Mutant Analysis and Computational Analysis of the Essential Light Chain and Regulatory Light Chain Interactions with Respect to Regulation of Smooth Muscle Myosin

Abstract

To understand how smooth muscle myosin's (SMM) light chains are involved in phosphorylation dependent regulation of ATP hydrolysis, we expressed single, double and triple amino acid mutants of the heavy chain, regulatory (RLC) or essential light chain (ELC) in the area of the Ca2+-binding loop of the ELC. We mutated this area because of similarities to scallop smooth myosin's Ca2+ regulatory mechanism. Since the ELC is not required to maintain the off state, we reasoned that mutations in this region would specifically alter the activity of the on (phosphorylated) state of SMM. Mutations were made to disrupt hydrogen bonding between the ELC and RLC and the heavy chain (HC) based on scallop crystal structures. All mutant ATPase activities and actin sliding velocities (ASVs) were essentially identical to wild type in the unphosphorylated. For the phosphorylated states, one class of mutant showed normal ATPase activity and ASVs; a second class showed similarly depressed ATPase activity and ASVs; and a third class with differentially depressed ATPase activity and ASVs. We also created recovery mutants that restored (and recoupled) ATPase rates with ASVs. We then used course grain discrete molecular dynamics and force constant profiling to reconcile changes in the interactions of ELC, RLC & HC in that region, with changes at the ATPase site. We created a prepowerstroke smooth muscle myosin model with an ELC Ca2+ loop from scallop that interacted weakly with the RLC and used FlexServe (http://mmb.pcb.ub.es/FlexServ/) to compute changes in flexibility between the native (1QVI) and modified model. We see that changes in the ELC RLC interaction have direct effects on lever arm flexibility and active site protein dynamics.