br Materials and Methods br Results br Discussion The

Materials and Methods
Results
Discussion The crystal structure of ClfA N2N3 in complex with the Fab fragment generated from tefibazumab defines the epitope for this inhibiting mAb, which to our surprise is located on top of the N3 subdomain. Through biochemical studies we demonstrate that the tefibazumab epitope partially overlaps with a second Fg binding site, which is required for high affinity binding of Fg to ClfA. A substantial variation in the amino smo inhibitor sequence of the identified tefibazumab epitope is apparent from examining ClfA sequences available in the public domain (Supplementary Table ST2). To evaluate the significance of these variations we expressed four of the more common tefibazumab epitope isoforms and determined their relative binding to immobilized tefibazumab by SPR. One of these isoforms (ClfAN463R) where Asn is replaced by the bulky charged residue Arg, showed a 60-fold reduced affinity for the mAb (KD=0.7nM (ClfA229–545) to KD=45nM (ClfAN463R) (Fig. 6A). Interestingly, this variant appears to have maintained its high affinity for Fg (Fig. 6B). These results demonstrate that tefibazumab recognizes some but not all naturally occurring ClfA variants of S. aureus. In addition, tefibazumab cannot completely block Fg binding to ClfA and although the recorded KD for the mAb\'s binding to ClfA is low it is not quite as low as that demonstrated for the corresponding mouse mAb before being “humanized” (Fig. 1). Furthermore, the observed IC50 is rather high (Fig. 1), which may be a consequence of the complex Fg binding mechanism employed by ClfA. Taken together these properties may explain the modest effect of tefibazumab as an anti-staphylococcal therapy in humans (Weems et al., 2006). Our earlier study showed that ClfA binding to Fg involves a variant of the Dock, Lock and Latch mechanism where the C-terminus of the Fg γ-chain docks in a trench formed between the N2 and N3 subdomains of the MSCRAMM (Ganesh et al., 2008). In the current report we demonstrate that the high affinity interaction of ClfA with intact Fg involves additional contacts between the MSCRAMM and the ligand protein. These additional interactions increase the overall affinity of the interaction and appear to involve residues in the γ-globular domain of the γ-chain of Fg that can be brought into close contact and interact with the “top” of the N3 domain according to our modeling experiments. Is it possible that this complex, multi-contact binding mechanism here shown for the ClfA/Fg interaction, also applies to other ligand/MSCRAMM interactions. The Fg binding MSCRAMMs FnBPA (Wann et al., 2000), FnBPB (Burke et al., 2011) of S. aureus and Fbl of S. lugdunensis (Geoghegan et al., 2010) all target the C-terminus of the Fg γ-chain using N2N3 segments with similar subdomain organization to ClfA. Thus Fg binding to these MSCRAMMs could also involve additional binding sites. A synthetic peptide corresponding to the linear sequence in Fg targeted by the related protein ClfB binds to the MSCRAMM with rather low affinity and the peptide is a poor inhibitor of Fg binding to ClfB (unpublished data). To account for the much higher affinity seen with intact Fg, ClfB like ClfA, may provide additional interactive sites outside the N2N3 trench. The Fg-binding MSCRAMM SdrG binds to a 15 amino acid linear Fg sequence in a rather high affinity interaction (Ponnuraj et al., 2003). In this case it is possible that additional sites are not required for an overall high affinity, although preliminary modeling experiments suggest that additional contacts are possible. Thus, although the DLL binding mechanism seems to be involved in most MSCRAMM ligand interactions additional contacts may be required for high affinity interactions. Furthermore, it is possible that sequence variations in the MSCRAMM at the second binding site could affect the affinity for Fg and thus the virulence potential of the ClfA variant. It is also possible that amino acid variations in human Fg sequences targeting the second site may bind with different affinities to the MSCRAMM and consequently affect the susceptibility of an individual to staphylococcal infections.