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 ...

Initiation of actin polymerization in cells requires nucleation factors. Here we describe an actinbinding protein, leiomodin, which acted as a strong filament nucleator in muscle cells. Leiomodin shared two actin-binding sites with the filament pointed-end capping protein tropomodulin; a flexible N-terminal region and a leucine-rich repeat domain. Leiomodin also contained a C-terminal extension of 150 residues. The smallest fragment with strong nucleation activity included the leucine-rich repeat and C-terminal extension. The N-terminal region enhanced the nucleation activity 3-fold and recruited tropomyosin, which weakly stimulated nucleation and mediated localization of leiomodin to the middle of muscle sarcomeres. Knocking down leiomodin severely compromised sarcomere assembly in cultured muscle cells, suggesting a role for leiomodin in the nucleation of tropomyosindecorated filaments in muscles.Actin binding proteins suppress the spontaneous nucleation of actin monomers into filaments, so cells use nucleation factors to initiate actin polymerization. In non-muscle cells, the bestcharacterized filament nucleators are Arp2/3 complex and formins (1). Less is known about the initiation of actin filaments in striated and smooth muscle cells, where specialized proteins may be used to assemble and remodel the tropomyosin-decorated filaments.We identified leiomodin (Lmod) as a potential filament nucleator in muscle cells because sequence analysis suggested that it contained at least three actin-binding sites and could possibly recruit three actin monomers to form a polymerization nucleus. Thus, the first ~340 residues of Lmod are ~40% identical to tropomodulin (Tmod) ( fig. S1), a protein that caps actin filament pointed ends (2,3). The N-terminal portion of Tmod is unstructured, except for three helical segments involved in binding tropomyosin (residues 24-35 and 126-135) and actin (residues 65-75) (4). This region of Tmod, caps the pointed end of actin filaments in a tropomyosin-dependent manner (5). Tmod has a second, tropomyosin-independent, actinbinding and capping site within the C-terminal region (residues 160-359) (5), consisting almost †To whom correspondence should be addressed. droberto@mail.med.upenn.edu. 5 Present address: Elan Pharmaceuticals,

Several neurological diseases, includingThe importance of ␣-synuclein to the pathogenesis of Parkinson disease (PD) 4 and the related disorder, dementia with Lewy bodies (DLB), is suggested by its association with Lewy bodies and Lewy neurites, the inclusions that characterize these diseases (1)(2)(3), and demonstrated by the existence of mutations that cause syndromes mimicking sporadic PD and DLB (4 -6). Furthermore, three separate mutations cause early onset forms of PD and DLB. It is particularly telling that duplications or triplications of the gene (7-9), which increase levels of ␣-synuclein with no alteration in sequence, also cause PD or DLB.␣-Synuclein has been reported to be phosphorylated on serine residues, at Ser-87 and Ser-129 (10), although to date only the Ser-129 phosphorylation has been identified in the central nervous system (11,12). Phosphorylation at tyrosine residues has been observed by some investigators (13,14) but not by others (10 -12). Phosphorylation at Ser-129 (p-Ser-129) is of particular interest because the majority of synuclein in Lewy bodies contains this modification (15). In addition, p-Ser-129 was found to be the most extensive and consistent modification in a survey of synuclein in Lewy bodies (11). Results have been mixed from studies investigating the function of phosphorylation using S129A and S129D mutations to respectively block and mimic the modification. Although the phosphorylation mimic was associated with pathology in studies in Drosophila (16) and in transgenic mouse models (17, 18), studies using adeno-associated virus vectors to overexpress ␣-synuclein in rat substantia nigra found an exacerbation of pathology with the S129A mutation, whereas the S129D mutation was benign, if not protective (19). Interpretation of these studies is complicated by a recent study showing that the S129D and S129A mutations themselves have effects on the aggregation properties of ␣-synuclein independent of their effects on phosphorylation, with the S129A mutation stimulating fibril formation (20). Clearly, determination of the role of p-Ser-129 phosphorylation would be helped by identification of the responsible kinase. In addition, identification will provide a pathologically relevant way to increase phosphorylation in a cell or animal model.Several kinases have been proposed to phosphorylate ␣-synuclein, including casein kinases 1 and 2 (10, 12, 21) and members of the G-protein-coupled receptor kinase family (22). In this report, we offer evidence that a member of the polo-like kinase (PLK) family, PLK2 (or serum-inducible kinase, SNK), functions as an ␣-synuclein kinase. The ability of PLK2 to directly phosphorylate ␣-synuclein at Ser-129 is established by overexpression in cell culture and by in vitro reaction with the purified kinase. We show that PLK2 phosphorylates ␣-synuclein in cells, including primary neuronal cultures, using a series of kinase inhibitors as well as inhibition of expression with RNA interference. In addition, inhibitor and knock-out studies in mouse brai...

