Loop 1's Role in a Novel Step on the ADP Release Pathway of Smooth Muscle

Abstract

Smooth muscle myosin has two N-terminal isoforms that result from alternative splicing of loop 1. Loop 1 contains a seven amino acid insert (QGPSFSY) in one isoform (SM-B) that is absent in the other (SM-A). It has been shown that the presence of the insert causes a two-fold increase in the rate of in-vitro actin sliding velocity and actin-activated ATPase activity (Rovner et al., 1997, Muscle Res Cell Motil 18:103). Based on these results and its proximity to the active site, it was hypothesized that loop 1 plays a role in modulating the release of ADP (Spudich, 1994, nature 372:515). However, little is known about the conformation of loop 1 in different nucleotide states, as it is absent in crystal structures. To further investigate the role of loop 1 in modulating ADP release we have inserted a single tryptophan residue into the interior of loop 1 in the SM-B isoform to monitor its dynamics. In combination with stopped-flow kinetics to monitor the release rate of mant-ADP from the motor domain, we have observed three steps in the ADP release mechanism, one of which is a unique transition that occurs before ADP release and following opening of the active site. Significantly, this previously undetected kinetic step appears to arise from a specific change in the state of loop 1. This is the first time a role of loop 1 in the ADP release mechanism has been directly identified and may account for the functional differences observed between two isoforms of smooth muscle myosin.