Tightly-controlled regulation of smooth muscle myosin's activity is essential for healthy cardiovascular, respiratory, and digestive function in humans. Recent structural data have provided insight into how smooth muscle myosin's activity is inhibited by dephosphorylation of the regulatory light chain (RLC).
In Aim #1 we propose to determine the molecular basis by which RLC phosphorylation activates smooth muscle myosin, a mechanism that has yet to be established. Functional studies (motility, actin-activated ATPase) of mutant myosins, expressed using the baculovirus insect/cell system, will test the hypothesis that the phosphorylated N-terminus of the RLC disrupts intramolecular head-head interactions and docks between the light chains to stiffen the lever arm for optimal activation. Analysis of 2-D arrays of phosphorylated heavy meromyosin, and crystallographic studies of relevant pieces of the myosin neck and rod, will complement the functional assays. We will also test the hypothesis that myosin with only one of its two heads phosphorylated can slowly cycle and bear a load, hence mimicking key properties of a latchbridge. Techniques include single molecule studies using an optical laser trap, and measurement of the force:velocity relationship, using small ensembles (10 molecules) of myosin in a force clamp laser trap assay. Recently, mutations in smooth muscle myosin have been linked for the first time to an inherited arterial disease, namely thoracic aortic aneurysm/aortic dissection and patent ductus arteriosis. We will (Aim #2) express mutant human smooth muscle myosin or rod with the known molecular defects, to deduce the molecular basis by which this initial insult leads to the disease phenotype. Electron microscopy will be used to assess filament assembly defects, while motility and enzymatic assays will test for functional defects. A broader, fundamental question addressed by this aim is how changes in the structure of the rod or the filament can be propagated at a distance to affect myosin's motor activity.
Aim #3 focuses on structural studies of actin and its binding partners. One goal is to crystallize dimers and higher oligomers of actin to understand the structural changes that must occur upon polymerization. A second goal is to crystallize an actin-motor domain complex, which will provide atomic resolution information on the fundamental unit needed to produce force and motion in muscle. These are not trivial undertakings, and many complexes will need to be tried, because crystallization is still part art and part science. This a high risk-high reward aim that we are well-positioned to pursue.
Recent statistics show that cardiovascular disease is still an underlying or contributing cause of death in over half of the mortalities in the United States. This staggering statistic illustrates the importance of understanding in detail the molecular mechanism by which myosin and actin interact, how in smooth muscle this interaction is tightly regulated by phosphorylation of a subunit in the head, and how mutations in the smooth muscle rod cause arterial disease.
