1. Introduction

The humoral immune system has the remarkable capability of recognizing a seemingly limitless number of antigens from a limited antibody repertoire. Antibody diversity stems firstly from combinatorial rearrangement of germline genes (Hozumi & Tonegawa, 1976), and the antibodies produced in this manner can be considered to be `germline'. Some types of antigen have the ability to stimulate mutations in these germline genes that can result in antibodies of much higher avidity through the process of affinity maturation (Jacob et al., 1991); however, even the generation of such antibodies must begin with germline-recognition events, which underscores their importance. The ability of germline antibodies to adapt their combining sites to bind a wide variety of antigens has been suggested as a mechanism to enhance the diversity of the humoral immune response (Wedemayer et al., 1997; Manivel et al., 2000; Nguyen et al., 2003).

A series of reports from our laboratories has described the molecular basis for cross-reactivity between antibodies and a panel of antigens based on chlamydial lipopolysaccharide (LPS). Antibody S25-2 and the related antibody S25-39 were remarkable in their ability to bind a range of chemically distinct antigens, which hinged on the flexible utilization of a pocket of amino-acid residues of germline origin to bind a single 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) residue in each antigen (Nguyen et al., 2003; Blackler et al., 2011; Brooks, Blackler et al., 2010; Brooks et al., 2008; Gerstenbruch et al., 2010; Evans et al., 2011).

S25-2 remains a valuable model in the study of germline-antibody recognition because of its remarkable cross-reactivity, and the use of synthetic Kdo analogues in particular provides a unique opportunity to probe the specificity of germline antibodies. The novel Kdo disaccharide analogue Kdo-(24)-5,6-dehydro-Kdo (Fig. 1a; Sixta et al., 2009) was synthesized in order to explore the importance of the 5-OH group on the second Kdo residue in recognition by S25-2.

Figure 1 (a) Ligand used for cocrystallization with S25-2: Kdo-(24)-5,6-dehydro-Kdo. (b) Structure of chlamydial LPS composed of Kdo-(28)-Kdo-(24)-Kdo-(26)-GlcN4P-(16)-GlcN1P. The Kdo-(24)-Kdo that previously cocrystallized with S25-2 (PDB entry 3t77 ; Nguyen et al., 2003) is highlighted in red. (c) 2Fo - Fc electron-density map contoured to 1 around Kdo-(24)-5,6-dehydro-Kdo cocrystallized with S25-2.

2. Materials and methods

2.3. Data collection and structure refinement

Crystals were flash-cooled to 113 K using an Oxford Cryostream 700 crystal cooler (Oxford Cryosystems). Data were collected using a Rigaku R-AXIS IV⁺⁺ area detector (Rigaku, Japan) coupled to a MicroMax-002 X-ray generator with Osmic Blue optics. The data were processed using CrystalClear/d*TREK (Rigaku). The structure of Fab S25-2 in complex with the ligand Kdo-(24)-5,6-dehydro-Kdo was solved by molecular replacement with PHENIX (Adams et al., 2010) using the structure of S25-2 in complex with Kdo as a search model (PDB entry 3t4y ; Nguyen et al., 2003), with Kdo, waters and ions being removed from the model prior to molecular replacement. Coot was used for manual fitting of the protein and ligand with 2F_o - F_c and F_o - F_c electron-density maps (Emsley & Cowtan, 2004). The structure was refined using PHENIX (Adams et al., 2010; phenix.refine v.1.8_1069). MolProbity (Chen et al., 2010) was used for Ramachandran analysis. The final refinement and model statistics are given in Table 1.

Table 1 Data-collection and refinement statistics for S25-2 in complex with Kdo-(24)-5,6-dehydro-Kdo Values in parentheses are for the highest resolution shell. Resolution 19.92-1.65 (1.71-1.65) Space group P212121 Unit-cell parameters (Å) a = 45.71, b = 81.30, c = 131.42 Observations 229551 Unique reflections 57408 Completeness (%) 95.9 (97.6) Rmerge 0.05 (0.253) <I/(I)> 12.4 (4.0) No. of non-H atoms   Polypeptide 3372   Ligand 33   Solvent 655   Ion 3 Rwork 0.176 Rfree 0.197 Average B factor (Å2)   Protein 21.9   Solvent 34.3   Ligand 17.9 Ramachandran plot, residues in (%)   Favoured regions 98.0   Additionally allowed regions 2.0 R.m.s.d., bonds (Å) 0.008 R.m.s.d., angles (°) 1.24

3. Results and discussion

The structure of Fab S25-2 was solved in complex with the non-natural Kdo derivative Kdo-(24)-5,6-dehydro-Kdo (Fig. 1a), which differs from the naturally occurring Kdo-(24)-Kdo disaccharide found in enterobacterial and chlamydial LPS (Fig. 1b). The residues in the structure were numbered according to the Kabat numbering scheme, which provides a common system of antibody residue numbering; letters are added to indicate insertions in CDR loops (Kabat et al., 1983). The ligand exhibited excellent electron density within the antibody-combining site (Fig. 1c).

The ligand is bound to S25-2 in the same electropositive pocket of conserved sequence as observed for other ligands (Fig. 2a), with the antibody forming a similar set of hydrogen bonds and charged-residue interactions with the terminal Kdo residue (Fig. 2b) to other liganded structures of S25-2 (Brooks et al., 2008; Nguyen et al., 2003). The 5,6-dehydro-Kdo is situated just outside the germline pocket, where it forms a hydrogen bond between the 7-OH group of Kdo and the side chain of Asn53 of the antibody light chain (Fig. 2b).