The adaptor protein missing-in-metastasis (MIM) contains independent F- and G-actin binding domains, consisting, respectively, of an N-terminal 250 aa IRSp53/MIM homology domain (IMD) and a C-terminal WASP-homology domain 2 (WH2). We determined the crystal structures of MIM's IMD and that of its WH2 bound to actin. The IMD forms a dimer, with each subunit folded as an antiparallel three-helix bundle. This fold is related to that of the BAR domain. Like the BAR domain, the IMD has been implicated in membrane binding. Yet, comparison of the structures reveals that the membrane binding surfaces of the two domains have opposite curvatures, which may determine the type of curvature of the interacting membrane. The WH2 of MIM is longer than the prototypical WH2, interacting with all four subdomains of actin. We characterize a similar WH2 at the C terminus of IRSp53 and propose that in these two proteins WH2 performs a scaffolding function.

α-synuclein is the major component of filamentous Lewy bodies found in the brains of patients diagnosed with Parkinson’s disease. Recent studies demonstrate that, in addition to the wild-type sequence, α-synuclein is found in several modified forms, including truncated and phosphorylated species. Although the mechanism by which the neuronal loss in Parkinson’s disease occurs is unknown, aggregation and fibril formation of α-synuclein is considered to be a key pathological feature. In this study we analyze the rates of fibril formation and the monomer-fibril equilibrium for eight disease-associated truncated and phosphorylated α-synuclein variants. Comparison of the relative rates of aggregation reveals a strong monotonic relationship between the C-terminal charge of α-synuclein and the lag time prior to the observation of fibril formation, with truncated species exhibiting the fastest aggregation rates. Moreover, we find that a decrease in C-terminal charge shifts the equilibrium to favor the fibrillar species. An analysis of these findings in the context of linear growth theories suggests that the loss of the charge-mediated stabilization of the soluble state is responsible for the enhanced aggregation rate and increased extent of fibril fraction. Therefore, C-terminal charge is kinetically and thermodynamically protective against α-synuclein polymerization and may provide a target for the treatment of Parkinson’s Disease.

Amino acid sequences of caspases 1, 3, 7, and 8 were aligned with their published three-dimensional (3D) structures. The resultant alignment was used as a template to compare the primary structures of caspases 2, 4-6, and 9-11 to build 3D homology models. The structural models were subsequently refined and validated using structure-activity relationship data obtained from an array of substrate-like inhibitors. All caspases were shown to have identical S1 and catalytic dyad architecture but diverse S2-S4 structures. S2 pockets of these 11 caspases can be briefly categorized into two groups: Csp3, -6, and -7 as one and Csp1, -2, -4, -5, -8, -9, -10, and -11 as the other. S2 pockets of Csp3, -6, and -7 are smaller than those of the other eight caspases, and are limited to binding small P2 residues such as Ala and Val. At the S3 site, the presence of a conserved Arg in all caspases suggests that Glu would be a universally preferred P3 residue. Csp8 and Csp9 have an additional Arg in this pocket that can further enhance the binding of a P3 Glu, whereas Csp2 has a Glu adjacent to the conserved Arg. As such, Csp2 is the only caspase that can accommodate both positively and negatively charged P3. At S4, Csp1, -4, -5, and -11 are closely related with respect to their structures and binder preferences; all have a large hydrophobic pocket and prefer large hydrophobic residues such as Trp. S4 of Csp2, -3, and -7 represents an opposite group with a conformation that is highly specific in binding an Asp. The S4 structures of Csp6, -8, -9, and -10 appear to be hybrids of the two extremes, and have little specificity for any P4. Information revealed from this work provides a guide for designing potent caspase inhibitors with desirable specificity.

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