Identification of Functionally Important Residues of Arp2/3 Complex by Analysis of Homology Models from Diverse Species

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1 doi: /j.jmb J. Mol. Biol. (2004) 336, Identification of Functionally Important Residues of Arp2/3 Complex by Analysis of Homology Models from Diverse Species Christopher C. Beltzner 1 and Thomas D. Pollard 1,2 * 1 Department of Molecular Cellular and Developmental Biology, Yale University, P.O. Box , New Haven, CT USA 2 Department of Cell Biology Yale University, P.O. Box , New Haven, CT USA *Corresponding author 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 solventexposed 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. q 2003 Elsevier Ltd. All rights reserved. Keywords: actin; cellular motility; dendritic nucleation; evolution; protein interactions Introduction Since its initial purification from Acanthamoeba by affinity chromatography on profilin-agarose, 1 Arp2/3 complex has been identified in many other organisms and implicated in the initiation of new actin filaments (reviewed by Pollard et al. 2 ). In every case, Arp2/3 complex has been shown to be composed of two actin-related proteins (Arps), a seven-bladed beta propeller (ARPC1), and four other subunits (ARPC2 5) with novel folds. 3 Supplementary data associated with this article can be found at doi: /j.jmb Abbreviations used: Arp, actin-related protein; WASp, Wiskott Aldrich syndrome protein; HUGO, Human Genome Organization; Bt, Bos torus; At, Arabidopsis thaliana; Ce, Caenorhabditis elegans; Dd, Dictyostelium discoideum; Dm, Drosophila melanogaster; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe. address of the corresponding author: thomas.pollard@yale.edu Homology modeling originally suggested that Arp2 and Arp3 have the surface features required to form a dimer and to initiate an actin filament growing in the barbed end direction. 4 Highly purified Arp2/3 complex caps the pointed ends of actin filaments and has weak nucleating activity 5 that is strongly activated by proteins called nucleation promoting factors 6 10 and by pre-existing filaments. 6,11,12 Daughter filaments grow at their barbed ends as 708 branches on the mother filament. 5,13 Branches formed by the purified proteins are identical with those at the leading edge of migrating eukaryotic cells. 14 The cell surface protein ActA of the intracellular bacterium Listeria 15 was the first nucleation promoting factor to be discovered (reviewed by Weaver et al. 16 ). Eukaryotic nucleation promoting factors related to WASp and Scar have a verprolin homology (V; also called WH2 for WASp homology) sequence that binds actin monomers and a C-terminal acidic sequence that binds Arp2/3 complex. A central sequence (called C) connects /$ - see front matter q 2003 Elsevier Ltd. All rights reserved.

2 552 Homology Models of Arp2/3 Complex the V and A motifs. 17 We refer to these regions as sequences or segments rather than domains, because free VCA polypeptides lack secondary structure but fold at least partially when they bind the WASp GTPase binding domain, 18 actin 19 or Arp2/3 complex. 20 All three regions of VCA contribute to efficient activation of Arp2/3 complex. Less active nucleation promoting factors such as App1p, Abp1p and cortactin, have an acidic sequence and an actin filament binding site rather than an actin monomer binding site, 16 but can stabilize actin filament branches. 21 These two classes of activators may cooperate to activate Arp2/3 complex by forming an active ternary complex. 22 Arp2, Arp3, ARPC1 and ARPC3 can be cross-linked to nucleation promoting factors. 22,23 A 2.0 Å crystal structure of bovine Arp2/3 complex 3 revealed that the two Arps are physically separated in the inactive complex. Activation was proposed to involve a conformational change that orients Arp2 and Arp3 like two subunits along the short-pitch helix of an actin filament, a conformation suitable to initiate polymerization in the barbed end direction. Binding to a mother filament and a nucleation promoting factor are both postulated to favor the same active conformation. 19 Arp2/3 complex contacts three consecutive actin subunits along one long-pitch helix of the mother filament in reconstructions of cryo-electron micrographs of branches. 24 Antibodies to the C terminus of ARPC2 inhibit binding of Arp2/3 complex to the side of actin filaments and branching. 25 Arp2, ARPC1, ARPC2 and ARPC5 can be cross-linked to actin subunits at branch points. 26 Investigators have modified individual amino acids of nucleation promoting factors to probe their functions, 19,20 but modifications of Arp2/3 complex have been confined to deletion of whole subunits. Subcomplexes lacking ARPC3 are stable but nucleate filaments inefficiently. 27 Pentameric subcomplexes lacking ARPC1 and ARPC5 are stable but have little nucleation activity. 27 A dimer of ARPC2/ARPC4 is soluble and binds the side of actin filaments similar to the whole complex. 