Localization and characterization of the inhibitory Ca2+-binding site of Physarum polycephalum Myosin II

László Farkas, A. Málnási-Csizmadia, Akio Nakamura, Kazuhiro Kohama, L. Nyitray

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)27399-27405
Number of pages7
JournalJournal of Biological Chemistry
Volume278
Issue number30
DOIs
Publication statusPublished - Jul 25 2003

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Physarum polycephalum
Myosin Type II
Myosins
Binding Sites
Light
Cytoplasmic Streaming
Physarum
EF Hand Motifs
Pectinidae
Plasmodium
Dialysis
Experiments
Divalent Cations
Eukaryota
Alanine
Glutamic Acid
Motor Activity
Fluorescence
Switches
Kinetics

ASJC Scopus subject areas

  • Biochemistry

Cite this

Localization and characterization of the inhibitory Ca2+-binding site of Physarum polycephalum Myosin II. / Farkas, László; Málnási-Csizmadia, A.; Nakamura, Akio; Kohama, Kazuhiro; Nyitray, L.

In: Journal of Biological Chemistry, Vol. 278, No. 30, 25.07.2003, p. 27399-27405.

Research output: Contribution to journalArticle

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