Sequences, structural models, and cellular localization of the actin- related proteins Arp2 and Arp3 from Acanthamoeba

We cloned and sequenced the two actin-related proteins (Arps) present in the profilin-binding complex of Acanthamoeba (Machesky, L. M., S. J. Atkinson, C. Ampe, J. Vandekerckhove, and T. D. Pollard. 1994, J. Cell Biol. 127:107-115). The sequence of Arp2 is more similar to other Arp2s than to actin, while the sequence of Arp3 is more similar to other Arp3s than to actin. Phylogenetic analysis of all known Arps demonstrates that most group into three major families, which are likely to be shared across all eukaryotic phyla. Together with conventional actins, the Arps form a larger family distinct from structurally related ATPases such as Hsp70's and sugar kinases. Atomic models of the Arps based on their sequences and the structure of actin provide some clues about function. Both Arps have atoms appropriately placed to bind ATP and divalent cation. Arp2, but not Arp3, has a conserved profilin-binding site. Neither Arp has the residues required to copolymerize with actin, but an Arp heterodimer present in the profilin-binding complex might serve as a pointed end nucleus for actin polymerization. Both Acanthamoeba Arps are soluble in cell homogenates, and both are concentrated in the cortex of Acanthamoeba. The cellular concentrations are 1.9 microM Arp2 and 5.1 microM Arp3, substoichiometric to actin (200 microM) but comparable to many actin- binding proteins.     

Tes, a specific Mena interacting partner, breaks the rules for EVH1 binding

The intracellular targeting of Ena/VASP family members is achieved via the interaction of their EVH1 domain with FPPPP sequence motifs found in a variety of cytoskeletal proteins, including lamellipodin, vinculin, and zyxin. Here we show that the LIM3 domain of Tes, which lacks the FPPPP motif, binds to the EVH1 domain of Mena, but not to those of VASP or Evl. The structure of the LIM3:EVH1 complex reveals that Tes occludes the FPPPP-binding site and competes with FPPPP-containing proteins for EVH1 binding. Structure-based gain-of-function experiments define the molecular basis for the specificity of the Tes-Mena interaction. Consistent with in vitro observations, the LIM3 domain displaces Mena, but not VASP, from the leading edge and focal adhesions. It also regulates cell migration through a Mena-dependent mechanism. Our observations identify Tes as an atypical EVH1 binding partner and a regulator specific to a single Ena/VASP family member.     

1H, 13C, and 15N assignment of the muscular LIM protein MLP/CRP3

The family of CRP proteins comprises three members, which are composed of two LIM domains separated by a long linker of more than 50 residues. We determined the structure of the muscle variant, MLP (CRP3), by nuclear magnetic resonance and show that the two LIM domains are independent of each other.     

Activation of the Arp2/3 complex by the Listeria acta protein. Acta binds two actin monomers and three subunits of the Arp2/3 complex

ActA is a bacterially encoded protein that enables Listeria monocytogenes to hijack the host cell actin cytoskeleton. It promotes Arp2/3-dependent actin nucleation, but its interactions with cellular components of the nucleation machinery are not well understood. Here we show that two domains of ActA (residues 85-104 and 121-138) with sequence similarity to WASP homology 2 domains bind two actin monomers with submicromolar affinity. ActA binds Arp2/3 with a K(d) of 0.6 microm and competes for binding with the WASP family proteins N-WASP and Scar1. By chemical cross-linking, ActA, N-WASP, and Scar1 contact the same three subunits of the Arp2/3 complex, p40, Arp2, and Arp3. Interestingly, profilin competes with ActA for binding of Arp2/3, but actophorin (cofilin) does not. The minimal Arp2/3-binding site of ActA (residues 144-170) is C-terminal to both actin-binding sites and shares sequence homology with Arp2/3-binding regions of WASP family proteins. The maximal activity at saturating concentrations of ActA is identical to the most active domains of the WASP family proteins. We propose that ActA and endogenous WASP family proteins promote Arp2/3-dependent nucleation by similar mechanisms and require simultaneous binding of Arp2 and Arp3.     