27 Mutagenesis might advance our understanding of the Arp2/3 complex, but this task is daunting given nearly 2000 residues in the complex. Fortunately, actin, nucleation promoting factors and Arp2/3 complex from protozoa, fungi and animals can be used in apparently any combination to reconstitute branching nucleation in biochemical assays (reviewed by Higgs & Pollard 17 ). Since this requires extensive interactions of Arp2/3 complex with multiple partners, we postulate that selective pressures have maintained the structure of contact sites since the divergence of protozoa, fungi and animals between 800 million and 1 billion years ago. Actin is an extreme example of this trend with 94% conservation of sequence between fungal and human actins, presumably due to extensive contacts between polymerized actin subunits and between actin and numerous binding proteins. Arp2/3 complex is less conserved, but we assumed that a survey of conserved surface residues would reveal those with functional significance. To enable this survey, we constructed homology models of Arp2/3 complex from six species (a protozoan, a plant, two fungi and two invertebrates) for comparison with each other and bovine Arp2/3 complex. We assume that residues that interact with partner proteins have less freedom to vary than those that do not. The limited number of conserved residues provided us with many clues about activation, actin filament nucleation and branching. Results and Discussion Construction of homology models and definitions of terms Homology modeling depends on an accurate alignment of model sequences with a reliable template. Bovine Arp2/3 complex (Figure 1) is a good template, since its structure is known at high resolution (with the exception of subdomains 1 and 2 of Arp2) and, where tested, it has biochemical properties similar to Arp2/3 complex from the model organisms. Most of the model sequences are sufficiently similar to the template for reliable alignment, especially elements of secondary structure. Automated sequence alignments based on secondary structure (carried out by What If) were most problematic for surface loops and turns with highly divergent sequences. Gaps in the alignment occurred when structural elements differed in length, large insertions caused uncertainty in the alignment of the surrounding region, or divergence complicated alignment. We rebuilt these gaps by hand when the insertions or deletions comprised six residues or fewer; other gaps were omitted from the model. After rebuilding gaps, the models Figure 1. Ribbon diagram of the crystal structure of inactive bovine Arp2/3 complex, PDB 1K8K. Subunits are named based on the HUGO nomenclature. The models of subdomains 1 and 2 of Arp2 (pink) are based on the uncomplexed actin structure (PDB: 1J6Z).

3 Homology Models of Arp2/3 Complex 553 were refined to remove steric clashes and improve bond geometries. Refinement was intentionally kept to a minimum. The crystal structure of bovine Arp2/3 complex lacked electron density for subdomains 1 and 2 of Arp2, so we used sequence alignments between the seven Arp2 sequences and human a-actin for our analysis. For Figures containing Arp2 we made a homology model of subdomains 1 and 2 of bovine Arp2 using uncomplexed rhodamine ADP actin (PDB 1J6Z) as the template. An important consideration when judging the quality of homology models is their intended use. Since our purpose is to locate potential sites of protein protein interactions and to identify residues favorable for mutagenesis, the most important aspect is the correct positioning of sidechains. Given that the C a positions of core residues of proteins in the same family having. 50% sequence identity deviate less than 1 Å rms, 28 and given that the sequences of many of the subunits of Arp2/3 complex are more than 50% identical with the bovine subunits (Table 1), it is highly likely that core residues are accurately placed in our homology models. This ensures that most exposed residues are correct as well. (See Guex et al. 29 and references within for a more detailed discussion of homology models and their reliability.) We used the sequence alignments employed for model building (Supplementary Material, Figures 1 7) to analyze the conservation of the proteins. For purposes of comparison, we classify each residue as core, subunit interacting, or exposed based on solvent exposure in the bovine crystal structure. Side-chains of core residues are less than 10% exposed to solvent. Subunit interacting residues have 10% or more of their side-chains shielded from solvent by interaction with another subunit. Side-chains of exposed residues are more than 10% exposed to solvent. Side-chain solvent exposure, expressed as a percentage, was determined by comparing the exposure in the protein with the same side-chain conformation in a GLY- XXX-GLY tripeptide, in vacuo. Since hydrogen atoms are not included in the calculation, glycine residues were classified based on the solvent exposure of the alpha carbon. We grouped the amino acids as follows: acidic (Asp, Glu); basic (Lys, Arg, His); uncharged polar (Ser, Thr, Cys, Asn, Gln); non-polar (Gly, Ala, Val, Leu, Ile, Met, Pro); or aromatic (Phe, Tyr, Trp). We use residue numbers for bovine Arp2/3 complex and human a-actin throughout. We use stringent