Crystal Structures of Monomeric Actin Bound to Cytochalasin D

Abstract

The fungal toxin cytochalasin D (CD) interferes with the normal dynamics of the actin cytoskeleton by binding to the barbed end of actin filaments. Despite its widespread use as a tool for studying actin-mediated processes, the exact location and nature of its binding to actin has not been previously determined. Here we describe two crystal structures of an expressed monomeric actin in complex with CD, one obtained by soaking preformed actin crystals with CD, and the other by cocrystallization. The binding site for CD, in the hydrophobic cleft between actin subdomains 1 and 3, is the same in the two structures. Polar and hydrophobic contacts play an equally important role in CD binding, and six hydrogen bonds stabilize the actin-CD complex. Many unrelated actin-binding proteins and marine toxins target this cleft, and the hydrophobic pocket at the front end of the cleft (viewing actin with subdomain 2 in the upper right corner). CD differs in that it binds to the back half of the cleft. The ability of CD to induce actin dimer formation and actin-catalyzed ATP hydrolysis may be related to its unique binding site, and the necessity to fit its bulky macrocycle into this cleft. Contacts with residues lining this cleft appear to be crucial to capping and/or severing. The cocrystallized actin-CD structure also revealed changes in actin conformation. A rotation of ~6° of the smaller actin domain (subdomains 1 and 2) with respect to the larger domain (subdomains 3 and 4) results in small changes in crystal packing that allow the D-loop to adopt an extended loop structure, instead of being disordered as it is in most crystal structures of actin. We speculate that these changes represent a potential conformation that the actin monomer can adopt on the pathway to polymerization or in the filament.

An actin cytoskeleton with evolutionarily conserved functions in the absence of canonical actin-binding proteins

“…S4B). The crystal structures of actin bound to cytochalasin D and latrunculin B are solved, so we examined the drug-binding pockets of giActin and found several substitutions consistent with reduced drug efficacy (31,32) (Fig. S4 C-E).…”

G-actin regulates the shuttling and PP1 binding of the RPEL protein Phactr1 to control actomyosin assembly

“…Increasing amounts of LatB-actin effectively competed for PP1 binding, and this was substantially impaired in the presence of cytochalasin D, whose binding site on actin overlaps that of the RPEL motif ( Fig. 4D) (Vartiainen et al, 2007;Mouilleron et al, 2008;Nair et al, 2008). Actin and PP1 thus compete for binding to Phactr1.…”

Mutation of Actin Tyr-53 Alters the Conformations of the DNase I-binding Loop and the Nucleotide-binding Cleft

“…There are five other reports of crystal structures of actin complexes with the D-loop in an ordered conformation, not necessarily ␣-helical (see references within Ref. 31), at least one of which (31) resembles the extended D-loop recently observed in F-actin (24). Independent molecular dynamic modeling studies of monomeric actin and trimeric actin (as a model of F-actin) (32)(33)(34) suggest that folded (helical) and unfolded D-loop conformations co-exist in equilibrium in ADP-G-actin, with equivalent free energy, but in the ADP-trimer the folded D-loop is stable at a free energy minimum (34).…”