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

Abstract

Using BIACORE SPR, we have examined the mechanism of temperature effects on the binding kinetics of two closely related antibody Fabs (H10 and H26) which recognize coincident epitopes on hen egg-white lysozyme (HEL), and whose association and dissociation kinetics are best described by the two-step conformational change model which we interpret as molecular encounter and docking. Time-course series data obtained at a series of six temperatures (6, 10, 15, 25, 30 and 37 degrees C) showed that temperature differentially affects the rate constants of the encounter and docking steps. Docking is more temperature-sensitive than the encounter step, and energetically less favorable at higher temperatures. At elevated temperatures, the time required for docking is longer and the apparent increase in off-rate reflects the greater proportion of the molecules failing to dock and remaining in the less stable encounter state. As a consequence, distribution of free energy change between the encounter and docking steps is altered. At physiological temperature (37 degrees C) the docking step of the H26 complex is energetically unfavorable and most complexes essentially do not dock. There is a significant decrease in total free energy change of the H26 complex at higher temperatures. Elevated temperature changes the rate-limiting step of H26--HEL association from the encounter to the docking step, but not that of H10--HEL. Our results indicate that the mechanism by which elevated temperature reduces the affinities of antigen--antibody complexes is to decrease the net docking rate, and/or stability of the docked complex; at higher temperatures, a smaller proportion of the complexes actually anneal to a more stable docked state. This mechanism may have broad applicability to other receptor--ligand complexes.

The Immunoglobulin Heavy Chain Constant Region Affects Kinetic and Thermodynamic Parameters of Antibody Variable Region Interactions with Antigen

“…The two-step model fitted better than one-step model to our experimental data ( Fig. 1S in supplemental data, and data not shown) and it has been shown to be a more precise representation of Ab-Ag interactions (23,24). The simulated curves generated with the two-state model were almost identical to the experimental curves (data not shown).…”

Mutations Designed to Destabilize the Receptor-Bound Conformation Increase MICA-NKG2D Association Rate and Affinity

“…The docking step is close to equilibrium and is enthalpy-driven. These thermodynamic signatures were also observed for two-step induced-fit antibody-antigen interactions: cases where molecules can approach quickly, driven by long range electrostatics, but must wait for flexible surfaces to shift before forming specific, enthalpy-driven contacts in the final complex (23).…”

“…In a two-step model, a fast "encounter" step precedes a slower "docking" step (22,23). If destabilized MICA mutants have lost local structure and become more induced-fit in mechanism, a slower docking step could result.…”

The Role of Antibody Polyspecificity and Lipid Reactivity in Binding of Broadly Neutralizing Anti-HIV-1 Envelope Human Monoclonal Antibodies 2F5 and 4E10 to Glycoprotein 41 Membrane Proximal Envelope Epitopes

“…3, D and E) and could be best defined by a two-step, conformational change model (A ϩ B 3 ABx; ABx 3 AB). This model has previously been used to describe conformational rearrangements associated with soluble CD4 binding to HIV-1 gp120 (46) and as well for anti-hen egg lysozyme Fabs to describe a linked two-step process involving molecular encounter and docking (47,48). In this model, the first step is described as an encounter step that results in the formation of the first less stable complex (ABx) with faster kinetics (k a1 and k d1 ).…”