Molecular dynamics force probe simulations of antibody/antigen unbinding: Entropic control and nonadditivity of unbinding forces

Unbinding of a spin-labeled dinitrophenyl (DNP) hapten from the monoclonal antibody AN02 F-ab fragment has been studied by force probe molecular dynam ics (FPMD) simulations. In our nanosecond simulations, unbinding was enforc ed by pulling the hapten molecule out of the binding pocket. Detailed inspe ction of the FPMD trajectories revealed a large heterogeneity of enforced u nbinding pathways and a correspondingly large flexibility of the binding po cket region, which exhibited induced fit motions. Principal component analy ses were used to estimate the resulting entropic contribution of similar to 6 kcal/mol to the AN02/DNP-hapten bond. This large contribution may explain the surprisingly large effect on binding kinetics found for mutation sites that are not directly involved in binding. We propose that such "entropic control" optimizes the binding kinetics of antibodies. Additional FPMD simu lations of two point mutants in the light chain, Y33F and I96K, provided fu rther support for a large flexibility of the binding pocket. Unbinding forc es were found to be unchanged for these two mutants. Structural analysis of the FPMD simulations suggests that, in contrast to free energies of unbind ing, the effect of mutations on unbinding forces is generally nonadditive.