Regulation Of Smooth and Nonmuscle Myosin

National Heart, Lung, And Blood Institute

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Abstract

Nonmuscle myosin 2 (NM2) molecules carry out a wide variety of functions within cells. There are three NM2 heavy chain genes in mammals. We are expressing full length NM2 proteins and fragments of these myosins in the baculovirus Sf9 system. We are studying their structure both at the level of the single molecule folded autoinhibited state as well as the structure of myosin filaments. We are particularly interested in how phosphorylation of both the heavy chain and light chain affects filament formation and activity. We use a single filament motility assay system wherein we can image the movement of fluorescently labeled myosin filaments over actin filaments fixed to the surface. We are examining the copolymerization of NM2A and NM2Bin vitro and wish to understand the dynamics of filament formation. embly. Optical trapping studies reveal that NM2A and NM2B are not processive as single molecules. Bipolar filaments of NM2B containing about 30 myosin molecules move processively along actin filaments attached to the surface. We can also image the movement of unlabeled NM2 filaments using iSCAT microscopy. We have reexamined the activity of an N93K mutant of NM2A which we previously published was inactive with regards to ATPase activity and in vitro motility. Recent work suggests that if properly expressed in Sf9 cells, the protein has substantial ATPase activity. We believe that the reason for the previous determination of inactivity may be related to problems in properly folding the molecule and this may explain some of the disease phenotypes in humans bearing this mutation. In collaboration with others, we have obtained high resolution structures of both NM2A and NM2B bound to actin in the presence and absence of ADP using cryo-electron microscopy and have crystallized NM2A in the the presence of ATP analogs. This will give us the ability to observe high resolution structures of myosin in all four cross-bridge states. We also have solved the structure of the folded, autoinhibited form of both smooth muscle myosin and nonmuscle myosin 2B at less than 4 Angstrom resolution. This has allowed us to determine the structural basis for this 10S structure and to speculate on why phosphorylation activates the myosin. A collaborating lab identified an alternatively spliced variant of NM2A containing 21 additional amino acids in loop 2. We expressed this myosin in Sf9 cells and have examined the effect of this splicing on enzymatic activity, motility and filament formation. Preliminary data suggest that the spliced form is much less active.