A conserved amphipathic helix in WASP/Scar proteins is essential for activation of Arp2/3 complex

Members of the Wiskott-Aldrich syndrome protein (WASP) family link Rho GTPase signaling pathways to the cytoskeleton through a multiprotein assembly called Arp2/3 complex. The C-terminal VCA regions (verprolin-homology, central hydrophobic, and acidic regions) of WASP and its relatives stimulate Arp2/3 complex to nucleate actin filament branches. Here we show by differential line broadening in NMR spectra that the C (central) and A (acidic) segments of VCA domains from WASP, N-WASP and Scar bind Arp2/3 complex. The C regions of these proteins have a conserved sequence motif consisting of hydrophobic residues and an arginine residue. Point mutations in this conserved sequence motif suggest that it forms an amphipathic helix that is required in biochemical assays for activation of Arp2/3 complex. Key residues in this motif are buried through contacts with the GTPase binding domain in the autoinhibited structure of WASP and N-WASP, indicating that sequestration of these residues is an important aspect of autoinhibition.     

Delineating the Tes Interaction Site in Zyxin and Studying Cellular Effects of Its Disruption

Focal adhesions are integrin-based structures that link the actin cytoskeleton and the extracellular matrix. They play an important role in various cellular functions such as cell signaling, cell motility and cell shape. To ensure and fine tune these different cellular functions, adhesions are regulated by a large number of proteins. The LIM domain protein zyxin localizes to focal adhesions where it participates in the regulation of the actin cytoskeleton. Because of its interactions with a variety of binding partners, zyxin has been proposed to act as a molecular scaffold. Here, we studied the interaction of zyxin with such a partner: Tes. Similar to zyxin, Tes harbors three highly conserved LIM domains of which the LIM1 domain directly interacts with zyxin. Using different zyxin variants in pull-down assays and ectopic recruitment experiments, we identified the Tes binding site in zyxin and showed that four highly conserved amino acids are crucial for its interaction with Tes. Based upon these findings, we used a zyxin mutant defective in Tes-binding to assess the functional consequences of abrogating the zyxin-Tes interaction in focal adhesions. Performing fluorescence recovery after photobleaching, we showed that zyxin recruits Tes to focal adhesions and modulates its turnover in these structures. However, we also provide evidence for zyxin-independent localization of Tes to focal adhesions. Zyxin increases focal adhesion numbers and reduces focal adhesion lifetimes, but does so independent of Tes. Quantitative analysis showed that the loss of interaction between zyxin and Tes affects the process of cell spreading. We conclude that zyxin influences focal adhesion dynamics, that it recruits Tes and that this interaction is functional in regulating cell spreading.     

Identification of functionally important residues of Arp2/3 complex by analysis of homology models from diverse species

We constructed homology models from the crystal structure of bovine Arp2/3 complex and sequences from six phylogenetically diverse species (Arabidopsis thaliana, Caenorhabditis elegans, Dictyostelium discoideum, Drosophila melanogaster, Saccharomyces cerevisiae, Schizosaccharomyces pombe) representing over 800 million years of evolution and used conserved surface residues to search for functionally important structural elements. The folds of the seven subunits and their core residues are well conserved, as well as residues at subunit interfaces. Only 45% of solvent-exposed surface residues are conserved and only 15% are identical across the seven species. Arp residues expected to interact with nucleotide and with the first and second actin subunits in a daughter filament are conserved and similar to actin. Arp residues required to form an Arp dimer differ from actin and may contribute to the dissociated state of the Arps in the unactivated complex. Conserved patches of surface residues guided us to candidate sites for nucleation promoting factors to interact with Arp3, Arp2, and ARPC3. Other conserved residues were used with experimental constraints to propose how residues on the subunits ARPC1, ARPC2, ARPC4 and ARPC5 might interact with the mother filament at branch junctions.     

The LIM-only protein DRAL/FHL2 binds to the cytoplasmic domain of several alpha and beta integrin chains and is recruited to adhesion complexes

LIM proteins contain one or more double zinc finger structures (LIM domains) mediating specific contacts between proteins that participate in the formation of multiprotein complexes. We report that the LIM-only protein DRAL/FHL2, with four and a half LIM domains, can associate with alpha(3A), alpha(3B), alpha(7A), and several beta integrin subunits as shown in yeast two-hybrid assays as well as after overexpression in human cells. The amino acid sequence immediately following the conserved membrane-proximal region in the integrin alpha subunits or the C-terminal region with the conserved NXXY motif of the integrin beta subunits are critical for binding DRAL/FHL2. Furthermore, the DRAL/FHL2 associates with itself and with other molecules that bind to the cytoplasmic domain of integrin alpha subunits. Deletion analysis of DRAL/FHL2 revealed that particular LIM domains or LIM domain combinations bind the different proteins. These results, together with the fact that full-length DRAL/FHL2 is found in cell adhesion complexes, suggest that it is an adaptor/docking protein involved in integrin signaling pathways.     

