Structural Evidence for Non-canonical Binding of Ca super(2+) to a Canonical EF-hand of a Conventional Myosin

• Published in: The Journal of biological chemistry 2005-12, Vol.280 (50), p.41458-41464
• Main Authors: Debreczeni, Judit E, Farkas, Laszlo, Harmat, Veronika, Hetenyi, Csaba, Hajdu, Istvan, Zavodszky, Peter, Kohama, Kazuhiro, Nyitray, Laszlo
• Format: Article
• Language: English
• Subjects: Physarum
• ISSN: 0021-9258; 1083-351X

We have previously identified a single inhibitory Ca super(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 super(2+)-bound closed domain in the essential light chain of the Ca super(2+)-activated scallop muscle myosin. Here we have reported the 1.8 Aa resolution structure of the regulatory domain (RD) of Physarum myosin II in which Ca super(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 super(2+) in the open state, is too far in the structure to participate in coordination of the ion. The structure includes a second Ca super(2+) that only mediates crystal contacts. To reveal the mechanism behind the regulatory effect of Ca super(2+), we compared conformational flexibilities of the liganded and unliganded RD. Our working hypothesis, i.e. the modulatory effect of Ca super(2+) on conformational flexibility of RD, is in line with the observed suppression of hydrogen-deuterium exchange rate in the Ca super(2+)-bound form, as well as with results of molecular dynamics calculations. Based on this evidence, we concluded that Ca super(2+)-induced change in structural dynamics of RD is a major factor in Ca super(2+)-mediated regulation of Physarum myosin II activity.