Mapping of the microvillar 110K-calmodulin complex (brush border myosin I). Identification of fragments containing the catalytic and F-actin-binding sites and demonstration of a calcium ion-dependent conformational change

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Abstract
In intestinal microvilli, the 110K-calmodulin complex is the major component of the cross-bridges which connect the core bundle of actin filaments to the membrane. Our previous work showed that 11-kDa polypeptide can be divided into three functional domains: a 78-kDa fragment that contains the ATPase activity and the ATP-reversible F-actin-binding site, a 12-kDa fragment required for binding calmodulin molecules, and a terminal 20-kDa domain of unknown function [Coluccio, L. M., and Bretscher, A. (1988) J. Cell Biol. 106, 367-374]. By analysis of limited .alpha.-chymotryptic cleavage products, we now show that the molecular organization is very similar to that described for the S1 fragment of myosin. The catalytic site was identified by photoaffinity labeling with [5,6-3H]UTP, and fragments binding F-actin were identified by cosedimentation assays. Cleavage of the 78-kDa fragment yielded major fragments of 32 and 45 kDa, followed by cleavage of the 45-kDa fragment to a 40-kDa fragment. Of these, only the 32-kDa fragment was labeled by [5,6-3H]UTP. Physical characterization revealed that the 45- and 32-kDa fragments exist as a complex that can bind F-actin, whereas the 40-kDa/32-kDa complex cannot bind actin. We conclude that the catalytic site is located in the 32-kDa fragment and the F-actin-binding site is present in the 45-kDa fragment; the ability to bind actin is lost upon further cleavage of the 45-kDa fragment to 40 kDa. Peptide sequence analysis revealed that the 45-kDa fragment lies within the molecule and suggests that the 32-kDa fragment is the amino terminus. Cleavage sites in the 78-kDa fragmetn which generate the 45-, 40-, and 32-kDa peptides are protected when the fragment is bound to F-actin; moreover, in the absence of Ca2+, the 110-kDa polypeptide is not readily cleaved by .alpha.-chymotrypsin to the 78-kDa fragment or beyond. These results suggest that the 12-kDa calmodulin-binding region confers a Ca2+-dependent accessibility to the F-actin-protectable cleavage site in the S1-like domain. This is the first evidence for a Ca2+-regulated structural change in the S1-like domain of the 110K-calmodulin complex.

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