MrkDrd and other adhesin domains share a jelly-roll β-barrel fold but present different receptor binding sites
MrkDrd shows a jelly-roll β-barrel fold comprising 17 β-strands (1a, 1b, 1c, 2a, 2b, 3, 4, 5, 6, 7, 8, 9a, 9b, 10a, 10b, 11a, 11b) (Fig. 1A). MrkDrd has a compact elongated shape with dimensions of 54.2 Å × 23.8 Å × 18.3 Å and a disulfide bridge between C22 (β1a) and C67 (β2b) that presumably confers rigidity to the structure and helps to stabilize a rather flexible N-terminus (Fig. 1B). A deep pocket is observed near the N-terminus (defined as the ‘top’ of the structure; clearly visible in Fig. 3B).
Figure 1. Crystal structure of the MrkD1P receptor binding domain.
A. Stereo view in ribbon representation of the model of MrkD1P receptor binding domain. β strands are numbered 1–11.
B. View of the MrkDrd structure showing the disulfide bridge between C22 and C67 coloured in blue and residues T52, G121 and T164 that were examined in a previous study (Sebghati and Clegg, 1999) in yellow and stick representation.
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Structure comparisons with the receptor binding domains of adhesins GafD (F17c-type and F17a-G) (pdb 1OIO and pdb 1O9W respectively), FimH (pdb 1TR7) and PapG (pdb 1J8R) (Dodson et al., 2001; Buts et al., 2003; Merckel et al., 2003; Bouckaert et al., 2005) showed that MrkDrd is most related to GafDrd/F17-G [Z score of 9.5 and root-mean square deviation (RMSD) of 4.1 Å for 141 Cα-positions] (Fig. 2), despite amino acid sequence homology of < 11% (sequence identity) (Holm et al., 2008). Alignment of the receptor binding domain structures of FimH and PapG with MrkDrd yielded lower Z scores of 4.1 and 3.3 and RMSD of 4.3 Å and 4.4 Å for 119 and 103 Cα-positions respectively.
Figure 2. Comparison of the receptor binding domain structures of adhesins MrkD1P and GafD.
A. Structure alignment of MrkD1P receptor binding domain (grey) and GafD receptor binding domain (light blue) bound to GlcNAc (magenta).
B. Topology diagrams of MrkD1P receptor binding domain (grey) and GafD receptor binding domain (light blue). Lines in orange (GafDrd) and light blue (MrkDrd) represent the location of the disulfide bridge in the two domains.
C. Sequence alignment of the MrkD1P and GafD receptor binding domains. The secondary structure elements for MrkD1P and GafD receptor binding domains are represented on the top and bottom of the protein sequences. Numbering for both protein sequences is indicated. Alignment was generated by ClustalW (Larkin et al., 2007) and conservation of amino acids visualized by the ESPript server (Gouet et al., 1999). White characters in red boxes represent strict amino acid identity, red characters represent type conserved amino acid substitutions and a blue frame represent semi-conserved amino acid substitutions.
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The receptor binding domains of adhesins MrkD1P, GafD/F17-G, FimH and PapG share a jelly-roll β-barrel fold (Figs 2 and S6). The receptor binding sites for GafD, FimH and PapG have been identified but locate in a very different region in each of the structures (Fig. S6): the d-mannopyranoside binding site of FimH is a deep negatively charged pocket at the tip of the receptor binding domain (Adams et al., 2002), the Gal(α1–4)Gal binding site of PapG is a shallow pocket on the side of the molecule (Dodson et al., 2001) and GafD/F17-G binds n-acetyl-d-glucosamine (GlcNAc) in a shallow pocket on top of the receptor binding domain (Buts et al., 2003; Merckel et al., 2003). This, together with the fact that sugar binding has not been described for any of the MrkD variants, suggests that the structural comparison of MrkDrd with the receptor binding domains of GafD, FimH or PapG might not provide helpful clues as to where the collagen binding site of MrkDrd might be located.
