Alanine-scanning mutagenesis of WH2 domains of VopF reveals residues important for conferring lethality in a Saccharomyces cerevisiae model

VopF, the type III effector molecule, has been implicated in the pathogenesis of non-O1, non-O139 strains of Vibrio cholerae. It is a protein of 530 amino acids, comprises of one formin homology 1-like (FH1-like) domain and three WASP homology 2 (WH2) domains. Previous works have demonstrated that WH2 domains are crucial for VopF function as a modulator of cellular actin homeostasis. Furthermore, domain deletion analysis also suggests that VopF variant constituted with only WH2 domain 3 is more efficient in restricting the growth of budding yeast than its congeners containing either only domain 1 or domain 2. Interestingly, a good degree of sequence diversity is present within each WH2 domain of VopF. In order to ascertain the importance of different amino acids in each WH2 domain, a systemic alanine scanning mutagenesis was employed. Using a yeast model system, the alanine derivatives of each amino acid of WH2 domain 1 and 3 of VopF were examined for growth restricting activity. Taken together, our mutagenesis results reveal the identification of critical residues of WH2 domain 1 and 3 of VopF.     

The C terminus of formin FMNL3 accelerates actin polymerization and contains a WH2 domain-like sequence that binds both monomers and filament barbed ends

Formin proteins are actin assembly factors that accelerate filament nucleation then remain on the elongating barbed end and modulate filament elongation. The formin homology 2 (FH2) domain is central to these activities, but recent work has suggested that additional sequences enhance FH2 domain function. Here we show that the C-terminal 76 amino acids of the formin FMNL3 have a dramatic effect on the ability of the FH2 domain to accelerate actin assembly. This C-terminal region contains a WASp homology 2 (WH2)-like sequence that binds actin monomers in a manner that is competitive with other WH2 domains and with profilin. In addition, the C terminus binds filament barbed ends. As a monomer, the FMNL3 C terminus inhibits actin polymerization and slows barbed end elongation with moderate affinity. As a dimer, the C terminus accelerates actin polymerization from monomers and displays high affinity inhibition of barbed end elongation. These properties are not common to all formin C termini, as those of mDia1 and INF2 do not behave similarly. Interestingly, mutation of two aliphatic residues, which blocks high affinity actin binding by the WH2-like sequence, has no effect on the ability of the C terminus to enhance FH2-mediated polymerization. However, mutation of three successive basic residues at the C terminus of the WH2-like sequence compromises polymerization enhancement. These results illustrate that the C termini of formins are highly diverse in their interactions with actin.     

Structural basis of actin sequestration by thymosin-beta4: implications for WH2 proteins

The WH2 (Wiscott-Aldridge syndrome protein homology domain 2) repeat is an actin interacting motif found in monomer sequestering and filament assembly proteins. We have stabilized the prototypical WH2 family member, thymosin-beta4 (Tbeta4), with respect to actin, by creating a hybrid between gelsolin domain 1 and the C-terminal half of Tbeta4 (G1-Tbeta4). This hybrid protein sequesters actin monomers, severs actin filaments and acts as a leaky barbed end cap. Here, we present the structure of the G1-Tbeta4:actin complex at 2 A resolution. The structure reveals that Tbeta4 sequesters by capping both ends of the actin monomer, and that exchange of actin between Tbeta4 and profilin is mediated by a minor overlap in binding sites. The structure implies that multiple WH2 motif-containing proteins will associate longitudinally with actin filaments. Finally, we discuss the role of the WH2 motif in arp2/3 activation.     

