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. 2009 Feb 13;17(2):255-65.
doi: 10.1016/j.str.2008.11.011.

Crystal structure of the N-terminal domain of the secretin GspD from ETEC determined with the assistance of a nanobody

Konstantin V Korotkov  1 , Els Pardon, Jan Steyaert, Wim G J Hol
Affiliations

Crystal structure of the N-terminal domain of the secretin GspD from ETEC determined with the assistance of a nanobody

Konstantin V Korotkov et al. Structure. .

Abstract

Secretins are among the largest bacterial outer membrane proteins known. Here we report the crystal structure of the periplasmic N-terminal domain of GspD (peri-GspD) from the type 2 secretion system (T2SS) secretin in complex with a nanobody, the VHH domain of a heavy-chain camelid antibody. Two different crystal forms contained the same compact peri-GspD:nanobody heterotetramer. The nanobody contacts peri-GspD mainly via CDR3 and framework residues. The peri-GspD structure reveals three subdomains, with the second and third subdomains exhibiting the KH fold which also occurs in ring-forming proteins of the type 3 secretion system. The first subdomain of GspD is related to domains in phage tail proteins and outer membrane TonB-dependent receptors. A dodecameric peri-GspD model is proposed in which a solvent-accessible beta strand of the first subdomain interacts with secreted proteins and/or T2SS partner proteins by beta strand complementation.