A hydrophobic patch composes the collagen V binding site on the MrkD1P receptor binding domain
To identify the collagen V binding site on MrkDrd we mutated 16 solvent exposed amino acids in various regions of the MrkDrd structure and measured their impact on collagen V binding (Fig. 3A and B, and Experimental procedures). These mutants aim to test most surfaces of MrkDrd. Among other mutations, substitutions to Gly were introduced and tested. These mutations were either in loops or β-strands and therefore would not be expected to be structurally disruptive (see http://www.bmrb.wisc.edu/referenc/choufas.html and associated references). The collagen V binding assay was carried out using E. coli HB101 that contains plasmid pFK68ΔmrkD1P encoding all Mrk proteins for pilus assembly except for adhesin MrkD1P and was transformed with empty plasmid pTrc99A (negative control) or with plasmid pTrc99AmrkD1P encoding either wild type MrkD1P (positive control) or MrkD1P variants (Experimental procedures). Collagen V binding of E. coli HB101 double transformants was quantified by enzyme-linked immunosorbent assay (ELISA) (Experimental procedures). Furthermore, expression of Mrk pili on the bacterial surface was examined by serum agglutination and adherence of MrkD1P variants by HA of tannic acid-treated bovine erythrocytes (Experimental procedures).
Figure 3. Localization of residues mutated on the MrkDrd structure.
A. MrkDrd structure as ribbon representation is showing mutated residues as stick model. Residues are colour schemed for its effect on collagen V binding. In yellow (T52, G121 and T164) are represented the residues first described by Sebghati and collaborators as residues involved in collagen V binding (Sebghati and Clegg, 1999). Mutagenesis of residues in red (V49, R105, I136 and Y155) abolished collagen V binding in this study. Mutagenesis of residues in orange (V91 and R102) affected collagen V binding severely. Residues in grey (W23, V39, T54, V85, K106, T130, T132, K163 and V174) did not have an effect on collagen V binding upon mutagenesis. The disulfide bridge between C22 and C67 is shown in blue.
B. Surface representation of the MrkDrd structure shown in panel A with same colour-coding of residues. The arrow points to the deep pocket identified as one of the two collagen binding sites.
C. Protein sequence alignment of the receptor binding domain of adhesin variants MrkD1P, MrkD1C1 and MrkD1C2. Residues mutated in MrkD1P shown to affect collagen V binding are numbered and maintain the same colour scheme. Cysteines forming disulfide bridges are also listed and coloured in cyan.
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Of 20 E. coli HB101 strains encoding MrkD1P pilus variants, nine strains (W23A, V39G, T54A, V85G, K106A, T130A, T132A, K163A and V174G) showed pilus expression, HA and collagen V binding activities comparable to the wild type control showing that these residues are not involved in collagen V binding (Table 2; Fig. 4). E. coli HB101 harbouring plasmids pFK68ΔmrkD1P and pTrc99A without mrkD1P insert (negative control) were not piliated and consequently did not exhibit HA activity nor collagen V binding showing that Mrk pilus assembly is dependent on incorporation of MrkD1P into the pilus tip. The remaining 11 E. coli HB101 strains expressing MrkD1P variants V49G, V49A, T52A, T54S, V91G, R102G, R105E, I136G, I136A, Y155F and Y155A produced pili at wild type level but the mutations affected HA (Table 2) and collagen V binding activity (Fig. 4) demonstrating that these residues are involved in collagen V binding.
Figure 4. Effect of MrkD1P mutations on collagen V binding ability. Bars show collagen V binding activity of MrkD1P variants or wild type MrkD1P incorporated into Mrk pili expressed by E. coli HB101. Wild type MrkD1P represents 100% collagen V binding activity. Statistical analyses were performed using a two-tailed Students t-test.
