The structure of the adeno-associated virus (AAV-2) has been determined to 3-Å resolution by x-ray crystallography. AAV is being developed as a vector for gene therapy to treat diseases including hemophilia, cancer, and cystic fibrosis. As in the distantly related autonomous parvoviruses, the capsid protein has a ␤-barrel fold, but long loops between the ␤-strands share little structural homology with other parvoviruses, leading to unique surface features. Most prominent are groups of threefold-related peaks, each an intimate association of loops from two neighboring subunits. Mutations affecting cell entry and receptor binding are clustered near the positively charged side of each peak, implicating the region in attachment to the cellular receptor, heparan sulfate proteoglycan. Amino acids involved in antibody binding are in the same general vicinity. The structure will guide rational engineering of vector capsids to tailor cellular targeting and to avoid immediate neutralization by an immune system sensitized by prior exposure to AAV.

Arginine kinase belongs to the family of enzymes, including creatine kinase, that catalyze the buffering of ATP in cells with f luctuating energy requirements and that has been a paradigm for classical enzymological studies. The 1.86-Å resolution structure of its transition-state analog complex, reported here, reveals its active site and offers direct evidence for the importance of precise substrate alignment in the catalysis of bimolecular reactions, in contrast to the unimolecular reactions studied previously. In the transitionstate analog complex studied here, a nitrate mimics the planar ␥-phosphoryl during associative in-line transfer between ATP and arginine. The active site is unperturbed, and the reactants are not constrained covalently as in a bisubstrate complex, so it is possible to measure how precisely they are pre-aligned by the enzyme. Alignment is exquisite. Entropic effects may contribute to catalysis, but the lone-pair orbitals are also aligned close enough to their optimal trajectories for orbital steering to be a factor during nucleophilic attack. The structure suggests that polarization, strain toward the transition state, and acid-base catalysis also contribute, but, in contrast to unimolecular enzyme reactions, their role appears to be secondary to substrate alignment in this bimolecular reaction.

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.

The three-dimensional crystal structure of an arginine kinase transition-state analogue complex has been re®ned at 1.2 A Ê resolution, with an overall R factor of 12.3%. The current model provides a unique opportunity to analyze the structure of a bimolecular (phosphagen kinase) enzyme in its transition state. This atomic resolution structure con®rms in-line transfer of the phosphoryl group and the catalytic importance of the precise alignment of the substrates. The structure is consistent with a concerted proton transfer that has been proposed for an unrelated kinase. Re®nement of anisotropic temperature factors and translation±libration±screw (TLS) analyses led to the identi®cation of four rigid groups and their prevalent modes of motion in the transition state. The relative magnitudes of the mobility of rigid groups are consistent with their proposed roles in catalysis.

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...