Crystal structure of human 53BP1 BRCT domains bound to p53 tumour suppressor

The BRCT (BRCA1 C-terminus) is an evolutionary conserved protein-protein interacting module found as single, tandem or multiple repeats in a diverse range of proteins known to play roles in the DNA-damage response. The BRCT domains of 53BP1 bind to the tumour suppressor p53. To investigate the nature of this interaction, we have determined the crystal structure of the 53BP1 BRCT tandem repeat in complex with the DNA-binding domain of p53. The structure of the 53BP1-p53 complex shows that the BRCT tandem repeats pack together through a conserved interface that also involves the inter-domain linker. A comparison of the structure of the BRCT region of 53BP1 with the BRCA1 BRCT tandem repeat reveals that the interdomain interface and linker regions are remarkably well conserved. 53BP1 binds to p53 through contacts with the N-terminal BRCT repeat and the inter-BRCT linker. The p53 residues involved in this binding are mutated in cancer and are also important for DNA binding. We propose that BRCT domains bind to cellular target proteins through a conserved structural element termed the 'BRCT recognition motif'.     

The conformational state of Tes regulates its zyxin-dependent recruitment to focal adhesions

The function of the human Tes protein, which has extensive similarity to zyxin in both sequence and domain organization, is currently unknown. We now show that Tes is a component of focal adhesions that, when expressed, negatively regulates proliferation of T47D breast carcinoma cells. Coimmunoprecipitations demonstrate that in vivo Tes is complexed with actin, Mena, and vasodilator-stimulated phosphoprotein (VASP). Interestingly, the isolated NH2-terminal half of Tes pulls out alpha-actinin and paxillin from cell extracts in addition to actin. The COOH-terminal half recruits zyxin as well as Mena and VASP from cell extracts. These differences suggest that the ability of Tes to associate with alpha-actinin, paxillin, and zyxin is dependent on the conformational state of the molecule. Consistent with this hypothesis, we demonstrate that the two halves of Tes interact with each other in vitro and in vivo. Using fibroblasts lacking Mena and VASP, we show that these proteins are not required to recruit Tes to focal adhesions. However, using RNAi ablation, we demonstrate that zyxin is required to recruit Tes, as well as Mena and VASP, but not vinculin or paxillin, to focal adhesions.     

Structural Variations in Protein Superfamilies: Actin and Tubulin

Structures of homologous proteins are usually conserved during evolution, as are critical active site residues. This is the case for actin and tubulin, the two most important cytoskeleton proteins in eukaryotes. Actins and their related proteins (Arps) constitute a large superfamily whereas the tubulin family has fewer members. Unaligned sequences of these two protein families were analysed by searching for short groups of family-specific amino acid residues, that we call motifs, and by counting the number of residues from one motif to the next. For each sequence, the set of motif-to-motif residue counts forms a subfamily-specific pattern (landmark pattern) allowing actin and tubulin superfamily members to be identified and sorted into subfamilies. The differences between patterns of individual subfamilies are due to inserts and deletions (indels). Inserts appear to have arisen at an early stage in eukaryote evolution as suggested by the small but consistent kingdom-dependent differences found within many Arp subfamilies and in γ-tubulins. Inserts tend to be in surface loops where they can influence subfamily-specific function without disturbing the core structure of the protein. The relatively few indels found for tubulins have similar positions to established results, whereas we find many previously unreported indel positions and lengths for the metazoan Arps.     

Tolerance to the substitution of buried apolar residues by charged residues in the homologous protein structures