criteria to define phylogenetically conserved residues as those with the same chemical type (i.e. acidic, basic, etc) at a particular position in six of the seven species. Due to gaps some residues are represented in less than seven species. In this case only residues that are chemically equivalent in six of six species are considered conserved. Residues with five out of six, five out of five, or fewer were classified as not conserved, because revision of the structures could result in an alignment where the residue would no longer be considered conserved. Conservation of residues in actin is based on standard sequence alignments with the sequences of the major isoform of actin from each of the seven species. In the text, we indicate conserved residues by capital letters of the single letter amino acid code and non-conserved residues by lower case. Residues identical in all seven species are in bold capital letters. For comparisons between actin and Arp2 or Arp3, we give the residue number, identity and conservation. For example a comparison of residues in actin and Arp2 denoted as 203T-205s refers to a conserved threonine at position 203 in human a-actin and a serine at position 205 of bovine Arp2 that is not conserved among the seven species. Additionally, we denote substitutions between species by the bovine residue number followed by the residue at the corresponding location of the other species. For example, substitution of phenylalanine 17 in bovine Arp2/3 complex for tyrosine at the analogous position in Arabidopsis is given as F17Y. Overall description of models After rebuilding gaps the Drosophila model has 1720 residues, Caenorhabditis elegans has 1700, Dictyostelium has 1677, Saccharomyces cerevisisae has 1669, S. pombe has 1665 and Arabidopsis has 1672, compared with the original bovine structure of 1709 residues. The extended N terminus of ARPC5 is generally incomplete in the models, since the sequences are poorly conserved and vary in length. The Arabidopsis and Dictyostelium models both lack the extended N terminus of ARPC5 that interacts with Arp2. The Arabidopsis ARPC5 sequence has 11 fewer N-terminal residues and it Table 1. Identity and similarity of sequences used for homology models versus bovine Arp2/3 Identity/similarity (versus bovine) (%) Species Arp3 Arp2 ARPC1 ARPC2 ARPC3 ARPC4 ARPC5 Whole complex Dm 80/90 81/88 49/63 74/82 58/75 83/88 52/68 70/80 Ce 76/85 76/85 48/64 70/80 54/69 74/85 42/55 62/74 Dd 69/79 71/79 41/53 40/58 42/60 67/82 36/55 55/68 Sp 60/72 64/75 42/57 48/64 52/67 68/79 26/44 53/67 Sc 63/71 66/76 37/53 41/56 46/63 69/78 27/48 52/65 At 59/72 61/73 41/53 27/48 46/64 61/77 30/50 47/61

4 554 Homology Models of Arp2/3 Complex is not clear if the Arp2 binding region is present. Both fungal models include the N-terminal residues of ARPC5 that bind Arp2, but lack the residues connecting this region to the globular part of the subunit. The models include the short helix inserted between blades 6 and 7 of ARPC1 but not the connecting residues, because they are disordered in the crystal structure and are poorly conserved. A loop consisting of residues of Arp3 was omitted from the models, since they are disordered in the crystal structure and not conserved among the seven species. Other sequences omitted from the models include several loops of more than ten residues: 35 residues in S. cerevisiae and 14 residues in S. pombe in subdomain 4 of Arp3; 19 residues in S. cerevisiae and 17 residues in S. pombe between the first helix and the first strand of the second a/b domain of ARPC2; 11 residues at the C terminus of S. cerevisiae. None of these inserts has significant sequence conservation with known proteins. General features Of 562 core residues, 80.0% are conserved; 20.5% of the conserved core residues are polar (charged and uncharged), 63.8% are non-polar and 15.7% are aromatic. Among the core residues the conservation of each chemical type is roughly the same with the non-polar residues being the highest at 83.0% and the uncharged polar residues being the lowest at 67.8%. Of the conserved charged polar residues (acidic and basic) 86.7% are identical. Of the conserved aromatic residues in the cores only 43.5% are identical and overall only 33.5% of the conserved core residues are identical. This demonstrates how poorly Arp2/3 complex tolerates conservative substitutions of charged polar residues in the protein cores. Residues comprising subunit interfaces are more conserved than residues on the free surfaces. Of the 335 residues at subunit interfaces 69.6% are conserved; 17.2% of the conserved residues are acidic, 22.7% are basic, 15.5% are uncharged polar, 30.9% are non-polar, and 13.7% are aromatic. The conservation of uncharged polar residues is lower at 57.1% than the other chemical types, which vary from 69.9 to 73.6%. Conserved basic, uncharged polar and non-polar residues are equally tolerant to conservative mutations with 41.5%, 36.1% and 36.1% of the conserved residues being identical. Conserved acidic and aromatic residues are less tolerant of conservative changes with 60.0% and 56.3% of the conserved residues being identical. The complementary surfaces of subunit interfaces tend to be equally conserved, while other parts of the subunits have diverged to various degrees (Table 2). For example, at the interface of Arp3 and ARPC3, 85% of the ARPC3 residues and 80% of the Arp3 residues are conserved, while overall only 54% of total ARPC3 residues and 75% of total Arp3 residues are conserved. The subunit interfaces of ARPC1 and ARPC5 are the least conserved. These two subunits depend on each other to bind to the complex. 