Two verprolin homology domains increase the Arp2/3 complex-mediated actin polymerization activities of N-WASP and WAVE1 C-terminal regions

WASP family proteins induce actin polymerization through a C-terminal verprolin homology, cofilin homology, and acidic (VCA) region by activating the Arp2/3 complex. The N-WASP VCA region is the most potent activator of the Arp2/3 complex. In addition, full-length WAVE1 and a WAVE1 VCA fragment show differential activity. The mechanisms underlying these differences are poorly understood. We examined the activities of various N-WASP and WAVE1 VCA mutant proteins with several types of fusion moieties. When fused to GST, maltose-binding protein, or the WAVE1 proline-rich domain, N-WASP VCA and WAVE1 VCA mutant proteins with two V motifs showed stronger activities than wild-type WAVE1 VCA with one V motif, demonstrating the importance of two V motifs for strong VCA activity. A WAVE1 VCA fragment tagged with six histidines (His) showed markedly reduced activity compared to GST-fused VCA, whereas His-tagged N-WASP VCA showed similar activity to GST-fused VCA. An additional V motif failed to enhance WAVE1 VCA activity in the His-tagged form. Thus, the WAVE1 VCA fragment may exist in an unfavorable conformation to activate the Arp2/3 complex, implying the existence of a structural difference between WAVE1 and N-WASP VCAs in addition to the number of V motifs.     

Clustering of VASP actively drives processive, WH2 domain-mediated actin filament elongation

Vasodilator-stimulated phosphoprotein (VASP) is a key regulator of dynamic actin structures like filopodia and lamellipodia, but its precise function in their formation is controversial. Using in vitro TIRF microscopy, we show for the first time that both human and Dictyostelium VASP are directly involved in accelerating filament elongation by delivering monomeric actin to the growing barbed end. In solution, DdVASP markedly accelerated actin filament elongation in a concentration-dependent manner but was inhibited by low concentrations of capping protein (CP). In striking contrast, VASP clustered on functionalized beads switched to processive filament elongation that became insensitive even to very high concentrations of CP. Supplemented with the in vivo analysis of VASP mutants and an EM structure of the protein, we propose a mechanism by which membrane-associated VASP oligomers use their WH2 domains to effect both the tethering of actin filaments and their processive elongation in sites of active actin assembly.     

Structure, dynamics, and binding thermodynamics of the v-Src SH2 domain: implications for drug design

SH2 domains provide fundamental recognition sites in tyrosine kinase-mediated signaling pathways which, when aberrant, give rise to disease states such as cancer, diabetes, and immune deficiency. Designing specific inhibitors that target the SH2 domain-binding site, however, have presented a major challenge. Despite well over a decade of intensive research, clinically useful SH2 domain inhibitors have yet to become available. A better understanding of the structural, dynamic, and thermodynamic contributions to ligand binding of individual SH2 domains will provide some insight as to whether inhibitor development is possible. We report the first high resolution solution structure of the apo-v-Src SH2 domain. This is accompanied by the analysis of backbone dynamics and pK(a) values within the apo- and peptide-bound states. Our results indicate that the phosphotyrosine (pY) pocket is tightly structured and hence not adaptable to exogenous ligands. On the other hand, the pocket which accommodates residues proximal and C-terminal of the pY (pY + 3) or so-called specificity determining region, is a large dynamic-binding surface. This appears to allow a high level of promiscuity in binding. Binding of a series of synthetic, phosphotyrosyl, peptidomimetic compounds designed to explore interactions in the pY + 3 pocket further demonstrates the ability of the SH2 domain to accommodate diverse ligands. The thermodynamic parameters of these interactions show dramatic enthalpy/entropy compensation. These data suggest that the v-Src SH2 domain does not have a highly specific secondary-binding site, which clearly presents a major hurdle to design selective inhibitors.     

The direction of actin polymerization for vesicle fission suggested from membranes tubulated by the EFC/F-BAR domain protein FBP17

Actin polymerization mediated by the Arp2/3 complex is essential for membrane tubulation, vesicle formation and fission during clathrin-dependent endocytosis. However, the mechanism by which the polymerizing actin filaments participate in vesicle formation and fission has remained unclear. Our analyses revealed that actin polymerization occurs toward FBP17-induced membrane tubules, which are considered to be generated during endocytic vesicle formation. The tubulated membrane between the future endocytic vesicle and the plasma membrane is proposed to form an arc upon scission of the endocytic vesicle. Therefore, the actin polymerization toward the tubulated membrane may be gradually converted to those toward both the vesicles and the plasma membrane.     