Figure 2 (a) Kdo-(24)-5,6-dehydro-Kdo binds in an electropositive pocket of the S25-2 combining site. (b) Interactions (green dashes) of S25-2 with Kdo-(24)-5,6-dehydro-Kdo. The terminal Kdo (bottom) makes a series of interactions that are conserved in other S25-2 structures (Brooks et al., 2008; Nguyen et al., 2003), while the 7-OH group of 5,6-dehydro-Kdo (top) forms a single hydrogen bond to residue Asn53 in the heavy chain. Amino-acid residues in the structure are numbered according to the Kabat numbering scheme for antibodies (Kabat et al., 1983); (H) refers to residues on the heavy chain and (L) refers to residues on the light chain.

The recent synthesis of Kdo analogues (Sixta et al., 2009) provides a convenient tool to probe the importance and the specificity of antibody-antigen interactions in the combining site of S25-2. The structure of S25-2 in complex with the antigen Kdo-(24)-Kdo (Fig. 1b) has been reported (PDB entry 3t77 ; Nguyen et al., 2003). The generation of the Kdo-(24)-5,6-dehydro-Kdo disaccharide allows direct examination of the importance of the 5-OH group on the second Kdo (Figs. 1a and 1b). In the structure with the Kdo-(24)-Kdo disaccharide, a single hydrogen bond is formed from the 5-OH group on the second Kdo residue to residue Asn53 in the heavy chain of S25-2 (Fig. 3a; Nguyen et al., 2003). The ligand 5,6-dehydro-Kdo is unable to form this interaction and only forms the hydrogen bond between the 7-OH group and the side chain of Asn53 (Figs. 2b and 3a). An overlay of S25-2 in complex with Kdo-(24)-Kdo and with Kdo-(24)-5,6-dehydro-Kdo reveals no significant movement (r.m.s.d. on C atoms of 0.2 Å) of the protein backbone and side chains in the combining site (Fig. 3). However, the two ligands are in different conformations, with a 55° rotation of the Kdo ring occurring about the glycosidic bond (Figs. 3a and 3b), with the effect that the 5,6-dehydro-Kdo does not form the salt bridge to residue Arg27f on the light chain of the antibody that is observed in the Kdo-(24)-Kdo disaccharide structure (Fig. 3a). Kdo-(24)-Kdo exists as a mixed population of two major conformers. Interestingly, S25-2 does not bind to either of these conformers, but rather binds to an energetically unfavourable conformation that does not exist in solution (Haselhorst et al., 1999). This specific bound orientation of Kdo-(24)-Kdo is created by a salt bridge between the carboxyl group of the second Kdo and Arg27f which twists the sugar ring in the binding site. This strong interaction does not form in the 5,6-dehydro-Kdo structure. The side chain of Arg27f is disordered in the 5,6-dehydro-Kdo structure, suggesting that the moiety is highly mobile. Initial correct positioning of the ligand in the binding site may be required to form the salt bridge and restrict the mobility of Arg27f. Thus, the interaction of the antibody with the 5-OH group on the second Kdo acts as an anchor and appears to be crucial in the formation of the salt bridge to Arg27f and hence the differences in the conformations of the Kdo-(24)-Kdo and 5,6-dehydro-Kdo disaccharides observed in the S25-2 binding sites (Haselhorst et al., 1999; Nguyen et al., 2003). The Kdo-(24)-Kdo disaccharide binds to S25-2 with a K_d of 1.1 × 10^-6 M, which is 15 times stronger than the binding of the Kdo monosaccharide (K_d = 15 × 10^-6 M), indicating the importance of the interactions of the second Kdo in the generation of high-affinity binding to S25-2 (Brooks et al., 2008). Although we do not have binding data for 5,6-dehydro-Kdo, we can hypothesize that the altered interactions to the modified second Kdo residue will result in a significantly lower affinity, similar to those observed for other Kdo analogue structures (Brooks et al., 2008).

Figure 3 (a) Overlay of binding sites of S25-2 in complex with Kdo-(24)-5,6-dehydro-Kdo (yellow; interactions shown in green) and Kdo-(24)-Kdo (white; interactions shown in red). In Kdo-(24)-Kdo the 5-OH group of the second Kdo residue forms a hydrogen bond to Asn53 (heavy chain) orienting the Kdo to form a salt bridge with Arg27f (light chain). The lack of the 5-OH group in the second Kdo of Kdo-(24)-5,6-dehydro-Kdo results in different interactions in the binding site. (b) Stereoview of Kdo-(24)-5,6-dehydro-Kdo (yellow) and Kdo-(24)-Kdo (white), which exhibit different orientations in the binding site. Amino-acid residues in the structure are numbered according to the Kabat numbering scheme for antibodies (Kabat et al., 1983); (H) refers to residues on the heavy chain and (L) refers to residues on the light chain.

This structure firstly underscores the importance of intermolecular hydrogen bonds in maintaining antigenic conformations, which certainly contribute to the affinity of binding of the Kdo-(24)-Kdo ligand. Secondly, this structure again demonstrates the remarkable ability of S25-2 to adapt and bind to the synthetic 5,6-dehydro-Kdo by forming new and different interactions in the antibody-combining site and highlights the diversity of the ability of the immune system to respond to novel antigenic challenges.