Transglutaminase-induced cross-linking between subdomain 2 of g-actin and the 636-642 lysine-rich loop of myosin subfragment 1

Luba Eligula, Li Chuang, Martin L. Phillips, Masao Motoki, Katsuya Seguro, Andras Muhlrad*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

25 Scopus citations

Abstract

G-actin was covalently cross-linked with S1 in a bacterial transglutaminase-catalyzed reaction. The crosslinking sites were identified with the help of fluorescent probes and limited proteolysis as the Gln-41 on the DNase I binding loop of subdomain 2 in G-actin and a lysine-rich loop (residues 636-642) on the S1 heavy chain. The same lysine-rich loop was cross-linked to another region of G-actin in a former study (Combeau, C., D. Didry, and M-F. Carlier. 1992. J. Biol. Chem. 267:14038-14048). This indicates the existence of more than one G-actin-S1 complex. In contrast to G-actin, no cross-linking was induced between F-actin and S1 by the transglutaminase reaction. This shows that in F-actin the inner part of the DNase I binding loop, where Gln-41 is located, is not accessible for S1. The cross-linked G-actin-S1 polymerized upon addition of 2 mM MgCl2 as indicated by electron microscopy and sedimentation experiments. The filaments obtained from the polymerization of cross-linked actin and S1 were much shorter than the control actin filaments. The ATpase activity of the cross-linked S1 was not activated by actin, whereas the K+(EDTA)-activated ATPase activity of S1 was unaffected by the cross-linking. The cross-linking between G-actin and S1 was not influenced by the exchange of the tightly bound calcium to magnesium; however, it was inhibited by the exchange of the actin-bound ATP to ADP. This finding supports the view that the structure of the DNase binding loop in ADP-G-actin is somewhere between the structures of ATP-G-actin and F-actin.

Original languageEnglish
Pages (from-to)953-963
Number of pages11
JournalBiophysical Journal
Volume74
Issue number2 I
DOIs
StatePublished - Feb 1998

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