A Rigorous Framework for Calculating Protein-Protein Binding Affinities in Membranes.

Calculating the binding free energy of integral transmembrane (TM) proteins is crucial for understanding the mechanisms by which they recognize one another and reversibly associate. The glycophorin A (GpA) homodimer, composed of two α-helical segments, has long served as a model system for studying TM protein reversible association. The present work establishes a methodological framework for calculating the binding affinity of the GpA homodimer in the heterogeneous environment of a membrane. Our investigation carefully considered a variety of protocols, including the appropriate choice of the force field, rigorous standardization reflecting the experimental conditions, sampling algorithm, anisotropic environment, and collective variables, to accurately describe GpA dimerization via molecular dynamics-based approaches. Specifically, two strategies were explored: (i) an unrestrained potential mean force (PMF) calculation, which merely enhances sampling along the separation of the two binding partners without any restraint, and (ii) a so-called "geometrical route", whereby the α-helices are progressively separated with imposed restraints on their orientational, positional, and conformational degrees of freedom to accelerate convergence. Our simulations reveal that the simplified, unrestrained PMF approach is inadequate for the description of GpA dimerization. Instead, the geometrical route, tailored specifically to GpA in a membrane environment, yields excellent agreement with experimental data within a reasonable computational time. A dimerization free energy of -10.7 kcal/mol is obtained, in fairly good agreement with available experimental data. The geometrical route further helps elucidate how environmental forces drive association before helical interactions stabilize it. Our simulations also brought to light a distinct, long-lived spatial arrangement that potentially serves as an intermediate state during dimer formation. The methodological advances in the generalized geometrical route provide a powerful tool for accurate and efficient binding-affinity calculations of intricate TM protein complexes in inhomogeneous environments.