Beyond the Binding Site: The Role of the β2 – β3 Loop and Extra-Domain Structures in PDZ Domains

A general paradigm to understand protein function is to look at properties of isolated well conserved domains, such as SH3 or PDZ domains. While common features of domain families are well understood, the role of subtle differences among members of these families is less clear. Here, molecular dynamics simulations indicate that the binding mechanism in PSD95-PDZ3 is critically regulated via interactions outside the canonical binding site, involving both the poorly conserved loop and an extra-domain helix. Using the CRIPT peptide as a prototypical ligand, our simulations suggest that a network of salt-bridges between the ligand and this loop is necessary for binding. These contacts interconvert between each other on a time scale of a few tens of nanoseconds, making them elusive to X-ray crystallography. The loop is stabilized by an extra-domain helix. The latter influences the global dynamics of the domain, considerably increasing binding affinity. We found that two key contacts between the helix and the domain, one involving the loop, provide an atomistic interpretation of the increased affinity. Our analysis indicates that both extra-domain segments and loosely conserved regions play critical roles in PDZ binding affinity and specificity. Author Summary: Protein interactions play crucial roles in all biological processes. A common way of studying them is to focus on sub-parts of proteins, called domains, that mediate specific types of interactions. For instance, it is known that most PDZ domains mediate protein interactions by binding to the C-terminus of other proteins. Humans have more than 200 slightly different copies of these domains. At the level of the binding site, PDZ domains look quite similar. This is in apparent contradiction with their heterogeneous binding specificity. Using detailed molecular dynamics simulations in conjunction with statistical analysis, we predict that contacts outside of the canonical binding site play important roles in regulating protein interactions. Some of these contacts influence the overall dynamics of PDZ domains, providing an explanation for their allosteric effect. These interactions involve regions of the PDZ domains that are much less conserved, suggesting that they can help in differentiating selectivity in this large domain family.