27 Exposed surface residues Of the 818 solvent-exposed residues in bovine Arp2/3 complex, 144 (17.6%) are acidic, 172 (21.0%) are basic, 195 (23.8%) are uncharged polar, 268 (32.8%) are non-polar, and 39 (4.8%) are aromatic. Less than half (44.6%) of these residues are conserved. Of the conserved exposed residues 16.4% are acidic, 21.9% are basic, 21.4% are uncharged polar, 34.0% are non-polar, and 6.3% are aromatic. Of the exposed residues, aromatic residues are more conserved (59.0%) than other chemical types, which vary from 40.5% to 46.5%. Conservative substitutions are frequent among conserved exposed uncharged polar residues with only 14 of the 79 conserved residues being identical. Overall only 33.2% of the conserved exposed residues (15% of the total surface residues) are identical across the seven species. The following sections explore the functional significance of conserved surface residues. Nucleotide binding to Arp2 and Arp3 The original crystal structure of bovine Arp2/3 complex lacked bound nucleotide but new structures have ADP or ATP bound to both Arps (R. Littlefield, B. Nolen & T.D.P., unpublished results). Hydrolyzable ATP bound to the Arps is required for nucleation of branches. 30,31 These studies agree that the affinity of Arp3 for ATP is in the low micromolar range, but disagree on the Table 2. % Identical and conserved residues at subunit interfaces of Arp2/3 complex Conserved/identical (%) Subunit Arp3/ARPC2 Arp3/ARPC3 Arp2/ARPC4 ARPC1/ARPC4 ARPC1/ARPC5 ARPC2/ARPC4 ARPC4/ARPC5 Arp3 88/46 80/45 Arp2 87/53 ARPC1 54/29 45/18 ARPC2 76/28 74/20 ARPC3 85/23 ARPC4 88/44 56/13 71/35 53/8 ARPC5 45/0 53/11

5 Homology Models of Arp2/3 Complex 555 Figure 2. Evolutionarily conserved residues exposed on the surface of Arp2/3 complex. Homology models of Arp2/ 3 complex were built from the sequences of six phylogenetically diverse species and bovine Arp2/3 complex crystal structure. Conserved residues are the same chemical type in at least six of the seven species. Exposed residues have 10% or more of their side-chain surface area exposed to solvent. (a) Three views of a space filling model of bovine Arp2/3 complex with subunits colored as in Figure 1. (b) Conserved exposed residues are colored blue and subunits are outlined. (c) Conserved exposed residues colored according to proposed functions. Residues identical in all seven species are bold in this legend. Orange, nucleotide binding by Arp2 and Arp3. Dark blue, potential mother filament binding residues on ARPC2 (M-1: Y153, D159, R160, T162, V164, Q183, E187, G188, R189, R190, T194, Q197, F200, S201, E204, P206, L207, E208, T224, F228, P229, R230), on ARPC4 (M-2: L5, L9, R13, R55, N56, E59, K77, Q78, D80, V132, D133, E140, D143, K144, K150, L151), on ARPC1 (M-4: A298, F302) and on ARPC5 (M-5: L46, S85, K87). Cyan, potential mother filament binding residues on ARPC1 (M-3: N17, E19, R20, T21, N52 (D in species other than bovine), D59, S64, N65, R66, N75, Y77, K87, P88, L90, I92, R94, P106, E108, S117 (A in species other than bovine), F125, E126, E128, N129, W131, V133, K135, K139, P152, N153, K165, K174, V176, E177, R179, P184, G186, P190, F191, G192, L194, E197, N213, S215, V237, E255, A340). Red, potential binding sites for C and A-segments of VCA nucleation promoting factors. C-1 (Arp2: I40, I41, R42, K46, I52, L55, M56, L64, S66, N71, D90), C-2 (Arp2: N26, F27, D346, R349, H352, A359), A-1 (Arp3: K228, M327, F328, R329, R333, R334, R337, K340, R341), A-2 (Arp3: A150, W153, R161, F379, M383, L384), and A-3 (ARPC3: P2, H5, S7, N18, E59, I60, K61, R66, I116). Green, potential contact sites between the Arps and between the Arps and the first two subunits of the daughter actin filament (enumerated in Figure 4). Table 3. Comparison of residues involved in nucleotide contacts Actin D12 S15 L17 K19 Q138 D158 V160 R211 K214 E215 T304 Y307 K337 Arp2 D12 T15 F17 K19 Q141 D161 V163 R214 K217 E218 S307 Y310 K351 Arp3 D11 T14 Y16 K18 Q144 D172 V174 k225 K228 E229 S325 F328 R374

6 556 Homology Models of Arp2/3 Complex Figure 3 (legend opposite)