MAP, a protein interacting with a tumor suppressor, merlin, through the run domain

Merlin (or schwannomin) is a tumor suppressor encoded by the neurofibromatosis type 2 gene. Many studies have suggested that merlin is involved in the regulation of cell growth and proliferation through interactions with various cellular proteins. To better understand the function of merlin, we tried to identify the proteins that bind to merlin using the yeast two-hybrid screening. Characterization of the positive clones revealed a protein of 749 amino acids named merlin-associated protein (MAP), which showed wide tissue distribution in Northern blot analysis. Sequence analysis revealed that MAP is a potential homologue of a yeast check-point protein, BUB2, and contains TBC, SH3, and RUN domains, thereby implicating its role in the Ras-like GTPase signal pathways. MAP and merlin were directly associated in vitro and in vivo, and colocalized in NIH3T3 cells. The RUN domain of MAP and the C-terminus of merlin appeared to be responsible for their interaction. MAP decreased the AP-1-dependent promoter activity additively with merlin in NIH3T3 cells. In addition, merlin and MAP synergistically reduced the colony formation of NIH3T3 cells. These results suggest that MAP may play a cooperative role in the merlin-mediated growth suppression of cells.     

RhoA-mediated MLC2 regulates actin dynamics for cytokinesis in meiosis

During oocyte meiosis, the bipolar spindle forms in the central cytoplasm and then migrates to the cortex. Subsequently, the oocyte extrudes the polar body through two successive asymmetric divisions, which are regulated primarily by actin filaments. Myosin light chain2 (MLC2) phosphorylation plays pivotal roles in smooth muscle contraction, stress fiber formation, cell motility and cytokinesis. However, whether MLC2 phosphorylation participates in the oocyte polarization and asymmetric division has not been clarified. The present study investigated the expression and functions of MLC2 during mouse oocyte meiosis. Our result showed that p-MLC2 was localized in the oocyte cortex, with a thickened cap above the chromosomes. Meanwhile, p-MLC2 was also localized in the poles of spindle. Disruption of MLC2 activity by MLC2 knock down (K_D) caused the failure of polar body extrusion. Immunofluorescent staining showed that a large proportion of oocytes arrested in telophase stage and failed to undergo cytokinesis after culturing for 12 hours. In the meantime, actin filament staining at oocyte membrane and cytoplasm were reduced in MLC2 K_D oocytes. Finally, we found that the phosphorylation of MLC2 protein levels was decreased after disruption of RhoA activity. Above all, our data indicated that the RhoA-mediated MLC2 regulates the actin organization for cytokinesis during mouse oocyte maturation.     

Grb7-SH2 domain dimerisation is affected by a single point mutation

Growth factor receptor bound protein 7 (Grb7) is an adaptor protein that is co-overexpressed and forms a tight complex with the ErbB2 receptor in a number of breast tumours and breast cancer cell lines. The interaction of Grb7 with the ErbB2 receptor is mediated via its Src homology 2 (SH2) domain. Whilst most SH2 domains exist as monomers, recently reported studies have suggested that the Grb7-SH2 domain exists as a homodimer. The self-association properties of the Grb7-SH2 domain were therefore studied using sedimentation equilibrium ultracentrifugation. Analysis of the data demonstrated that the Grb7-SH2 domain is dimeric with a dissociation constant of approximately 11 M. We also demonstrate, using size-exclusion chromatography, that mutation of phenylalanine 511 to an arginine produces a monomeric form of the Grb7-SH2 domain. This mutation represents the first step in the engineering of a Grb7-SH2 domain with good solution properties for further biophysical and structural investigation.     

