The dynamics and polarity of actin filaments are controlled by a conformational change coupled to the hydrolysis of adenosine 5'-triphosphate (ATP) by a mechanism that remains to be elucidated. Actin modified to block polymerization was crystallized in the adenosine 5'-diphosphate (ADP) state, and the structure was solved to 1.54 angstrom resolution. Compared with previous ATP-actin structures from complexes with deoxyribonuclease I, profilin, and gelsolin, monomeric ADP-actin is characterized by a marked conformational change in subdomain 2. The successful crystallization of monomeric actin opens the way to future structure determinations of actin complexes with actin-binding proteins such as myosin.

Wiskott-Aldrich syndrome protein (WASP)-homology domain 2 (WH2) is a small and widespread actin-binding motif. In the WASP family, WH2 plays a role in filament nucleation by Arp2͞3 complex. Here we describe the crystal structures of complexes of actin with the WH2 domains of WASP, WASP-family verprolin homologous protein, and WASP-interacting protein. Despite low sequence identity, WH2 shares structural similarity with the N-terminal portion of the actin monomer-sequestering thymosin ␤ domain (T␤). We show that both domains inhibit nucleotide exchange by targeting the cleft between actin subdomains 1 and 3, a common binding site for many unrelated actin-binding proteins. Importantly, WH2 is significantly shorter than T␤ but binds actin with Ϸ10-fold higher affinity. WH2 lacks a C-terminal extension that in T␤4 becomes involved in monomer sequestration by interfering with intersubunit contacts in F-actin. Owing to their shorter length, WH2 domains connected in tandem by short linkers can coexist with intersubunit contacts in F-actin and are proposed to function in filament nucleation by lining up actin subunits along a filament strand. The WH2-central region of WASP-family proteins is proposed to function in an analogous way by forming a special class of tandem repeats whose function is to line up actin and Arp2 during Arp2͞3 nucleation. The structures also suggest a mechanism for how profilin-binding Pro-rich sequences positioned N-terminal to WH2 could feed actin monomers directly to WH2, thereby playing a role in filament elongation.x-ray crystallography ͉ isothermal titration calorimetry ͉ nucleotide exchange T he actin cytoskeleton plays an essential role in many cellular functions, including intracellular transport and the control of cell shape and polarity (1). In the cell, a vast number of actin-binding proteins (ABPs) direct the location, rate, and timing for actin assembly into different structures, such as filopodia, lamellipodia, stress fibers, and focal adhesions. ABPs are commonly multidomain proteins, containing signaling domains and structurally conserved actin-binding motifs. One of the most abundant actin-binding motifs is Wiskott-Aldrich syndrome protein (WASP)-homology domain 2 (WH2) (2). The hematopoietic-specific protein, WASP, and its ubiquitously expressed ortholog N-WASP form part of a family that also includes the three WASP-family verprolin homologous protein (WAVE͞SCAR) isoforms: WAVE1, WAVE2, and WAVE3 (1, 3). Members of this family activate Arp2͞3-dependent actin nucleation and branching in response to signals mediated by Rho-family GTPases. Although the domain structure of these proteins varies, reflecting different modes of regulation, they all share a common C-terminal WH2 central-acidic region (CA region) (Fig. 1A), which constitutes the smallest fragment necessary for Arp2͞3 activation (4). WH2 is also present in members of the WASP-interacting protein (WIP) family, which form complexes with WASP͞N-WASP and modulate their functions in vivo (5, 6). Members of this family include ...

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Actin is the most abundant protein in eukaryotic cells, but its release from cells into blood vessels can be lethal, being associated with clinical situations including hepatic necrosis and septic shock. A homeostatic mechanism, termed the actin-scavenger system, is responsible for the depolymerization and removal of actin from the circulation. During the first phase of this mechanism, gelsolin severs the actin filaments. In the second phase, the vitamin Dbinding protein (DBP) traps the actin monomers, which accelerates their clearance. We have determined the crystal structures of DBP by itself and complexed with actin to 2.1 Å resolution. Similar to its homologue serum albumin, DBP consists of three related domains. Yet, in DBP a strikingly different organization of the domains gives rise to a large actin-binding cavity. After complex formation the three domains of DBP move slightly to ''clamp'' onto actin subdomain 3 and to a lesser extent subdomain 1. Contacts between actin and DBP throughout their extensive 3,454-Å 2 intermolecular interface involve a mixture of hydrophobic, electrostatic, and solventmediated interactions. The area of actin covered by DBP within the complex approximately equals the sum of those covered by gelsolin and profilin. Moreover, certain interactions of DBP with actin mirror those observed in the actin-gelsolin complex, which may explain how DBP can compete effectively with gelsolin for actin binding. Formation of the strong actin-DBP complex proceeds with limited conformational changes to both proteins, demonstrating how DBP has evolved to become an effective actin-scavenger protein.