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Table 2. Type 3 pilus surface expression and haemagglutination of tannic acid-treated bovine erythrocytes
|E. coli strain (plasmid)||Type 3 pilus expression||Haemagglutination|
|HB101 (pFK68ΔmrkD) (pTrc99mrkD)||++++||+++|
|HB101 (pFK68ΔmrkD) (pTrc99A)||–||–|
|HB101 (pFK68ΔmrkD) (pTrc99mrkDW23A)||++++||+++|
|HB101 (pFK68ΔmrkD) (pTrc99mrkDV39G)||++++||+++|
|HB101 (pFK68ΔmrkD) (pTrc99mrkDV49G)||++++||–|
|HB101 (pFK68ΔmrkD) (pTrc99mrkDV49A)||++++||+++|
|HB101 (pFK68ΔmrkD) (pTrc99mrkDT52A)||++++||+++|
|HB101 (pFK68ΔmrkD) (pTrc99mrkDT52S)||++++||+++|
|HB101 (pFK68ΔmrkD) (pTrc99mrkDT54A)||++++||+++|
|HB101 (pFK68ΔmrkD) (pTrc99mrkDV85G)||++++||+++|
|HB101 (pFK68ΔmrkD) (pTrc99mrkDV91G)||++++||–|
|HB101 (pFK68ΔmrkD) (pTrc99mrkDR102G)||++++||+ (weak)|
|HB101 (pFK68ΔmrkD) (pTrc99mrkDR105E)||++++||–|
|HB101 (pFK68ΔmrkD) (pTrc99mrkDK106A)||++++||+++|
|HB101 (pFK68ΔmrkD) (pTrc99mrkDT130A)||++++||+++|
|HB101 (pFK68ΔmrkD) (pTrc99mrkDT132A)||++++||+++|
|HB101 (pFK68ΔmrkD) (pTrc99mrkDI136G)||++++||–|
|HB101 (pFK68ΔmrkD) (pTrc99mrkDI136A)||++++||–|
|HB101 (pFK68ΔmrkD) (pTrc99mrkDY155A)||++++||–|
|HB101 (pFK68ΔmrkD) (pTrc99mrkDY155F)||++++||–|
|HB101 (pFK68ΔmrkD) (pTrc99mrkDK163A)||++++||+++|
|HB101 (pFK68ΔmrkD) (pTrc99mrkDV174G)||++++||+++|
Collagen V binding amino acids cluster around two regions on MrkDrd, a deep pocket, where residues R105 and Y155 are found at the bottom, that aligns approximately with the GlcNAc binding site of GafD, and a transversally oriented patch that spans strands β2a, β9b and β6 including residues V49, T52, V91, R102 and I136 (Fig. 3A and B). Residues V49 and I136 make up the hydrophobic core of the binding patch on MrkDrd and are surrounded by residues T52, V91 and R102 (Fig. 3A). When mutated to alanine, residue V49 still mediates HA of tannic acid treated erythocytes (Table 2), but collagen V binding activity of MrkD1P is decreased to 90%. In contrast, MrkD1P variant I136A could not trigger HA and its collagen V binding activity was only 40% of the wild type MrkD1P (Table 2). The influence of hydrophobicity of the hydrophobic patch on collagen V binding of MrkD1P became more apparent when residues V49 and I136 were mutated to glycine in order to exclude the hydrophobic effect that the alanine methyl side-chain exerts. This time, both MrkD1P variants V49G and I136G failed to mediate HA (Table 2). Collagen V binding activity of MrkD1P variant V49G was reduced even further to 30% of the wild type MrkD1P level while collagen V binding activity of variant I136G did not exceed the basal level of the negative control (E. coli HB101 transformed with plasmids pFK68ΔmrkD1P and pTrc99A without insert) (Fig. 4). MrkD1P variants V91G and R102G showed less HA activity compared with wild type MrkD1P (Table 2) and their ability to bind collagen V corresponded to ∼ 65% and ∼ 70% respectively of wild type MrkD1P level (Fig. 4). Mutation of residue T52 to the more hydrophobic and bulkier isoleucine was reported in a previous study to abolish collagen V binding of piliated bacteria expressing this MrkD1P variant (Sebghati and Clegg, 1999). Residue T52 lies in a stretch of conserved amino acids among all three MrkD (1C1, 1C2 and 1P) variants and is conserved between MrkD1C1 and MrkD1P (Fig. 3C). However, in the amino acid sequence of the inactive MrkD1C2 variant a serine is found in position 52. Since this is only a small change in an otherwise conserved stretch of the amino acid sequence and given the fact that variant T52I can eliminate collagen V binding of MrkD1P, we tested weather a T52S mutation could also abolish collagen V binding of MrkD1P. Surprisingly, MrkD1P variant T52S showed increased collagen V binding activity by 40% while mutation T52A did not affect collagen V binding. The mutagenesis of T52 to S, A or I shows, that collagen V binding of MrkD1P is sensitive to small side-chain changes of T52.