Association of alphaB-crystallin, a small heat shock protein, with actin: role in modulating actin filament dynamics in vivo

Disruption of cytoskeletal assembly is one of the early effects of any stress that can ultimately lead to cell death. Stabilization of cytoskeletal assembly, therefore, is a critical event that regulates cell survival under stress. alphaB-crystallin, a small heat shock protein, has been shown to associate with cytoskeletal proteins under normal and stress conditions. Earlier reports suggest that alphaB-crystallin could prevent stress-induced aggregation of actin in vitro. However, the molecular mechanisms by which alphaB-crystallin stabilizes actin filaments in vivo are not known. Using the H9C2 rat cardiomyoblast cell line as a model system, we show that upon heat stress, alphaB-crystallin preferentially partitions from the soluble cytosolic fraction to the insoluble cytoskeletal protein-rich fraction. Confocal microscopic analysis shows that alphaB-crystallin associates with actin filaments during heat stress and the extent of association increases with time. Further, immunoprecipitation experiments show that alphaB-crystallin interacts directly with actin. Treatment of heat-stressed H9C2 cells with the actin depolymerzing agent, cytochalasin B, failed to disorganize actin. We show that this association of alphaB-crystallin with actin is dependent on its phosphorylation status, as treatment of cells with MAPK inhibitors SB202190 or PD98059 results in abrogation of this association. Our results indicate that alphaB-crystallin regulates actin filament dynamics in vivo and protects cells from stress-induced death. Further, our studies suggest that the association of alphaB-crystallin with actin helps maintenance of pinocytosis, a physiological function essential for survival of cells.     

Synaptotagmin-like protein 5: a novel Rab27A effector with C-terminal tandem C2 domains

Synaptotagmin-like proteins 1-4 (Slp1-4) are new members of the carboxyl-terminal-type (C-type) tandem C2 proteins and are classified as a subfamily distinct from the synaptotagmin and the Doc2 families, because the Slp family contains a unique homology domain at the amino terminus, referred to as the Slp homology domain (SHD). We previously showed that the SHD functions as a binding site for Rab27A, which is associated with human hemophagocytic syndrome (Griscelli syndrome) [J. Biol. Chem. 277 (2002) 9212; J. Biol. Chem. 277 (2002) 12432]. In the present study, we identified a novel member of the Slp family, Slp5. The same as other Slp family members, the SHD of Slp5 preferentially interacted with the GTP-bound form of Rab27A and marginally with Rab3A and Rab6A, both in vitro and in intact cells, but not with other Rabs tested (Rab1, Rab2, Rab4A, Rab5A, Rab7, Rab8, Rab9, Rab10, Rab11A, Rab17, Rab18, Rab20, Rab22, Rab23, Rab25, Rab28, and Rab37). However, unlike other members of the Slp family, expression of Slp5 mRNA was highly restricted to human placenta and liver. Expression of Slp5 protein and in vivo association of Slp5 with Rab27A in the mouse liver were further confirmed by immunoprecipitation. The results suggest that Slp5 might be involved in Rab27A-dependent membrane trafficking in specific tissues.     

Interaction of D-lactate dehydrogenase protein 2 (Dld2p) with F-actin: implication for an alternative function of Dld2p

D-Lactate dehydrogenase protein 2 [Yeast 15 (1999) 1377; Biochem. Biophys. Res. Commun. 295 (2002) 910] was initially identified as the actin interacting protein 2 (Aip2p) using a two-hybrid screen to search for proteins that interact with actin [Nat. Struct. Biol. 2 (1995) 28], but no other evidence indicating an interaction between Aip2p and actin cytoskeleton has been reported so far. During our search for the protein conformation modifying activity, we serendipitously identified Aip2p isolated from Saccharomyces cerevisiae as exhibiting an interaction with F-actin both in vitro and in vivo. Incubation with Aip2p facilitated the formation of the circular form of F-actin in vitro, which exhibited an aberrant trypsin susceptibility. Overexpression of Aip2p induced multi-buds in yeast cells, whereas reduced expression interfered with the formation of the cleavage furrow for the cell division, which was rescued by the introduction of wild-type Aip2p. While Aip2p-treated F-actin in the circular form was negligibly stained by rhodamine-labeled phalloidin (rhodamine-phalloidin) in vitro, rhodamine-phalloidin staining profiles in actin interacting protein 2 gene (AIP2)-modified cells suggested a correlation between the conformation of F-actin and the expression of Aip2p in vivo. AIP2-deleted cells became sensitive to osmotic conditions, a hallmark of actin dysfunction. Finally, immunoprecipitation of yeast cells using anti-Aip2p antibody demonstrated that Aip2p associates with actin. These properties suggest that Aip2p may interact with F-actin in vivo and play an important role in the yeast cell morphology.     

