HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 278, Issue 30, 27399-27405, July 25, 2003

This Article Full Text Full Text (PDF) All Versions of this Article: 278/30/27399    most recent M304220200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Farkas, L. Articles by Nyitray, L. Search for Related Content PubMed PubMed Citation Articles by Farkas, L. Articles by Nyitray, L. Social Bookmarking                      What's this?

Localization and Characterization of the Inhibitory Ca²⁺-binding Site of Physarum polycephalum Myosin II^*

László Farkas , András Málnási-Csizmadia , Akio Nakamura , Kazuhiro Kohama  and László Nyitray  ¶

From the Department of Biochemistry, Eötvös Loránd University, Budapest 1117, Hungary and Department of Pharmacology, Gunma University School of Medicine, Maebashi, Gunma 371, Japan

A myosin II is thought to be the driving force of the fast cytoplasmic streaming in the plasmodium of Physarum polycephalum. This regulated myosin, unique among conventional myosins, is inhibited by direct Ca²⁺ binding. Here we report that Ca²⁺ binds to the first EF-hand of the essential light chain (ELC) subunit of Physarum myosin. Flow dialysis experiments of wild-type and mutant light chains and the regulatory domain revealed a single binding site that shows moderate specificity for Ca²⁺. The regulatory light chain, in contrast to regulatory light chains of higher eukaryotes, is unable to bind divalent cations. Although the Ca²⁺-binding loop of ELC has a canonical sequence, replacement of glutamic acid to alanine in the –z coordinating position only slightly decreased the Ca²⁺ affinity of the site, suggesting that the Ca²⁺ coordination is different from classical EF-hands; namely, the specific "closed-to-open" conformational transition does not occur in the ELC in response to Ca²⁺. Ca²⁺- and Mg²⁺-dependent conformational changes in the microenvironment of the binding site were detected by fluorescence experiments. Transient kinetic experiments showed that the displacement of Mg²⁺ by Ca²⁺ is faster than the change in direction of cytoplasmic streaming; therefore, we conclude that Ca²⁺ inhibition could operate in physiological conditions. By comparing the Physarum Ca²⁺ site with the well studied Ca²⁺ switch of scallop myosin, we surmise that despite the opposite effect of Ca²⁺ binding on the motor activity, the two conventional myosins could have a common structural basis for Ca²⁺ regulation.

Received for publication, April 22, 2003

^* This work was supported by National Scientific Research Fund Grant OTKA T32443 and the Japan Society for the Promotion of Science. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Biochemistry, Eötvös Loránd University, Budapest, PázmányP.sétány 1/C, H-1117. Tel.: 361-381-2171; Fax: 361-381-2172; E-mail: nyitray{at}cerberus.elte.hu.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				J. E. Debreczeni, L. Farkas, V. Harmat, C. Hetenyi, I. Hajdu, P. Zavodszky, K. Kohama, and L. Nyitray Structural Evidence for Non-canonical Binding of Ca2+ to a Canonical EF-hand of a Conventional Myosin J. Biol. Chem., December 16, 2005; 280(50): 41458 - 41464. [Abstract] [Full Text] [PDF]