7 Homology Models of Arp2/3 Complex 557 affinity of Arp2 for ATP. The K d is in the low nanomolar range according to LeClainche et al. 30 and in the low micromolar range according to Dayel et al. 31 The affinity of actin is 0.12 nm for Ca-ATP and 1.2 nm for Mg-ATP. 32 To explore nucleotide binding by Arp2 and Arp3 we made a structure-based alignment of the bovine Arps with rabbit skeletal muscle a-actin (PDB 1ATN) and compared the residues known to participate in nucleotide binding to actin. Of the residues present in the recently determined ATP and ADP structures, all are correctly predicted by this method (orange in Figure 2(c)) (R. Littlefield & T.D.P., unpublished results). For subdomains 1 and 2 of Arp2 we used sequence alignments between bovine Arp2 and human a-actin. These residues are shown in italics (Table 3). Most residues making side-chain interactions with ATP are conserved among actin, Arp2 and Arp3. Four residues (D12, K19, D158, and K214) are identical among the three proteins across the sampled species. Of the Arp2 residues included in the bovine crystal structure, the only substitution between the seven species is S307T in C. elegans; otherwise all the residues are identical among the seven species. The only substitution that occurs between actin and residues in the bovine crystal structure of Arp2 is T304-S307. In actin, T304 makes a side-chain hydrogen bond to the carbonyl oxygen of G157. This contact is not maintained by S307 in bovine Arp2; however, G160 is located at the same position as G157 in actin and the hydroxyl group is oriented in the proper direction. This hydrogen bond may simply be weakened in Arp2. Of the Arp2 residues missing from the bovine crystal structure, there are two substitutions between the species; F17Y in Arabidopsis and K351R in S. pombe. This region has two substitutions between Arp2 and actin, one conservative and one non-conservative. The conservative substitution is S15-T15. In actin, the side-chain of this residue makes a hydrogen bond to an oxygen on the betaphosphate. The non-conservative substitution is L17-F17. In actin the side-chain of this residue is near the alpha and beta phosphate groups. Arp3 has several conservative differences from actin among residues that participate in nucleotide binding: S15-T14, R211-k225, T304-S325, Y307- F328, and K337-R374. In actin, R211 forms a hydrogen bond with the side-chain of E215, which hydrogen bonds to the 2 0 -OH of the ribose ring. In bovine Arp3 k225 makes a hydrogen bond with the hydroxyl group of Y16. Y307 interacts with positions 1 and 2 of the adenine ring. K337 is near position 7 of the adenine ring, but does not form a hydrogen bond. In bovine Arp3 R374 is oriented toward the adenine ring and does not make any hydrogen bonds. The contributions of S15 and T304 to nucleotide binding in actin are described above. T14 of Arp3 does not maintain the hydrogen bond of actin S15. S325 of Arp3 does maintain the hydrogen bond of actin T304. Position 225 of Arp3 varies between the species, where Arabidopsis has a K225R substitution, and both fungi have a K225E substitution. L17-Y16 is a non-conservative substitution between actin and Arp3; however, S. cerevisiae Arp3 has L like actin, presumably a reverse substitution. The remaining positions are conserved and the same as actin, except for substitutions of Q144N and V174A in Arabidopsis and E229Q in S. cerevisiae. The lack of nucleotide in the bovine crystal structure and biochemical data showed that both Arps have a lower affinity for nucleotide than actin, although one group reports a significantly lower affinity only for Arp3. Our study does not reveal a basis for a lower affinity of Arp2 for nucleotide, because most residues in direct contact with nucleotide are identical in the seven species of Arp2 and identical with actin. Interactions with other subunits or other features of Arp2 may favor the open conformation captured in the crystals lacking nucleotide. Our study does reveal a potential basis for lower affinity of Arp3 for nucleotide. Five conservative differences and one non-conservative difference between Arp3 and actin are maintained across most of the seven species sampled. While most of these substitutions are conservative, they change the structure of the nucleotide-binding cleft and could contribute to lower affinity. Only one position in Arp3, 225, is not conserved among the species studied. The charge reversal mutations, R to E, found in the fungal species suggest that either this residue has little effect on nucleotide binding or that fungal Arp3 has even lower affinity for nucleotide. The insert from in Arp3 between subdomains 1 and 3 may influence the transition from the open to closed conformations and therefore affect nucleotide affinity. Only 154T, 155S and 161R are conserved in this insert and the length of the insert varies, with Arabidopsis having three fewer residues and Dictyostelium having one less residue. Figure 3. Ribbon diagrams of models of an Arp2 Arp3 short-pitch filament dimer. (a) Two stereo views of the Arp2 Arp3 short-pitch filament dimer with ARPC3 and the first subunit of the daughter actin filament. The molecules were aligned on the Holmes actin filament model with Arp2/3 complex subunits colored as in Figure 1 and actin in blue. The arrowhead indicates a steric clash between subdomain 2 of Arp2 and ARPC3. The arrow indicates a steric clash between subdomain 3 of Arp3 and subdomain 4 of actin. (b) Two stereo views of the Arp2 and Arp3 with ARPC3 from the structure of inactive complex shown in Figure 1. Green, conserved residues with potential to bind the C-segment (C-1, C-2) and the A-segment (A-1, A-2, A-3) of VCA nucleation promoting factors (enumerated in Figure 2(c)).