Occurrence and accommodation of charged amino acid residues in proteins that are structurally equivalent to buried non-polar residues in homologues have been investigated. Using a dataset of 1,852 homologous pairs of crystal structures of proteins available at 2A or better resolution, 14,024 examples of apolar residues in the structurally conserved regions replaced by charged residues in homologues have been identified. Out of 2,530 cases of buried apolar residues, 1,677 of the equivalent charged residues in homologues are exposed and the rest of the charged residues are buried. These drastic substitutions are most often observed in homologous protein pairs with low sequence identity (<30%) and in large protein domains (>300 residues). Such buried charged residues in the large proteins are often located in the interface of sub-domains or in the interface of structural repeats, Beyond 7A of residue depth of buried apolar residues, or less than 4% of solvent accessibility, almost all the substituting charged residues are buried. It is also observed that acidic sidechains have higher preference to get buried than the positively charged residues. There is a preference for buried charged residues to get accommodated in the interior by forming hydrogen bonds with another sidechain than the main chain. The sidechains interacting with a buried charged residue are most often located in the structurally conserved regions of the alignment. About 50% of the observations involving hydrogen bond between buried charged sidechain and another sidechain correspond to salt bridges. Among the buried charged residues interacting with the main chain, positively charged sidechains form hydrogen bonds commonly with main chain carbonyls while the negatively charged residues are accommodated by hydrogen bonding with the main chain amides. These carbonyls and amides are usually located in the loops that are structurally variable among homologous proteins.     

Involvement of actin-related proteins in ATP-dependent chromatin remodeling

Actin-related proteins (Arps) and conventional actin are enigmatic components of many chromatin-remodeling enzyme complexes. The yeast INO80 ATP-dependent chromatin-remodeling complex contains stoichiometric amounts of Arp4, Arp5, Arp8, and actin. Here we have revealed functions of Arp5 and Arp8 by analysis of mutants. arp5 Delta and arp8 Delta mutants display an ino80 Delta phenotype. Purification of INO80 complexes from arp5 Delta and arp8 Delta cells shows that protein complexes remain intact but are compromised for INO80 ATPase activity, DNA binding, and nucleosome mobilization. The INO80 (arp8 Delta) complex is strikingly deficient, not only for the Arp8 subunit, but also for Arp4 and actin, suggesting an ordered assembly of Arps. Binding of Arp8 to the INO80 complex requires an N-terminal region of Ino80 adjacent to the conserved ATPase domain. GST-Arp8 binds preferentially to histones H3 and H4 in vitro, suggesting a histone chaperone function. These findings show direct involvement of Arps in the chromatin-remodeling process.     

Vertebrate Arp6, a novel nuclear actin-related protein, interacts with heterochromatin protein 1

Actin-related proteins (Arps) were recently shown to contribute to the organization and regulation of chromatin structures. The nuclear functions of Arps have been investigated principally in budding yeast in which six of the ten Arp subfamilies are localized in the nucleus. In vertebrates, only two isoforms of Arp4 have so far been identified as showing localization to the nucleus. Here we show the predominant nuclear localization of another Arp subfamily, Arp6, in vertebrate cells. Vertebrate Arp6 directly interacted with heterochromatin protein 1 (HP1) orthologs and the two proteins colocalized in pericentric heterochromatin. Yeast Arp6 is involved in telomere silencing, while Drosophila Arp6 is localized in the pericentric heterochromatin. Our data strongly suggest that Arp6 has an evolutionarily conserved role in heterochromatin formation and also provide new insights into the molecular organization of heterochromatin.     

Identification and analysis of novel amino acid sequence repeats and domains in Pyrobaculum aerophilum using computational tools

We have identified four repeats and five domains that are novel in proteins encoded by the Pyrobaculum aerophilum str. IM2 proteome using automated in silico methods. A "repeat" corresponds to a region comprising less than 55 amino acid residues that occurs more than once in the protein sequence and sometimes present in tandem. A "domain" corresponds to a conserved region comprising greater than 55 amino acid residues and may be present as single or multiple copies in the protein sequence. These correspond to (1) 85 amino acid residues AAG domain, (2) 72 amino acid residues GFGN domain, (3) 43 amino acid residues KGG repeat, (4) 25 amino acid residues RWE repeat, (5) 25 amino acid residues RID repeat, (6) 108 amino acid residues NDFA domain, (7) 140 amino acid residues VxY domain, (8) 35 amino acid residues LLPN repeat and (9) 98 amino acid residues GxY domain. A repeat or domain is characterized by specific conserved sequence motifs. We discuss the presence of these repeats and domains in proteins from other genomes and their probable secondary structure.     

