Structural evidence for non-canonical binding of Ca 2+ to a canonical EF-hand of a conventional myosin

Abstract

We have previously identified a single inhibitory Ca 2+-binding site in the first EF-hand of the essential light chain of Physarum conventional myosin (Farkas, L., Malnasi-Csizmadia, A., Nakamura, A., Kohama, K., and Nyitray, L. (2003) J. Biol. Chem. 278, 27399-27405). As a general rule, conformation of the EF-hand-containing domains in the calmodulin family is "closed" in the absence and "open" in the presence of bound cations; a notable exception is the unusual Ca 2+-bound closed domain in the essential light chain of the Ca 2+-activated scallop muscle myosin. Here we have reported the 1.8 Å resolution structure of the regulatory domain (RD) of Physarum myosin II in which Ca 2+ is bound to a canonical EF-hand that is also in a closed state. The 12th position of the EF-hand loop, which normally provides a bidentate ligand for Ca 2+ in the open state, is too far in the structure to participate in coordination of the ion. The structure includes a second Ca 2+ that only mediates crystal contacts. To reveal the mechanism behind the regulatory effect of Ca 2+, we compared conformational flexibilities of the liganded and unliganded RD. Our working hypothesis, i.e. the modulatory effect of Ca 2+ on conformational flexibility of RD, is in line with the observed suppression of hydrogen-deuterium exchange rate in the Ca 2+-bound form, as well as with results of molecular dynamics calculations. Based on this evidence, we concluded that Ca 2+ -induced change in structural dynamics of RD is a major factor in Ca 2+-mediated regulation of Physarum myosin II activity. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.