8 558 Homology Models of Arp2/3 Complex Potential contacts between the Arps and with the daughter actin filament Actin filaments are polar owing to the common orientation of the asymmetrical subunits along the double helical polymer. The ends are designated as barbed and pointed from the arrowhead-like pattern created by bound myosin heads. 33 Subdomains 1 and 3 of actin are exposed at the barbed end. 34 In solution the rate-limiting step for pure actin monomers to initiate a new filament is the formation of a nucleus consisting of an actin trimer. Both possible actin dimers are unstable, but the short-pitch dimer is thought to be more stable than the long-pitch dimer and on the pathway to trimers. 35 We used the actin filament model proposed by Holmes 34 to construct a model of Arp3 (associated with ARPC3), Arp2 and the first subunit of the daughter filament (Figure 3(a)). We positioned Arp2 and Arp3 by creating a least-squares fit of the C a positions of each Arp and actin and noted the residues in the Arps corresponding to the actin residues predicted to make contacts in the Holmes model. We used standard sequence alignments with actin for the parts of Arp2 not in the model. In this simple model subdomain 2 of Arp2 clashes with ARPC3, so in reality some rearrangement is required. This model assumes a conformational change in the inactive complex that rearranges Arp2 and Arp3 to adopt orientations similar to two subunits along the short-pitch helix of an actin filament. 3,24 Arp2 is at the barbed end of the short-pitch Arp2 Arp3 dimer, because that is the arrangement of the Arps in the crystal structure and ARPC3 blocks the pointed end of Arp3. In this model, Arp3 contacts Arp2 and the first actin subunit in the daughter filament, while Arp2 contacts Arp 3 and the first two actin subunits in the daughter filament. The daughter filament nucleus is completed adding an actin monomer to the Arp2 Arp3 dimer. This arrangement is favored over formation of a long-pitch dimer by Arp2 and Arp3, which would require massive rearrangement of the complex. Another model of activation involving the incorporation of one of the Arps into the mother filament 12 is not supported by electron microscopy 24 or observations of branching in real time by fluorescence microscopy. We used this model to identify potential contacts between the Arps (called R sites, green in Figures 2(c), 4(a) and (b)) and between the Arps and the first two actin subunits of the daughter filament (called D sites, green in Figures 2(c), 4(c) (e)). Most surface residues of Arp2 and Arp3 that are proposed to make R site contacts in the short-pitch dimer in the activated complex are conserved among the Arps, but only 58% of these key residues are of the same chemical type as the corresponding residues of actin. Six Arp2 residues and five Arp3 residues conserved among the Arps are the same chemical type as actin (blue in Figure 4(a) and (b)). Three Arp2 residues and five Arp3 residues conserved among the Arps differ in chemical type from the corresponding residues in actin (red in Figure 4(a) and (b)). Three Arp2 residues and five Arp3 residues are not conserved (green in Figure 4(a) and (b)). We propose that these differences in the Arp2 Arp3 dimer interface relative to actin favor the dissociated, inactive conformation of the Arps in spite of their close proximity in the complex. In the absence of nucleation promoting factors, this inactive, dissociated conformation is captured in the crystals. Crystallography, electron microscopy and biochemistry all suggest that elongation, in the barbed end direction, from an Arp2 Arp3 dimer is the most plausible mechanism for Arp2/3 complex to initiate a daughter filament. The Arp residues with potential to contact actin are highly conserved and identical with the corresponding residues of actin. Eleven residues at the barbed end of Arp2 that are predicted to interact with the first subunit in the actin filament (D1 site) are conserved and identical with actin (blue in Figure 4(c)). Eight residues at the barbed end of Arp3 expected to contact actin (D2 site) are conserved and identical with actin (blue in Figure 4(d)). Three Arp2 D1 residues (red in Figure 4(c)) and two Arp3 D2 residues (red in Figure 4(d)) are conserved among the Arps, but differ in chemical type from actin. One D1 residue of Arp2 and one D2 residue of Arp3 are not conserved (green in Figure 4(c) and (d)). Given the low tendency of the Arps to dimerize, we propose that interactions of the first subunit in the daughter filament with the D1 and D2 sites of the two Arps stabilize the active