Interactions of Isolated C-terminal Fragments of Neural Wiskott-Aldrich Syndrome Protein (N-WASP) with Actin and Arp2/3 Complex

Wiskott-Aldrich syndrome proteins (WASP) are a family of proteins that all catalyze actin filament branching with the Arp2/3 complex in a variety of actin-based motile processes. The constitutively active C-terminal domain, called VCA, harbors one or more WASP homology 2 (WH2) domains that bind G-actin, whereas the CA extension binds the Arp2/3 complex. The VCA·actin·Arp2/3 entity associates with a mother filament to form a branched junction from which a daughter filament is initiated. The number and function of WH2-bound actin(s) in the branching process are not known, and the stoichiometry of the VCA·actin·Arp2/3 complex is debated. We have expressed the tandem WH2 repeats of N-WASP, either alone (V) or associated with the C (VC) and CA (VCA) extensions. We analyzed the structure of actin in complex with V, VC, and VCA using protein crystallography and hydrodynamic and spectrofluorimetric methods. The partial crystal structure of the VC·actin 1:1 complex shows two actins in the asymmetric unit with extensive actin-actin contacts. In solution, each of the two WH2 domains in V, VC, and VCA binds G-actin in 1:2 complexes that participate in barbed end assembly. V, VC, and VCA enhance barbed end depolymerization like profilin but neither nucleate nor sever filaments, in contrast with other WH2 repeats. VCA binds the Arp2/3 complex in a 1:1 complex even in the presence of a large excess of VCA. VCA·Arp2/3 binds one actin in a latrunculin A-sensitive fashion, in a 1:1:1 complex, indicating that binding of the second actin to VCA is weakened in the ternary complex.     

Actin binding to the central domain of WASP/Scar proteins plays a critical role in the activation of the Arp2/3 complex

The Arp2/3 complex nucleates and cross-links actin filaments at the leading edge of motile cells, and its activity is stimulated by C-terminal regions of WASP/Scar proteins, called VCA domains. VCA domains contain a verprolin homology sequence (V) that binds monomeric actin and central (C) and acidic sequences (A) that bind the Arp2/3 complex. Here we show that the C domain binds to monomeric actin with higher affinity (K(d) = 10 microm) than to the Arp2/3 complex (K(d) > 200 microm). Nuclear magnetic resonance spectroscopy reveals that actin binds to the N-terminal half of the C domain and that both the V and C domains can bind actin independently and simultaneously, indicating that they interact with different sites. Mutation of conserved hydrophobic residues in the actin-binding interface of the C domain disrupts activation of the Arp2/3 complex but does not alter affinity for the complex. By chemical cross-linking the C domain interacts with the p40 subunit of the Arp2/3 complex and, by fluorescence polarization anisotropy, the binding of actin and the Arp2/3 complex are mutually exclusive. Our results indicate that both actin and Arp2/3 binding are important for C domain function but that the C domain does not form a static bridge between the two. We propose a model for activation of the Arp2/3 complex in which the C domain first primes the complex by inducing a necessary conformational change and then initiates nucleus assembly by bringing an actin monomer into proximity of the primed complex.     

Armadillo: domain boundary prediction by amino acid composition

The identification and annotation of protein domains provides a critical step in the accurate determination of molecular function. Both computational and experimental methods of protein structure determination may be deterred by large multi-domain proteins or flexible linker regions. Knowledge of domains and their boundaries may reduce the experimental cost of protein structure determination by allowing researchers to work on a set of smaller and possibly more successful alternatives. Current domain prediction methods often rely on sequence similarity to conserved domains and as such are poorly suited to detect domain structure in poorly conserved or orphan proteins. We present here a simple computational method to identify protein domain linkers and their boundaries from sequence information alone. Our domain predictor, Armadillo (http://armadillo.blueprint.org), uses any amino acid index to convert a protein sequence to a smoothed numeric profile from which domains and domain boundaries may be predicted. We derived an amino acid index called the domain linker propensity index (DLI) from the amino acid composition of domain linkers using a non-redundant structure dataset. The index indicates that Pro and Gly show a propensity for linker residues while small hydrophobic residues do not. Armadillo predicts domain linker boundaries from Z-score distributions and obtains 35% sensitivity with DLI in a two-domain, single-linker dataset (within +/-20 residues from linker). The combination of DLI and an entropy-based amino acid index increases the overall Armadillo sensitivity to 56% for two domain proteins. Moreover, Armadillo achieves 37% sensitivity for multi-domain proteins, surpassing most other prediction methods. Armadillo provides a simple, but effective method by which prediction of domain boundaries can be obtained with reasonable sensitivity. Armadillo should prove to be a valuable tool for rapidly delineating protein domains in poorly conserved proteins or those with no sequence neighbors. As a first-line predictor, domain meta-predictors could yield improved results with Armadillo predictions.