Cross-linking studies and molecular modeling of the regulated tropomyosin-actin-myosin S1 crossbridge cycle

In vertebrate striated muscles, crossbridges extend from thick filaments which contain myosin to thin filaments which contain actin, tropomyosin (TM), and troponin (TN). Muscle contraction results from cyclic interactions between thin and thick filaments, coupled with enzymatic hydrolysis of adenosine 5-triphosphate (ATP) by myosin. Myosin may be proteolysed to subfragment-1 (S1), a soluble head region, which retains ATPase and actin binding properties of myosin. Kinetic studies indicate weak crossbridges, (A·M·ATP and A·M·ADP·Pi) and strong crossbridges (A·M·ADP and A·M). Actin activates myosin S1 MgATPase by enhancing Pi release from A·M·ADP·Pi. Movement and tension are believed to occur as a power-stroke in strong crossbridges. Actomyosin systems are regulated by Ca2+, which binds to troponin, resulting in a shift of tropomyosin along the actin filament. However, the mechanism of tropomyosin change is controversial, with conflicting evidence for steric and kinetic blocks.; Our understanding of the crossbridge cycle was raised to a new level by recent determinations of crystal structures of rabbit actin and myosin S1. Crystal structures of truncated Dictyostelium S1 with nucleotide analogs simulate different transition states, and recent work has focused on changes in the nucleotide binding pocket and the S1 lever arm during ATP cycling. The initial formation of actin-S1 crossbridges appears to involve electrostatic interactions between S1 sites and N-terminal actin. These sites are identified by carbodiimide cross-linking with EDC to yield 170kD and 180kD bands on SDS polyacrylamide gel electrophoresis. The S1 EDC sites are located in flexible regions without crystal structure.; This project involves combined use of EDC cross-linking experiments and molecular modeling to explore the tropomyosin-actin-S1 crossbridge cycle with special reference to mechanisms of actin activation and tropomyosin regulation. The strategy follows: (1) EDC cross-linking studies of actin-S1 in different nucleotide induced transition states. The date are treated as a kinetic system to characterize binding affinities in different transition states, and study the docking interactions of S1 with actin. (2) EDC cross-linking studies of Ca2+ regulated TN-TM-actin-S1 in different transition states confirm the presence of weak and strong crossbridge, and indicate that strong crossbridges move between 3 discrete states during Ca2+ regulation. (3) An atomic model is constructed for tropomyosin, based on the known low-resolution Cα skeleton. Analysis of this structure reveals axial and azimuthal repeats consistent with a series of electrostatic interactions with actin monomers at equivalent sites along the actin filament, coupled with runs of strongly hydrophobic regions and flexible regions in the tropomyosin core. (4) Molecular modeling of the tropomyosin construct and actin filament leads to a simple picture in which tropomyosin rotates on its long axis across the actin surface, passing through three energetically favorable positional states during Ca2+ regulation. (5) Atomic models are developed for the flexible S1 loops with EDC cross-linked sites. The S1 structures with loops in different conformational states are used to simulate weak and strong crossbridges, consistent with EDC data. The docking interactions of S1 with actin suggest a mechanism for actin activation of myosin ATPase. (6) A molecular model is developed for Ca2+-regulation of tropomyosin-actin-S1 in 3 different positional states, combining the simulated structures for actin-tropomyosin and actin-S1. This model reconciles the previous EM and kinetic evidence.