The Affinity-Enhancing Roles of Flexible Linkers in Two-Domain DNA-Binding Proteins

Abstract

Recently many attempts have been made to design high-affinity DNA-binding proteins by linking two domains. Here a theory for guiding these designs is presented. Flexible linkers may play three types of roles: (a) linking domains which by themselves are unfolded and bind to DNA only as a folded dimer (as in a designed single-chain Arc repressor), (b) connecting domains which can separately bind to DNA (as in the Oct-1 POU domain), and (c) linking a DNA-binding domain with a dimerization domain (as in the λ repressor). In (a), the linker keeps the protein as a folded dimer so that it is always DNA-binding-competent. In (b), the linker is predicted to enhance DNA-binding affinity over those of the individual domains (with dissociation constants K_A and K_B) by p(d₀)/K_B or p(d₀)/K_A, where p(d₀) = (3/4πl_pbL)^3/2 exp(−3d₀²/4l_pbL)(1 − 5l_p/4bL + ...) is the probability density for the end-to-end vector of the linker with L residues to have a distance d₀. In (c), the linker is predicted to enhance the binding affinity by K_d^C/p(d₀), where K_d^C is the dimer dissociation constant for the dimerization domain. The predicted affinity enhancements are found to be actually reached by the Oct-1 POU domain and λ repressor. However, there is room for improvement in many of the recently designed proteins. The theoretical limits presented should provide a useful guide for current efforts of designing DNA-binding proteins.