Mutations at the structure's bottom (V39G, V174G), top (W23A), backside (K163A), face (T54A, K106A, T130A and T132A) or on a potential extension of the hydrophobic patch (V85G) were carried out to delimit the collagen V binding site of MrkDrd and showed HA and collagen V binding activities comparable to that of wild type MrkD1P indicating that these residues do not contribute to collagen V binding (Table 2; Figs 3A and 4). The non-collagen V binding residues T54, T130, T132, K106 lie in between the collagen V binding hydrophobic patch and the small pocket of MrkDrd. There is no direct connection of collagen V binding residues from the hydrophobic patch to the small pocket on MrkD. Thus, collagen V must be bound by two distinct sites of the MrkDrd.
The transversally oriented receptor binding patch on MrkDrd has an estimated area of approximately 80 Å2 (∼ 16 Å length and ∼ 5 Å width) and therefore meets the requirement for binding a fibre shaped molecule like a collagen V triple helix. Interestingly, collagen V binding residues V49, V91 and I136 of MrkD1P are structurally conserved in GafD (V127, V31, I73; GafD numbering) but their function has not been examined nor has collagen binding been described for GafD/F17-G.
The MrkDrd crystal structure also revealed the structural basis for the effects of mutations G121D and T164I shown in a previous study to abolish collagen V binding of MrkD1P (Fig. 1B) (Sebghati and Clegg, 1999): indeed, mutation of G121D abolishes the flexibility that G121 confers to a short loop between β-strands β8 and β9a and will therefore hamper the correct folding of MrkDrd, while residue T164 is buried in the core of the molecule and mutation to isoleucine will most likely destabilize the MrkDrd fold.
A deep pocket on the MrkD1P receptor binding domain also affects the binding of collagen V
Our study revealed a deep pocket at the top of the molecule (Fig. 3B) as a potential collagen V binding site on MrkDrd. This pocket is lined at the bottom by the hydroxyl group of Y155 and the guanidinium group of R105. This pocket is too small to recognize larger parts of the collagen V triple helix structure. Therefore, we suspected that a single side-chain of collagen V (possibly a negatively charged residue given the positively charged nature of the pocket) could be inserted into the MrkDrd pocket. To test this hypothesis, mutations R105E, Y155A and Y155F were introduced and their ability to bind collagen V tested. All E. coli HB101 transformants expressed Mrk pili but the mutations R105E, Y155A and Y155F of MrkDrd clearly affected HA and collagen V binding activities (Table 2; Fig. 4). Mutations Y155A and Y155F abolished HA activity of MrkD1P and decreased its collagen V binding activity to ∼ 30% of the wild type MrkD1P level (Table 2; Fig. 4). The effect on collagen V binding of mutation Y155A could also be attributed to structural destabilization of the MrkDrd pocket or even of the whole MrkDrd domain because the small methyl side-chain of Y155A cannot replace the bulky phenyl ring of Y155. However, removing the hydroxyl group of Y155 (Y155F mutation) had a similar effect on HA and collagen V binding activities of MrkD1P (Table 2; Fig. 4) like mutation Y155A, indicating that the polar hydroxyl group of Y155 is crucial for collagen V binding rather than its phenyl group. Because of its small size it seems unlikely that the missing hydroxyl group of mutant Y155F could destabilize the MrkDrd structure.
Replacement of the positively charged R105 by a negatively charged shorter glutamate in the pocket of MrkDrd abolished HA and collagen V binding activities (Table 2; Fig. 4). We cannot rule out that mutation R105E caused structural instability of the MrkDrd domain (Sebghati and Clegg, 1999). On the other hand, a negatively charged collagen residue like glutamate could be repulsed by an equally charged R105E in the MrkDrd pocket.