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

Subdomain 2 of actin is a dynamic segment of the molecule. The cross-linkin g of Gln-41 on subdomain 2 to Cys-374 on an adjacent monomer in F-actin inh ibits actomyosin motility and force generation (Kim et al., 1998; Biochemis try 37, 17,801-17,809). To shed light on this effect, additional modificati ons 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 bindi ng loop of subdomain 2 were treated with bacterial transglutaminase. Accord ing to the results of Edman degradation, transglutaminase introduced an int ramolecular zero-length cross-linking between Gln-41 and Lys-50 in both int act and subtilisin cleaved actins. This cross-linking perturbs G-actin stru cture as shown by the inhibition of subtilisin and tryptic cleavage in subd omain 2, an allosteric inhibition of tryptic cleavage at the C-terminus and decrease of modification rate of Cys-374. The cross-linking increases whil e the subtilisin cleavage dramatically decreases the thermostability of F-a ctin. The Mg- and S1-induced polymerizations of both intact and subtilisin cleaved actins were only slightly influenced by the cross-linking. The acti vation of S1 ATPase by actin and the sliding speeds of actin filaments in t he in vitro motility assays were essentially unchanged by the cross-linking . Thus, although intramolecular cross-linking between Gln-41 and Lys-50 per turbs the structure of the actin monomer, it has only a small effect on act in polymerization and its interaction with myosin. These results suggest th at the new cross-linking does not alter the intermonomer interface in F-act in 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 pe rturbations at the actin's C-terminus.