Structural Evidence for Non-canonical Binding of Ca2+to a Canonical EF-hand of a Conventional Myosin

Judit É. Debreczeni, László Farkas, Veronika Harmat, Csaba Hetényi, István Hajdú, Péter Závodszky, Kazuhiro Kohama, László Nyitray
2005 Journal of Biological Chemistry  
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
more » ... nd 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. Conventional myosins (class II) have a fundamental role in muscle contraction and in a variety of cellular motilities. The globular heads of myosins consist of the motor domain responsible for the enzymatic activity and actin binding, and the so-called regulatory domain (RD), 4 FIGURE 2. Structure and comparison of the Ca 2؉ binding EF-hands of ELC and CaM. A, octahedral coordination of Ca 2ϩ ion in the first EF-hand of ELC showing an unusual closed conformation. Ca 2ϩ is coordinated by and two water molecules (red dots). Glu-26, which normally provides a bidentate ligand for Ca 2ϩ in the open state, is too far in the structure to participate in coordination of the ion. B, comparison of the Ca 2ϩ binding EF-hand I of the Physarum (red) and scallop myosins (blue; Protein Data Bank accession code 1wdc) and the mutant CaM structure (green; 1y6w) reveals high similarity between the Physarum and the mutant CaM structure. Note the extra turn in the first helix of scallop ELC. r.m.s. deviations of the backbone carbons, after superimposing the scallop and calmodulin structures, are 0.53 Å.
doi:10.1074/jbc.m506315200 pmid:16227209 fatcat:ufusg4iwazhhpp57zpttyeh224