Intrastrand Cross-Linked Actin between Gln-41 and Cys-374. III. Inhibition of Motion and Force Generation with Myosin

Abstract

Structural and functional properties of intrastrand, ANP (N-(4-azido-2-nitrophenyl)-putrescine) cross-linked actin filaments, between Gln-41 and Cys-374 on adjacent monomers, were examined for several preparations of such actin. Extensively cross-linked F-actin (with 12% un-cross-linked monomers) lost at 60 degrees C the ability to activate myosin ATPase at a 100-fold slower rate and unfolded in CD melting experiments at a temperature higher by 11 degrees C than the un-cross-linked actin. Electron microscopy and image reconstruction of these filaments did not reveal any gross changes in F-actin structure but showed a change in the orientation of subdomain 2 and a decrease in interstrand connectivity. Rigor and weak (in the presence of ATP) myosin subfragment (S1) binding and acto-S1 ATPase did not show major changes upon 50% and 90% ANP cross-linking of F-actin; the Kd and Km values were little affected by the cross-linking, and the Vmax decreased by 50% for the extensively cross-linked actin. The cross-linking of actin (50%) decreased the mean speed and the number of sliding filaments in the in vitro motility assays by approximately 35% while the relative force, as measured by using external load in these assays, was inhibited by approximately 25%. The mean speed of actin filaments decreased with the increase in their cross-linking and approached 0 for the 90% cross-linked actin. Also examined were actin filaments reassembled from cross-linked and purified ANP cross-linked dimers, trimers, and oligomers. All of these filaments had the same acto-S1 ATPase and rigor S1 binding properties but different behavior in the in vitro motility assays. Filaments made of cross-linked dimers moved at approximately 50% of the speed of the un-cross-linked actin. The movement of filaments made of cross-linked trimers was inhibited more severely, and the oligomer-made filaments did not move at all. These results show the uncoupling between force generation and other events in actomyosin interactions and emphasize the role of actin filament structure and dynamics in the contractile process.

The crystal structure of a cross-linked actin dimer suggests a detailed molecular interface in F-actin

“…Previously, we reported that an actin longitudinal dimer can be stabilized by cross-linking the Gln-41 and Cys-374 residues of two adjacent actin protomers with a heterobifunctional photo-activated reagent, ANP (21,31,36). The purified ANP cross-linked dimers can be readily reassembled into filaments (in the presence of Mg 2ϩ ).…”

“…The longitudinal dimer of rabbit skeletal actin was prepared by cross-linking Gln-41 and Cys-374 residues of two adjacent actin protomers with a photo-activated heterobifunctional reagent N-(4-azido-2-nitrophenyl) putrescine (ANP), as described earlier (21,31). To decrease the amount of higher-order actin oligomers, cross-linking was carried out with copolymers made from equal amounts of ANP-labeled (by means of transglutaminase reaction) and unlabeled actin.…”

Effects of post-translational modifications catalysed by pollen transglutaminase on the functional properties of microtubules and actin filaments

“…Rather than forming true microtubules, monomer aggregates could induce the formation of microtubule bundles, not necessarily formed only by covalent aggregates of tubulin but also by monomeric tubulin "entrapped" into the aggregates. [39,40]. In addition, the TGase-mediated intramolecular cross-linking between Gln 41 and Lys 50 disturbed the structure of G-actin but apparently had small effects on actin polymerization and on myosin interaction [32] or could even activate the ATPase activity of myosin subfragment 1 [9].…”

Multiple crystal structures of actin dimers and their implications for interactions in the actin filament

“…The interface through which they interact is the longitudinal interface. While variable twist angles of filaments have been well documented (Egelman et al, 1982;Stokes & DeRosier, 1987;Kim, Bobkova et al, 1998;Galkin et al, 2001), in general the two protofilaments in the F-actin filament are staggered so that two adjacent monomers from different protofilaments are related to each other by a rotation of about $166…”

“…Defined oligomers of actin can be obtained by limited cross-linking in the filamentous state, followed by depolymerization and purification. Chemical strategies have been developed for specific cross-linking in either the longitudinal or lateral directions Kim, Bobkova et al, 1998;Kim, Phillips et al, 1998;Knight & Offer, 1978). The first structural study of a cross-linked actin oligomer was of a trimer held together by two lateral cross-links, with polymerization blocked by a gelsolin segment 1 (GS1) bound to each protomer (Dawson et al, 2003).…”