conformation. This provides a mechanism for the actin monomer bound to nucleation promoting factors to contribute to efficient activation of Arp2/3 complex. Arp2 residues forming the D3 site proposed to interact with the pointed end of the second subunit in the daughter filament diverge more from actin than those that contact the first subunit. Five Arp2 D3 residues are conserved and identical with actin (blue in Figure 4(e)). One D3 residue is conserved among the Arp2 proteins but differs chemically from actin (red in Figure 4(e)) and four D3 residues are not conserved among Arp2 proteins (green in Figure 4(e)). Consequently, the first actin subunit of the daughter filament may contribute more than Arp2 to binding the second daughter subunit. Conserved inserts in the Arps predicted to interact with the daughter filament Both Arps have conserved inserts relative to actin, particularly a prominent insert corresponding to actin residues (see Figure 2 of Robinson et al. 3 ). These inserts extend an alphahelix at the base of subdomain 3 and the following extended chain that leads to subdomain 1. These S3 inserts are predicted to interact with the first and second actin subunits of the daughter filament.

9 Homology Models of Arp2/3 Complex 559 Figure 4. Space filling models of Arp2 and Arp3 with residues predicted to participate in interactions between Arp2, Arp3 and actin monomers. Arp2 and Arp3 structures were aligned with human a-actin (PDB: 1ATN) to identify residues corresponding to those mediating interactions between actin monomers in the Holmes actin filament model. Blue, conserved Arp residues of the same chemical type as actin. Red, residues conserved among the seven species of Arp2 or Arp3 but differing in chemical type from actin. Green, residues not conserved among the seven species of Arp2 or Arp3. Pink, conserved residues in the S3 inserts. Cyan: non-conserved residues in the S3 inserts. (a) Back view of Arp2 with R-1 residues predicted to interact with Arp3 in a short-pitch helix. Residues conserved among Arp2 proteins and of the same chemical type as actin (196R-200R, 197G- 201G, 201T p -205N ( p residue 201 is V in bovine and T or S in the other species), 267I-271I, 269M-273V, 270E-274E). Residues conserved among Arp2 proteins but differing in chemical type from actin (199S- 203A, 206R-210F, 266f-270L). Residues that are not conserved (191K-195k, 195E-199l, 268G-272n). (b) Back view of Arp3 with R-2 residues predicted to interact with Arp2 in a short-pitch helix. Residues conserved among Arp3 proteins and of the same chemical type as actin (110L-117L, 111N-118N, 171L-186I, 270E-291D, 285C-307C). Residues conserved among Arp3 proteins but differing in chemical type from actin (173H-188S, 176M-191K, 179D-194P, 268G-289N, and 286D- 308P). Residues that are not conserved (112P-119t, 177R-192h, 266f-287f, 267I-288a, 269M-290p). (c) Back view of Arp2 with D-1 residues predicted to interact with the first actin subunit of the daughter filament. Residues conserved among Arp2 proteins and of the same chemical type as actin (110L-114M, 111N-115N, 112P-116P, 171L-175L, 173H- 177H, 177R-181R, 179D-183D, 267I-271I, 269M-273V, 270E-274E, 286D-290D). Residues conserved among Arp2 proteins but differing in chemical type from actin (176M-180R, 266f-270L, 285C-289A). Position 268G-272n is not conserved. (d) Bottom view of Arp3 with D-2 residues predicted to interact with the first actin subunit of the daughter filament. Residues conserved among Arp3 proteins and of the same chemical type as actin (167E-182E, 168G-183G, 169Y-184Y, 286D-308P, 287I-309I, 288D-310D, 291K-313R, 325M-363I). Residues conserved among Arp3 proteins but differing in chemical type from actin (166Y-181A, 324T-362P). Position 289I-311v is not conserved. (e) Bottom view of Arp2 with D-3 residues predicted to interact with the second actin subunit of the daughter filament. Residues conserved among Arp2 proteins and of the same chemical type as actin (166Y-170Y, 167E-171E, 286D-290D, 287I-291I, 288D-292D). Position 322P-325Y is conserved among Arp2 proteins but differs chemically from actin. Positions 168G-172g, 169Y- 173y, 289I-293t, and 291K-295s are not conserved among Arp2 proteins. The Arp2 S3 insert consisting of residues extends the helix by four residues (Figure 4(e)). All of the inserted residues are conserved across the sampled species except for 326, where both fungi have F rather than L. This short insert contacts ARPC5, but leaves the conserved, solvent-exposed residues 328R, 329V, 330L and 331K (pink in Figure 4(e)) to interact with the second actin subunit without