Intrastrand Cross-Linked Actin between Gln-41 and Cys-374. I. Mapping of Sites Cross-Linked in F-actin by N-(4-azido-2-nitrophenyl) Putrescine

Abstract

A new heterobifunctional photo-cross-linking reagent, N-(4-azido-2-nitrophenyl)-putrescine (ANP), was synthesized and covalently bound to Gln-41 of rabbit skeletal muscle actin by a bacterial transglutaminase-mediated reaction. Up to 1.0 mol of the reagent was incorporated per mole of G-actin; at least 90% of it was bound to Gln-41 while a minor fraction (about 8%) was attached to Gln-59. The labeled G-actin was polymerized, and the resulting F-actin was intermolecularly cross-linked by irradiation with UV light. The labeled and cross-linked peptides were isolated from either a complete or limited tryptic digest of cross-linked actin. In the limited digest the tryptic cleavage was restricted to arginine by succinylation of the lysyl residues. N-terminal sequencing and mass spectrometry indicated that the cross-linked peptides contained residues 40-50 (or 40-62 in the arginine limited digest) and residues 373-375, and that the actual cross-linking took place between Gln-41 and Cys-374. This latter finding was also supported by the inhibition of Cys-374 labeling with a fluorescent probe in the cross-linked actin. The dynamic length of ANP, between 11.1 and 12.5 A, constrains to that range the distance between the gamma-carboxyl group of Gln-41 in one monomer and the sulfur atom of Cys-374 in an adjacent monomer. This is consistent with the distances between these two residues on adjacent monomers of the same strand in the long-pitch helix in the structural models of F-actin [Holmes, K. C., Popp, D., Gebhard, W., and Kabsch, W. (1990) Nature 347, 44-49 and Lorenz, M., Popp, D., and Holmes, K. C. (1993) J. Mol. Biol. 234, 826-836]. The effect of cross-linking on the function of actin is described in the companion papers.

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

“…Numerous cross-linking experiments provide supporting evidence for residues expected to be proximal based on models of F-actin. Cross-linking data are available both for protomers related in the lateral direction [i.e., sideways between the two helical strands (18)(19)(20)] and for protomers related in the longitudinal direction [i.e., along one vertical strand of the two stranded F-actin helix (19,21)]. Data from synchrotron x-ray radiolysis experiments, probing the reactivity of solvent-accessible residues, are also consistent with structural models (22).…”

“…Longitudinal dimers are of particular interest for structure determination because many actin-binding proteins attach to two longitudinally adjacent protomers in F-actin. Some information is already available regarding the approximate regions of contact between molecules at this interface (18)(19)(20)(21). A more detailed view of the interface would effectively define the strand structure in F-actin and also could constrain models of the complete double-stranded filament.…”

“…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.…”

X-ray scattering study of actin polymerization nuclei assembled by tandem W domains

“…This proposal, however, has not been directly demonstrated. An apparent contradiction is that the cleft between actin subdomains 1 and 3, where the N-terminal helix of the W domain binds, is also thought to participate in intersubunit contacts in the actin filament (11)(12)(13). Therefore, it remains to be demonstrated whether and how tandem W domains stabilize actin monomers into a filament-like structure.…”