A nucleotide-dependent conformational change regulates actin filament dynamics. Yet, the structural basis of this mechanism remains controversial. The x-ray crystal structure of tetramethylrhodamine-5-maleimide-actin with bound AMPPNP, a non-hydrolyzable ATP analog, was determined to 1.85-Å resolution. A comparison of this structure to that of tetramethylrhodamine-5-maleimide-actin with bound ADP, determined previously under similar conditions, reveals how the release of the nucleotide ␥-phosphate sets in motion a sequence of events leading to a conformational change in subdo- The actin filament (F-actin) is asymmetric, undergoing net incorporation of ATP-actin monomers to the barbed end and dissociation of ADP-actin monomers from the pointed end (1, 2). This dynamic process, known as actin filament treadmilling, is an essential part of many forms of cell motility. A driving force behind actin treadmilling is the hydrolysis of ATP by actin. However, in vivo treadmilling is further regulated by a number of factors including a battery of actin-binding proteins. Actin-depolymerizing factor/cofilin, for instance, binds preferentially to the ADP-actin monomers that accumulate toward the pointed end of the filament, accelerating their dissociation. Other proteins such as profilin and thymosin-␤4 bind ATPactin with higher affinity than ADP-actin, maintaining a pool of ATP-actin monomers ready for incorporation into the barbed end of the filament. The fact that these proteins can "distinguish" between ATP-and ADP-actin suggests that these two states are structurally different. Consistent with this view, biochemical (3-5), spectroscopic (6 -8), and electron microscopic (9) evidence has suggested that a conformational change in actin subdomain 2 accompanies the hydrolysis of ATP and the release of inorganic phosphate.

The molecular chaperone Hsp27 exists as a distribution of large oligomers that are disassembled by phosphorylation at Ser-15, -78, and -82. It is controversial whether the unphosphorylated Hsp27 or the widely used triple Ser-to-Asp phosphomimic mutant is the more active molecular chaperone in vitro. This question was investigated here by correlating chaperone activity, as measured by the aggregation of reduced insulin or ␣-lactalbumin, with Hsp27 self-association as monitored by analytical ultracentrifugation. Furthermore, because the phospho-mimic is generally assumed to reproduce the phosphorylated molecule, the size and chaperone activity of phosphorylated Hsp27 were compared with that of the phospho-mimic. Hsp27 was triply phosphorylated by MAPKAP-2 kinase, and phosphorylation was tracked by urea-PAGE. An increasing degree of suppression of insulin or ␣-lactalbumin aggregation correlated with a decreasing Hsp27 self-association, which was the least for phosphorylated Hsp27 followed by the mimic followed by the unphosphorylated protein. It was also found that Hsp27 added to pre-aggregated insulin did not reverse aggregation but did inhibit these aggregates from assembling into even larger aggregates. This chaperone activity appears to be independent of Hsp27 phosphorylation. In conclusion, the most active chaperone of insulin and ␣-lactalbumin was the Hsp27 (elongated) dimer, the smallest Hsp27 subunit observed under physiological conditions. Next, the Hsp27 phospho-mimic is only a partial mimic of phosphorylated Hsp27, both in self-association and in chaperone function. Finally, the efficient inhibition of insulin aggregation by Hsp27 dimer led to the proposal of two models for this chaperone activity.

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Our group has previously shown that vasoconstrictors increase net actin polymerization in differentiated vascular smooth muscle cells (dVSMC) and that increased actin polymerization is linked to contractility of vascular tissue (Kim et al., Am J Physiol Cell Physiol 295: C768-778, 2008). However, the underlying mechanisms are largely unknown. Here, we evaluated the possible functions of the Ena/vasodilator-stimulated phosphoprotein (VASP) family of actin filament elongation factors in dVSMC. Inhibition of actin filament elongation by cytochalasin D decreases contractility without changing myosin light-chain phosphorylation levels, suggesting that actin filament elongation is necessary for dVSM contraction. VASP is the only Ena/VASP protein highly expressed in aorta tissues, and VASP knockdown decreased smooth muscle contractility. VASP partially colocalizes with alpha-actinin and vinculin in dVSMC. Profilin, known to associate with G actin and VASP, also colocalizes with alpha-actinin and vinculin, potentially identifying the dense bodies and the adhesion plaques as hot spots of actin polymerization. The EVH1 domain of Ena/VASP is known to target these proteins to their sites of action. Introduction of an expressed EVH1 domain as a dominant negative inhibits stimulus-induced increases in actin polymerization. VASP phosphorylation, known to inhibit actin polymerization, is decreased during phenylephrine stimulation in dVSMC. We also directly visualized, for the first time, rhodamine-labeled actin incorporation in dVSMC and identified hot spots of actin polymerization in the cell cortex that colocalize with VASP. These results indicate a role for VASP in actin filament assembly, specifically at the cell cortex, that modulates contractility in dVSMC.