oxytoca GspG. These authors propose that the 4th strand under physiological conditions is provided by the same subunit as the three other strands. b) Topology Diagrams of the three T4BP pilins and the two T2SS pseudopilins of known structure. c) A structure-based alignment of 67 residues of EpsH and PilAPAK that can be superimposed with an r.m.s.d. of 2.5 Å for 67 Cα. The region that is most conserved between PilAPAK and EpsH is indicated by a pink box. This corresponds to the same region that has the highest sequence conservation within the EpsH30-188 structure (Figure 2). Three residues that have their numbers highlighted in red (Pro139 in EpsH, Gly175 in EpsH and Gly125 in PilAPAK) are conserved within the EpsH and PilA families, respectively. The black arrows denote two cis prolines in PilA that are in the same location in EpsH but are in the trans conformation. d) N-terminal alignment of EpsH and PilAPAK. Shown are the 29 N-terminal residues in EpsH, and the 33 N-terminal residues in PilAPAK, that were deleted from these proteins to obtain the respective crystal structures.
Figure 4
Figure 4. Comparison of Vibrio cholerae EpsH with all known Type 4a-like Pilin structures to date
a) EpsH is shown schematically along side the structures of equivalent structures of GspG from K. oxytoca (PDB code 1T92), PilAPAK from P. aeruginosa (PDB code 1DZO), PilE from N. gonorrhoeae (PDB code 2HI2), and PilAK1224 from P. aeruginosa (PDB code 1RGO). To facilitate the comparison, all structures are shown in the same orientation of the conserved β-sheet colored in green. The fourth β-strand of K. oxytoca GspG is colored yellow to indicate a strand exchange between two equivalent β-strands of two chains observed in the asymmetric unit of K. oxytoca GspG. These authors propose that the 4th strand under physiological conditions is provided by the same subunit as the three other strands. b) Topology Diagrams of the three T4BP pilins and the two T2SS pseudopilins of known structure. c) A structure-based alignment of 67 residues of EpsH and PilAPAK that can be superimposed with an r.m.s.d. of 2.5 Å for 67 Cα. The region that is most conserved between PilAPAK and EpsH is indicated by a pink box. This corresponds to the same region that has the highest sequence conservation within the EpsH30-188 structure (Figure 2). Three residues that have their numbers highlighted in red (Pro139 in EpsH, Gly175 in EpsH and Gly125 in PilAPAK) are conserved within the EpsH and PilA families, respectively. The black arrows denote two cis prolines in PilA that are in the same location in EpsH but are in the trans conformation. d) N-terminal alignment of EpsH and PilAPAK. Shown are the 29 N-terminal residues in EpsH, and the 33 N-terminal residues in PilAPAK, that were deleted from these proteins to obtain the respective crystal structures.
Figure 4
Figure 4. Comparison of Vibrio cholerae EpsH with all known Type 4a-like Pilin structures to date
a) EpsH is shown schematically along side the structures of equivalent structures of GspG from K. oxytoca (PDB code 1T92), PilAPAK from P. aeruginosa (PDB code 1DZO), PilE from N. gonorrhoeae (PDB code 2HI2), and PilAK1224 from P. aeruginosa (PDB code 1RGO). To facilitate the comparison, all structures are shown in the same orientation of the conserved β-sheet colored in green. The fourth β-strand of K. oxytoca GspG is colored yellow to indicate a strand exchange between two equivalent β-strands of two chains observed in the asymmetric unit of K. oxytoca GspG. These authors propose that the 4th strand under physiological conditions is provided by the same subunit as the three other strands. b) Topology Diagrams of the three T4BP pilins and the two T2SS pseudopilins of known structure. c) A structure-based alignment of 67 residues of EpsH and PilAPAK that can be superimposed with an r.m.s.d. of 2.5 Å for 67 Cα. The region that is most conserved between PilAPAK and EpsH is indicated by a pink box. This corresponds to the same region that has the highest sequence conservation within the EpsH30-188 structure (Figure 2). Three residues that have their numbers highlighted in red (Pro139 in EpsH, Gly175 in EpsH and Gly125 in PilAPAK) are conserved within the EpsH and PilA families, respectively. The black arrows denote two cis prolines in PilA that are in the same location in EpsH but are in the trans conformation. d) N-terminal alignment of EpsH and PilAPAK. Shown are the 29 N-terminal residues in EpsH, and the 33 N-terminal residues in PilAPAK, that were deleted from these proteins to obtain the respective crystal structures.
Figure 5
Figure 5. A possible position of EpsH at the tip of the T2SS pseudopilus
The conserved hydrophobic crevice of Vibrio cholerae EpsH facing outwards when EpsH is placed at the tip of a tentative pseudopilus model (for details see Materials and Methods). The model T2SS pseudopilus was constructed from EpsG homology-modeled subunits, based on the K. oxytoca GspG structure, that are superimposed onto PilE subunits within the assembled PilE pilus. The modeled EpsG subunits are colored dark green. The helix of PilE is colored light green. EpsH superimposed onto the tip of the PilE pilus is colored red. The 18 residues that form the hydrophobic crevice on the surface of EpsH are colored yellow.
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