PARG1, a protein-tyrosine phosphatase-associated RhoGAP, as a putative Rap2 effector

Rap2 belongs to the Ras family of small GTP-binding proteins, but its specific signaling role is unclear. By yeast two-hybrid screening, we have found that the Caenorhabditis elegans ortholog of Rap2 interacts with a protein containing a Rho-GTPase-activating protein (RhoGAP) domain, ZK669.1a, whose human ortholog PARG1 exhibits RhoGAP activity in vitro. ZK669.1a and PARG1 share a homology region with previously unknown function, designated the ZK669.1a and PARG1 homology (ZPH) region. Here we show that the ZPH region of PARG1 mediates interaction with Rap2. PARG1 interacted with Rap2 in a GTP-dependent manner but not with Ras or Rap1. We also show that PARG1 and its mutant lacking the ZPH region induce typical cytoskeletal changes for Rho inactivation in fibroblasts. Rap2 suppressed this in vivo action of PARG1 but not that of the mutant PARG1. These results suggest that PARG1 is a putative specific effector of Rap2 to regulate Rho.     

EDD, a novel phosphotransferase domain common to mannose transporter EIIA, dihydroxyacetone kinase, and DegV

Using a recently developed program (SCOPmap) designed to automatically assign new protein structures to existing evolutionary-based classification schemes, we identify a evolutionarily conserved domain (EDD) common to three different folds: mannose transporter EIIA domain (EIIA-man), dihydroxyacetone kinase (Dak), and DegV. Several lines of evidence support unification of these three folds into a single superfamily: statistically significant sequence similarity detected by PSI-BLAST; "closed structural grouping" using DALI Z-scores (each protein inside a group finds all other group members with scores higher than those to proteins outside the group) that includes only these proteins sharing a unique alpha-helical hairpin at the C-terminus and excludes all other proteins with similar topology; similar domain fusions connect Dak and DegV, and genomic neighborhood organizations connect Dak and EIIA-man. Finally, both Dak and EIIA-man perform similar phosphotransfer reactions, suggesting a phosphotransferase activity for the DegV-like family of proteins, whose function other than lipid binding revealed in the crystal structure remains unknown.     

Gcn1 and actin binding to Yih1: implications for activation of the eIF2 kinase GCN2

Yeast Yih1 protein and its mammalian ortholog IMPACT, abundant in neurons, are inhibitors of Gcn2, a kinase involved in amino acid homeostasis, stress response, and memory formation. Like Gcn2, Yih1/IMPACT harbors an N-terminal RWD domain that mediates binding to the Gcn2 activator Gcn1. Yih1 competes with Gcn2 for Gcn1 binding, thus inhibiting Gcn2. Yih1 also binds G-actin. Here, we show that Yih1-actin interaction is independent of Gcn1 and that Yih1-Gcn1 binding does not require actin. The Yih1 RWD (residues 1-132) was sufficient for Gcn2 inhibition and Gcn1 binding, but not for actin binding, showing that actin binding is dispensable for inhibiting Gcn2. Actin binding required Yih1 residues 68-258, encompassing part of the RWD and the C-terminal "ancient domain"; however, residues Asp-102 and Glu-106 in helix3 of the RWD were essential for Gcn1 binding and Gcn2 inhibition but dispensable for actin binding. Thus, the Gcn1- and actin-binding sites overlap in the RWD but have distinct binding determinants. Unexpectedly, Yih1 segment 68-258 was defective for inhibiting Gcn2 even though it binds Gcn1 at higher levels than does full-length Yih1. This and other results suggest that Yih1 binds with different requirements to distinct populations of Gcn1 molecules, and its ability to disrupt Gcn1-Gcn2 complexes is dependent on a complete RWD and hindered by actin binding. Modeling of the ancient domain on the bacterial protein YigZ showed peculiarities to the eukaryotic and prokaryotic lineages, suggesting binding sites for conserved cellular components. Our results support a role for Yih1 in a cross-talk between the cytoskeleton and translation.