2 SUMMARYLatrunculin A is used extensively as an agent to sequester monomeric actin in living cells. We hypothesize that additional activities of latrunculin A may be important for its biological activity. Our data are consistent with the formation of a one to one stoichiometric complex with equilibrium dissociation constant of 0.2 to 0.4 µM, and provide no evidence that the actin-latrunculin A complex participates in the elongation of actin filaments. Profilin and latrunculin A bind independently to actin, whereas binding of thymosin β 4 to actin is inhibited by latrunculin A.Potential implications of this differential effect on actin-binding proteins are discussed. From a structural perspective, if latrunculin A binds to actin at a site that sterically influences binding by thymosin β 4 , then the observation that latrunculin A inhibits nucleotide exchange on actin implies an allosteric effect on the nucleotide binding cleft. Alternatively, if as previously postulated, latrunculin A binds in the nucleotide cleft of actin, then its ability to inhibit binding by thymosin β 4 is a surprising result that suggests that significant allosteric changes affect the thymosin β 4 binding site. We show that latrunculin A and actin form a crystalline structure with orthorhombic space group P2 1 2 1 2 1 and diffraction to 3.10 Å. A highresolution structure with optimized crystallization conditions should provide insight regarding these remarkable allosteric properties.

An antiparallel actin dimer has been proposed to be an intermediate species during actin filament nucleation. We now show that latrunculin A, a marine natural product that inhibits actin polymerization, arrests polylysine-induced nucleation at the level of an antiparallel dimer, resulting in its accumulation. These dimers, when composed of pyrene-labeled actin subunits, give rise to a fluorescent excimer, permitting detection during polymerization in vitro. We report the crystallographic structure of the polylysine-actin-latrunculin A complex at 3.5-Å resolution. The non-crystallographic contact is consistent with a dimeric structure and confirms the antiparallel orientation of its subunits. The crystallographic contacts reveal that the mobile DNase I binding loop of one subunit of a symmetry-related antiparallel actin dimer is partially stabilized in the interface between the two subunits of a second antiparallel dimer. These results provide a potential explanation for the paradoxical nucleation of actin filaments that have exclusively parallel subunits by a dimer containing antiparallel subunits.Actin filament nucleation occurs very slowly de novo, but it occurs rapidly as a necessary step in actin-based motility (1). The formation of a dimer from monomeric subunits is the most thermodynamically unfavorable nucleation step with an estimated equilibrium dissociation constant of 4.6 M (in contrast to 0.6 mM for conversion of dimer to trimer) in a recent molecular dynamic simulation of nucleation (2). The formation of an effective nucleus may be accelerated in vivo by an actin-binding protein such as gelsolin, which can stabilize dimeric actin, or by a protein complex such as Arp2/3 that is thought to contain two actin-like molecules constrained in an orientation that promotes nucleation (3, 4). Antiparallel actin dimers have been identified as a precursor to actin filament polymerization by covalent cross-linking during polymerization induced with divalent cations (5). A gelsolin-actin complex capable of nucleating filament growth at the slow growing, pointed end of filaments has also been shown by covalent cross-linking to contain two actin subunits in the antiparallel configuration (6). The assumption of an antiparallel configuration of subunits is based on evidence that Cys-374 in the C terminus of actin is the only residue involved in the cross-linking reaction. In contrast, when polymerization is complete, intrafilament cross-linking yields a parallel dimer. More recently, electron microscopy has revealed that newly formed actin filaments show evidence of incorporation of antiparallel dimers. This incorporation results in a branched filament network, implying that the dimers have nucleating activity (7). Interestingly, analysis of a Listeria model of cell motility using high-resolution laser tracking provides evidence that filaments elongate in 5.4 nm steps, consistent with in vivo incorporation of dimeric actin (8).In the current work, we provide evidence that polylysine nucleates actin polymerization by e...

Regulation of the actin cytoskeleton by filament capping proteins is critical to myriad dynamic cellular functions. The ability of these proteins to bind both filaments as well as monomers is often central to their cellular functions. The ubiquitous pointed end capping protein Tmod3 (tropomodulin 3) acts as a negative regulator of cell migration, yet mechanisms behind its cellular functions are not understood. Analysis of Tmod3 effects on kinetics of actin polymerization and steady state monomer levels revealed that Tmod3, unlike previously characterized tropomodulins, sequesters actin monomers with an affinity similar to its affinity for capping pointed ends. Furthermore, Tmod3 is found bound to actin in high speed supernatant cytosolic extracts, suggesting that Tmod3 can bind to monomers in the context of other cytosolic monomer binding proteins. The Tmod3-actin complex can be efficiently cross-linked with 1-ethyl-3-(dimethylaminopropyl)carbodiimide/N-hydroxylsulfosuccinimide in a 1:1 complex. Subsequent tryptic digestion and liquid chromatography/tandem mass spectrometry revealed two binding interfaces on actin, one distinct from other actin monomer binding proteins, and two potential binding sites in Tmod3, which are independent of the previously characterized leucine-rich repeat structure involved in pointed end capping. These data suggest that the Tmod3 isoform may regulate actin dynamics differently in cells than the previously described tropomodulin isoforms.

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