Abstract
Elucidation of x-ray crystal structures for the S1 subfragment of myosin afforded atomic resolution of the nucleotide and actin binding sites of the enzyme. The structures have led to more detailed hypotheses regarding the mechanisms by which force generation is coupled to ATP hydrolysis. However, the three-dimensional structure of double-headed myosin consisting of two S1 subfragments has not yet been solved. Therefore, to investigate the overall shape and relative orientations of the two heads of myosin, we performed small-angle x-ray and neutron scattering measurements of heavy meromyosin containing all three light chains (LC(1-3)) in solution. The resulting small-angle scattering intensity profiles were best fit by models of the heavy meromyosin head-tail junction in which the angular separation between heads was less than 180 degrees. The S1 heads of the best fit models are not related by an axis of symmetry, and one of the two S1 heads is bent back along the rod. These results provide new information on the structure of the head-tail junction of myosin and indicate that combining scattering measurements with high resolution structural modeling is a feasible approach for investigating myosin head-head interactions in solution.
Highlights
Myosin II, the major contractile protein of muscle, consists of two globular catalytic domains, referred to as S1 subfragments, attached to a long coiled-coil rod
Experimental smallangle scattering intensity profiles were reproduced by simple models in which the S1 heads were joined asymmetrically to a coiled-coil rod
In contrast to heavy meromyosin (HMM) produced by tryptic digestion, HMM produced by mild ␣-chymotryptic digestion is relatively homogenous [15]
Summary
Myosin II, the major contractile protein of muscle, consists of two globular catalytic domains, referred to as S1 subfragments, attached to a long coiled-coil rod. Insights into the mechanisms of contraction were provided by x-ray crystal structures of individual S1 fragments from several myosin isoforms [1,2,3] From these studies it is apparent that each S1 can be divided into two subdomains; that is, a globular motor domain that contains separate actin and nucleotide binding sites and an unusual ϳ8.5-nm ␣-helical segment, the so-called light chain binding domain, which connects the motor domain to the coiled-coil rod. In smooth and invertebrate myosins, activation of ATPase activity is dependent on events occurring at this junction, such as Ca2ϩ binding or phosphorylation of the regulatory light chains, accessory proteins that bind the light chain binding domain near the S1/S2 junction For these regulated myosins, activation appears to involve a significant conformational change that encompasses both head-head and head-tail interactions [5]. Experimental smallangle scattering intensity profiles were reproduced by simple models in which the S1 heads were joined asymmetrically to a coiled-coil rod
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