Effect of intramolecular cross-linking between glutamine-41 and lysine-50 on actin structure and function

被引:19
作者
Eli-Berchoer, L
Hegyi, G
Patthy, A
Reisler, E
Muhlrad, A [1 ]
机构
[1] Hebrew Univ Jerusalem, Hadassah Sch Dent Med, Inst Dent Sci, Dept Oral Biol, IL-91120 Jerusalem, Israel
[2] Eotvos Lorand Univ, Dept Biochem, H-1088 Budapest, Hungary
[3] Agr Biotechnol Ctr, H-2101 Godollo, Hungary
[4] Univ Calif Los Angeles, Dept Chem & Biochem, Los Angeles, CA 90095 USA
[5] Univ Calif Los Angeles, Inst Mol Biol, Los Angeles, CA 90095 USA
关键词
D O I
10.1023/A:1005649604515
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
Subdomain 2 of actin is a dynamic segment of the molecule. The cross-linking of Gln-41 on subdomain 2 to Cys-374 on an adjacent monomer in F-actin inhibits actomyosin motility and force generation (Kim et al., 1998; Biochemistry 37, 17,801-17,809). To shed light on this effect, additional modifications of the Gln-41 site on actin were carried out. Both intact G-actin and G-actin cleaved by subtilisin between Met-47 and Gly-48 in the DNase 1 binding loop of subdomain 2 were treated with bacterial transglutaminase. According to the results of Edman degradation, transglutaminase introduced an intramolecular zero-length cross-linking between Gln-41 and Lys-50 in both intact and subtilisin cleaved actins. This cross-linking perturbs G-actin structure as shown by the inhibition of subtilisin and tryptic cleavage in subdomain 2, an allosteric inhibition of tryptic cleavage at the C-terminus and decrease of modification rate of Cys-374. The cross-linking increases while the subtilisin cleavage dramatically decreases the thermostability of F-actin. The Mg- and S1-induced polymerizations of both intact and subtilisin cleaved actins were only slightly influenced by the cross-linking. The activation of S1 ATPase by actin and the sliding speeds of actin filaments in the in vitro motility assays were essentially unchanged by the cross-linking. Thus, although intramolecular cross-linking between Gln-41 and Lys-50 perturbs the structure of the actin monomer, it has only a small effect on actin polymerization and its interaction with myosin. These results suggest that the new cross-linking does not alter the intermonomer interface in F-actin and that changes in actomyosin motility reported for the Gln-41-Cys-374 intrastrand cross-linked actin are not due to decreased flexibility of loop 38-52 but to constrains introduced into the F-actin structure and/or to perturbations at the actin's C-terminus.
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页码:405 / 414
页数:10
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