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Figures

Figure 1
Figure 1. Structure of the peri-GspD:Nb7 complex
(A) Alignment of peri-GspD sequences from selected species. The secondary structure elements corresponding to the crystal structure of peri-GspD:Nb7 are colored in black and predicted secondary structure elements for N3 domain are colored in magenta. Dashed lines indicate disordered regions. Triangles (van der Waals) and stars (salt bridges) indicate contacts between N0 and N1 domains colored according to the interacting partner. Circles (van der Waals) and stars (salt bridges) indicate contacts with the nanobody Nb7. Red and orange symbols indicate contacts with CDR3 and framework residues respectively. Residues of PulD susceptible to trypsin proteolysis are highlighted in green. Vertical black arrows indicate the beginning and end of ETEC peri-GspD used in this study. (B) Two views of the heterotetramer (peri-GspD)2:(Nb7)2. Note the interface between the two nanobodies formed by two-anti-parallel β-strands. (C) A stereoview of the peri-GspD structure. The three subdomains N0, N1 and N2 are colored cyan, light blue and blue, respectively. Note strand β2 at the bottom which is solvent accessible and may play a key role in T2SS functioning. (D) Topology diagram of peri-GspD with the subdomains in the same colors as in (C).
Figure 1
Figure 1. Structure of the peri-GspD:Nb7 complex
(A) Alignment of peri-GspD sequences from selected species. The secondary structure elements corresponding to the crystal structure of peri-GspD:Nb7 are colored in black and predicted secondary structure elements for N3 domain are colored in magenta. Dashed lines indicate disordered regions. Triangles (van der Waals) and stars (salt bridges) indicate contacts between N0 and N1 domains colored according to the interacting partner. Circles (van der Waals) and stars (salt bridges) indicate contacts with the nanobody Nb7. Red and orange symbols indicate contacts with CDR3 and framework residues respectively. Residues of PulD susceptible to trypsin proteolysis are highlighted in green. Vertical black arrows indicate the beginning and end of ETEC peri-GspD used in this study. (B) Two views of the heterotetramer (peri-GspD)2:(Nb7)2. Note the interface between the two nanobodies formed by two-anti-parallel β-strands. (C) A stereoview of the peri-GspD structure. The three subdomains N0, N1 and N2 are colored cyan, light blue and blue, respectively. Note strand β2 at the bottom which is solvent accessible and may play a key role in T2SS functioning. (D) Topology diagram of peri-GspD with the subdomains in the same colors as in (C).
Figure 2
Figure 2. Comparison of peri-GspD N0 domains with structural homologs
(A) The N0 subdomain (cyan) of peri-GspD superimposed onto the signaling domain (red) of the TonB-dependent outer membrane receptor FpvA from Pseudomonas aeruginosa (PDB: 2O5P) (Brillet et al., 2007). TonB-box residues of FpvA are shown in yellow. (B) Superposition of the N0 subdomain onto the second domain (pink) of prophage MuSo 43 kDa tail protein from Shewanella oneidensis (PDB: 3CDD). (C) Structure-based sequence alignment of N0 domain with structural homologs shown in (A) and (B). The yellow arrow indicates residues participating in TonB-box contacts in the signaling domain of FpvA.
Figure 3
Figure 3. Comparison of the peri-GspD N1 and N2 subdomains with each other and structural homologs
(A) Superposition of the N1 and N2 subdomains of peri-GspD colored in light blue and blue, respectively. (B) The N1 subdomain superimposed with the KH-domain (orange) of the neuronal splicing factor Nova-1 (PDB: 2ANR). (C) The N1 subdomain superimposed with the first domain (green) of the type 3 secretion system protein EscJ from EPEC (PDB: 1YJ7) (Yip et al., 2005). (D) The N2 subdomain superimposed with the KH-domain (yellow) of Nova-1 RNA-binding protein (PDB: 1DTJ) (Lewis et al., 1999). (E) The N2 subdomain superimposed with a ferredoxin-like domain (lime) of the Menkes protein ATP7A (PDB: 1YJR) (Banci et al., 2005). (F) The N2 subdomain superimposed with the first domain (green) of the type 3 secretion system protein EscJ from EPEC (PDB: 1YJ7) (Yip et al., 2005). (G) Structure-based sequence alignment of N1and N2 subdomains of ETEC GspD, with a sequence-based N3 domain alignment in the center. Secondary structure elements correspond to the structure of N1 (top) and N2 (bottom) subdomains. Residues that are identical in any pair-wise comparison are highlighted in red.
Figure 4
Figure 4. Interactions within peri-GspD and with the nanobody Nb7
(A) Stereo figure of the interface between the N0 and N1 subdomains. Atoms of interface residues are in cyan for N0 and in light blue for N1. (B) Structure of the peri-GspD:Nb7 dimer with Nb7 in orange, CDR1 in green, CDR2 purple; CDR3 red, and the peri-GspD subdomains in the same colors as in Figure 1C. (C) An “open book” representation of the peri-GspD:Nb7 interface with footprints in colors according to interacting partner (CDR3 footprint in red; framework orange; N0 subdomain cyan: N1 subdomain light blue).
Figure 5
Figure 5. A model of a dodecameric ring of peri-GspD domains
(A) A model of peri-GspD with C12 symmetry, obtained as described in the text, plus a model of the C12 ring of the N2 subdomain assembly, with the 12-fold symmetry axes of the two rings coinciding. The N2 ring is positioned slightly above the N0-N1 ring to indicate that the loop connecting the N1 and N2 subdomains (Figure 1C) allows one translational and one rotational degree of freedom which still have to be determined. The structure of heat-labile nterotoxin (LT B5, PDB 1LTA) is shown for size comparison. (B) Bottom view of the C12 ring of N0-N1 subdomains shown in (A) with the LT B5 toxin structure. (C) Stereo figure of the contacts between neighboring subunits in the structure of EPEC EscJ viewed perpendicular to the 6-fold screw axis (Yip et al., 2005). (D) Stereo figure of the N0-N1 interface, viewed perpendicular to the C12 symmetry axis, in the ETEC GspD model shown in (A). Strand β2 that might participate in β-strand exchange with other proteins is highlighted in purple. The yellow β-strand is homologous to the TonB box β-strand interacting with the signaling domain of apo-FpvA (Brillet et al., 2007) illustrating how secreted proteins or T2SS partner proteins might interact with GspD. (E) Stereo figure of the subunit interface in the ETEC GspD ring of N2 domains shown in (B), viewed parallel to the C12 symmetry axis.
Figure 5
Figure 5. A model of a dodecameric ring of peri-GspD domains
(A) A model of peri-GspD with C12 symmetry, obtained as described in the text, plus a model of the C12 ring of the N2 subdomain assembly, with the 12-fold symmetry axes of the two rings coinciding. The N2 ring is positioned slightly above the N0-N1 ring to indicate that the loop connecting the N1 and N2 subdomains (Figure 1C) allows one translational and one rotational degree of freedom which still have to be determined. The structure of heat-labile nterotoxin (LT B5, PDB 1LTA) is shown for size comparison. (B) Bottom view of the C12 ring of N0-N1 subdomains shown in (A) with the LT B5 toxin structure. (C) Stereo figure of the contacts between neighboring subunits in the structure of EPEC EscJ viewed perpendicular to the 6-fold screw axis (Yip et al., 2005). (D) Stereo figure of the N0-N1 interface, viewed perpendicular to the C12 symmetry axis, in the ETEC GspD model shown in (A). Strand β2 that might participate in β-strand exchange with other proteins is highlighted in purple. The yellow β-strand is homologous to the TonB box β-strand interacting with the signaling domain of apo-FpvA (Brillet et al., 2007) illustrating how secreted proteins or T2SS partner proteins might interact with GspD. (E) Stereo figure of the subunit interface in the ETEC GspD ring of N2 domains shown in (B), viewed parallel to the C12 symmetry axis.
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