Mutations of an Epitope Hot-Spot Residue Alter Rate Limiting Steps of Antigen−Antibody Protein−Protein Associations

Abstract

The antibodies, HyHEL-10 and HyHEL-26 (H10 and H26, respectively), share over 90% sequence homology and recognize with high affinity the same epitope on hen egg white lysozyme (HEL) but differ in degree of cross-reactivity with mutant lysozymes. The binding kinetics, as measured by BIAcore surface plasmon resonance, of monovalent Fab from both Abs (Fab10 and Fab26) to HEL and mutant lysozymes are best described by a two-step association model consistent with an encounter followed by docking that may include conformational changes. In their complexes with HEL, both Abs make the transition to the docked phase rapidly. For H10, the encounter step is rate limiting, whereas docking is also partially rate limiting for H26. The forward rate constants of H10 are higher than those of H26. The docking equilibrium as well as the overall equilibrium constant are also higher for H10 than for H26. Most of the free energy change of association (Delta G degrees) occurs during the encounter phase (Delta G1) of both Abs. H10 derives a greater amount and proportion of free energy change from the docking phase (Delta G2) than does H26. In the H10--HEL(R21Q) complex, a significant slowing of docking results in lowered affinity, a loss of most of Delta G2, and apparently faster dissociation. Slower encounter and docking cause lowered affinity and a loss of free energy change primarily in the encounter step (Delta G1) of H26 with mutant HEL(R21Q). Overall, in the process of complex formation with lysozyme, the mutations HEL(R21X) affect primarily the docking phase of H10 association and both phases of H26. Our results are consistent with the interpretation that the free energy barriers to conformational rearrangement are highest in H26, especially with mutant antigen.

Conformational Abs recognizing a generic amyloid fibril epitope

“…Another possibility is main chain elements involved in some unusual turn or chain reversal within the amyloid motif. Detailed structures of the epitopes of anti-protein Abs are normally characterized either by protein crystallography (42) or by mutational analysis of antigen fragments (43) or intact protein (44). Because amyloid has yet to be crystallized, and because WO1 and WO2 bind to many amyloids regardless of amino acid sequence, it is clear that the further structural analysis of the WO1͞WO2 epitope(s) will be challenging.…”

Temperature differentially affects encounter and docking thermodynamics of antibody–antigen association

“…Recombinant monovalent Fab (rFab) to be used as analyte was expressed in secretory form from cDNA clones using the commercially available vector pET-22b() (Novagen, Madison, WI) as described previously (Lavoie et al, 1990(Lavoie et al, , 1999Li et al, 2001). rFab was affinity purified, monitored for homogeneity and lack of dimers on FPLC and spun before use to remove possible aggregates (Li et al, 2001).…”

“…Recombinant monovalent Fab (rFab) to be used as analyte was expressed in secretory form from cDNA clones using the commercially available vector pET-22b() (Novagen, Madison, WI) as described previously (Lavoie et al, 1990(Lavoie et al, , 1999Li et al, 2001). rFab was affinity purified, monitored for homogeneity and lack of dimers on FPLC and spun before use to remove possible aggregates (Li et al, 2001). Fab samples at concentrations based upon UV absorption were stored as a 50-100 mg/ml substock to discourage aggregation and diluted in HBS-EP buffer (10 mM HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% P-20 surfactant, pH7.4; Biacore Inc., Piscataway, NJ, USA) to the desired concentration (rFab10 at 500 ng or 1 mg/ml and rFab26 at 4.4 mg/ml).…”

“…Newman et al, 1992). The two antibodies differ in their sensitivities to antigen mutation in the epitope and in the effects of antigenic mutations on their binding kinetics (Smith-Gill et al, 1984a;Lavoie et al, 1990;Newman et al, 1992;Li et al, 2001). Antigenic mutations increase the net dissociation rates of complexes with both antibodies, and they also significantly slow apparent association rates of H26 but not H10 complexes (Lavoie et al, 1999).…”

“…These structure-based behavior differences can be resolved further into a two-step model of association and conformational change when analyzed using BIACORE biosensor surface plasmon resonance (SPR) technology and the experimental approach we have developed for analysis of two-step binding (Lipschultz et al, 2000). The binding kinetics of both antibodies with native and mutant HEL are best described by a two-step bimolecular association model which we interpret as a rapid encounter step, with forward rate constants that approach diffusion limitation (Janin, 1995), followed by a slower docking step (Lipschultz et al, 2000;Li et al, 2001; Plate 1). In their respective complexes with HEL, the formation of the encounter complex is accompanied by the majority of free energy change (À10 to À11 kcal/mol).…”