Functional Analysis of Freeman-Sheldon Syndrome Causing Mutations on Embryonic Myosin

Abstract

Freeman-Sheldon Syndrome (FSS) is a rare genetic disorder characterized primarily by multiple congenital contractures of facial muscles and distal joints, craniofacial abnormalities among other phenotypes. Mutations in embryonic skeletal myosin (MYH3) are the only known cause of FSS, and the vast majority of patients have one of three mutations: R672C, R672H, and T178I. The goal of this study was to determine the functional effects of these FSS- causing mutations on the human embryonic myosin motor domain. Preliminary data show a 10-fold reduction in ATP binding for both S1 (k2+) and actin-S1 (k′2+) for the R672C mutation. ADP affinity appears unaffected for this mutant. In the case of R672H, measurements of ATP binding were difficult because there was such a small change in tryptophan fluorescence (1.5% compared to 7-8% for Emb-WT). There was a 30 fold reduction in ATP binding to S1. Taken together with the reduction in tryptophan fluorescence change, we speculate a disruption in the conformational change in the recovery stroke. However when measured in the presence of actin we see values similar to Emb-WT. Interestingly, the affinity of ADP for actin-S1 is >3 fold tighter in this mutant with the rate constant for ADP release being reduced >3 fold. The affinity for actin is ∼20 fold weaker in the rigor complex and 10 fold tighter in the presence of ADP. For the T178I mutant which in the myosin structure forms a hydrogen bond with the R672 residue, we expect a similar effect in nucleotide binding. Our preliminary data shows a 5 fold reduction in ATP binding to S1 and 3 fold to actin-S1. When comparing to Emb-WT, the functional characteristics of these mutants are severely disrupted specifically in ATP induction of cross-bridges, which can explain the disease phenotype.