Abstract
The actin binding and ATPase properties, as well as the functional domain structure of chick brain myosin-V, a two-headed, unconventional myosin, is reported here. Compared to conventional myosin from skeletal muscle, brain myosin-V exhibits low K-EDTA- and Ca-ATPase activities (1.8 and 0.8 ATP/s per head). The physiologically relevant Mg-ATPase is also low (approximately 0.3 ATP/s), unless activated by the presence of both F-actin and Ca2+ (Vmax of 27 ATP/s). Ca2+ stimulates the actin-activated Mg-ATPase over a narrow concentration range between 1 and 3 microM. In the presence of saturating Ca2+ and 75 mM KCl, surprisingly low concentrations of F-actin activate the Mg-ATPase in a hyperbolic manner (KATPase of 1.3 microM). Brain myosin-V also binds with relatively high affinity (compared to other known myosins) to F-actin in the presence of ATP, as assayed by cosedimentation. Digestion of brain myosin-V with calpain yielded a 65-kDa head domain fragment that cosediments with actin in an ATP-sensitive manner and a 80-kDa tail fragment that does not interact with F-actin. The 80-kDa fragment results from cleavage one residue beyond the proline-, glutamate-, serine-, threonine-rich region. Our data indicate that the Mg-ATPase cycle of brain myosin-V is tightly regulated by Ca2+, probably via direct binding to the calmodulin light chains in the neck domain, which like brush border myosin-I, results in partial (approximately 30%) dissociation of the calmodulin associated with brain myosin-V. The effect of Ca2+ binding, which appears to relieve suppression by the neck domain, can be mimicked by calpain cleavage near the head/neck junction.
Highlights
The conventional myosins responsible for processes such as muscle contraction and cytokinesis have been intensively studied, relatively little is known about the properties of the unconventional myosins
The class V myosins share a common domain structure consisting of an N-terminal head domain containing the actin-binding and ATP hydrolysis sites, an extended neck domain containing six IQ motifs, and a tail domain consisting of a region predicted to form coiled coils attached to a globular region of unknown function [7, 8]
In addition to myosin-II and myosin-I, which have been extensively studied as native proteins, there is enough biochemical data on BM-V to allow for an initial description of the domain functions and mechanochemical properties of a class V myosin, as well as a comparison with specific class I and class II myosins
Summary
Materials—Electrophoresis chemicals, molecular mass standards, imidazole, ATP (grade II), EDTA, EGTA, dithiothreitol (DTT), aprotinin, benzamidine, bovine brain CaM, and rabbit skeletal muscle calpain were purchased from Sigma. Proteins—Myosin-V was purified from chick brain essentially as described by Cheney et al [7], except for a change in the initial centrifugations from 18,000 to 40,000 ϫ g and the addition of the protease inhibitors Pefabloc (1 mM), benzamidine (1 mM), and aprotinin (2 g/ ml) These modifications and improved assay conditions resulted in higher yields and higher maximum ATPase activity than previously obtained. The binding of calpain-generated fragments to actin was analyzed by incubating F-actin (9 M) and BM-V (0.1 M) or fragments in 10 mM imidazole, pH 7.4, containing 75 mM KCl, 2.5 mM MgCl2, 0.1 mM EGTA, and 2 mM DTT, in the presence or absence of 2 mM ATP, for 10 min at room temperature. Protein concentrations of mixtures were determined by the method of Bradford [22]
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