LAUSR

Calcineurin (CaN) is a serine/threonine phosphatase that regulates a variety of physiological and pathophysiological processes in most mammalian tissue. In its inactive state, the CaN catalytic domain is inhibited by the auto-inhibitory domain (AID); the active state is obtained upon CaM binding to the CaN regulatory domain (RD). It has been established that the RD is highly disordered when inhibiting CaN, yet it undergoes a disorder-to-order transition upon binding calmodulin (CaM) to activate the phosphatase. Given that the RD is richly populated with polar and charged amino acids, arguably electrostatic interactions may influence the rate of CaM association. However, it is likely that properties of the RD conformational ensemble, such as its ‘effective volume’ and accessibility of its CaM binding motif, influence the CaM/CaN association rate. In present study, we investigated via computational modeling the extent to which electrostatics and structural disorder co-facilitate or hinder CaM/CaN binding kinetics. We examined several peptides containing the CaM binding motif, for which lengths and amino acid charge distributions were varied, to isolate the contributions of electrostatics versus conformational diversity to predicted, diffusion-limited association rates. These rates were predicted using Replica Exchange Molecular Dynamics (REMD) and Brownian Dynamics (BD) simulations. Our results indicate that association rates vary as a function of increasing CaN RD length (beyond the required CaM recognition sequence), thus indicating that RD conformational ensemble properties influence CaM binding. Second, we found that increasing the solvent ionic strength generally depressed CaM/CaN association rates, owing to the attenuation of long-range electrostatic interactions that would normally accelerate protein-protein association. Finally, CaN peptides with positively-charged amino acids substituted at native negatively-charged sites had complex effects on the predicted association rate, owing to ‘off-target’ interactions that in some cases competed with the intending binding site. Our findings detail the interplay between conformational diversity and electrostatically-driven protein-protein association involving CaN, which are likely to extend to wide-ranging processes regulated by intrinsically-disordered proteins.