Computational and mutagenesis studies of the streptavidin native dimer interface

[Display omitted] ▶ Estimating the stability of a mutant protein complex using molecular dynamics simulation is difficult because the modeled structures may differ significantly from the equilibrium structures. ▶ The degree of solvation of the buried interface is a convenient metric to predict which interfacial mutations will result in a stable complex. ▶ Steric complementarity plays an important role in streptavidin dimer association and the mutations that change side chain complementarity and polarity can significantly change the dimer stability. ▶ Biotin binding is coupled to the interactions at the streptavidin dimer interface and induces changes in side chain mobility and water hydrogen bonds. Furthermore, it can provide significant folding energy to overcome destabilizing interfacial mutations to form a stable tetramer. Wt streptavidin forms a domain swapped tetramer consisting of two native dimers. The role of tetramerization has been studied previously and is known to contribute to biotin binding by allowing the exchange of W120 between adjacent subunits. However, the role of dimer formation in streptavidin folding and function has been largely overlooked to date, although native dimers are necessary for tetramer formation and thus for high affinity biotin binding. To understand how the side chain interactions at the dimer interface stabilize the subunit association, we studied the structural and functional consequences of introducing interfacial mutations by a combination of molecular dynamics (MD) simulation and biochemical characterization. In particular, we introduced rational mutations at the dimer interface to engineer new side chain interactions and measured the stability and function of the resulting mutants. We focused on two residues that form a “knob” and a “hole” pair, G74 and T76, since steric complementarity plays an important role at these positions. We introduced mutations that would change the polarity and side chain packing to test if the interface can be rationally redesigned. Both energy calculation and geometric parameterization were used to interpret the simulated structures and predict how the mutations affect the dimer stability. In this regard, obtaining precise energy estimates was difficult because the simulated structures have large stochastic variations and some mutants did not reach an equilibrium by the end of the simulation. In contrast, comparing the wt and mutants to one another and parameterizing the simulation us