steric interference. This interaction may stabilize the base of the daughter filament. The Arp3 S3 insert consisting of residues 347 to 358 extends the helix by two turns and the following chain by about 15 Å (Figure 4(d)). This insert includes nine conserved residues (344D, 346R, 347L, 350S, 351E, 353L, 354S, 356G, and 362P (pink in Figure 4(d))) but varies in length, with both fungi having two fewer residues. In the current model of nucleation this large insert must rearrange to allow binding of the first actin subunit of the daughter filament (arrow in Figure 3(a)). The absence of density for residues from the electron density map indicates some flexibility. In particular, conserved residues 347L, 350S, 351E, 353L, 354S, 356G, and 362P may have a critical

10 560 Homology Models of Arp2/3 Complex role in structural rearrangements in this region, since they are conserved and present a possible steric clash with the first actin subunit of the daughter filament (cyan in Figure 3(a), pink in Figure 4(d)). Hydrophobic plugs Like actin, Arps have hydrophobic plugs with interesting species-specific variation. Although it was beyond the resolution of their fiber diffraction data, Holmes et al. 34 postulated that residues 266F, 267I, 268G, 269M swing out from the surface of the monomer to insert between two subunits in the adjacent long-pitch helix of a filament. These hydrophobic interactions along the axis of the filament were suggested to stabilize the polymer. Mutagenesis confirmed the importance of these residues for polymerization. 39 Assuming that the Holmes mechanism applies to Arp2/3 complex, the hydrophobic plug of Arp3 would interact with Arp2 and the hydrophobic plug of Arp2 would interact with Arp3 and the first actin subunit of the daughter filament. Residues of Arp3 correspond to the actin hydrophobic plug. The three metazoan Arp3 molecules have a consensus sequence FxNP, while Arabidopsis, Dictyostelium, S. cerevisiae, and S. pombe have a consensus sequence IaSS. The hydrophobic plugs of Arp2, residues , have consensus sequences of LINV in the three metazoans and Lvdv in Arabidopsis, Dictyostelium, S. cerevisiae, and S. pombe. Residue 266 of actin is also variable, being F in bovine, C. elegans, Dictyostelium, and Drosophila, MinArabidopsis, VinS. cerevisiae and AinS. pombe. An additional position in Arp3 119, which corresponds to position 112 in actin, is proposed to interact with Arp2 and varies between species in a similar pattern: T in metazoans; A in Dictyostelium; and P in Arabidopsis, S. cerevisiae, and S. pombe. The actin plug is more hydrophobic than the plugs of Arp2 and Arp3 and the Arp plugs from Arabidopsis, Dictyostelium, S. cerevisiae and S. pombe are even less hydrophobic than the three metazoan Arps. Experimental mutations that reduce the hydrophobicity of the yeast actin plug cause a cold-sensitive growth defect and compromise actin filament stability. 39 We propose that the polar character in the plugs of the Arps in non-metazoan species is a second factor (besides the noncomplementary interfaces) that contributes to a low affinity of Arp2 and Arp3 in the inactive complex. Accordingly, S. pombe Arp2/3 complex is tenfold less efficient than bovine Arp2/3 complex in nucleating actin filaments in the presence of saturating amounts of nucleation promoting factors (V. Sirotkin & T.D.P., unpublished observations). Potential binding sites for nucleation promoting factors Much has been learned about interactions of VCA-type nucleation promoting factors with Arp2/3 complex, but their binding sites on the complex are still unknown. A yeast two-hybrid assay identified ARPC3 as the first potential contact of Scar-VCA. 40 VCA has also been chemically cross-linked to ARPC3, ARPC1, Arp2 and Arp3, 22,23 but the crosslinked residues have not been identified. Spectroscopic, calorimetric and hydrodynamic evidence suggest that free VCA is unstructured, 19 but the C segment can fold into short alpha-helices bound intramolecularly to either the WASp GBD (GTPase binding domain) 18 or intermolecularly to Arp2/3 complex. 20 The C segment contributes energetically to binding of the V segment to actin monomers and to binding of the A segment to Arp2/3 complex, 19 so the C segment may interact with both actin and Arp2/3 complex in the activated complex with a bound actin monomer (modeled in Figure 3(a)). The following paragraphs discuss what is known about the parts of VCA. Human profilin-i and thymosin-b4 compete with VCA for binding to actin monomers, 19 suggesting that their binding sites at least partially overlap. This would place the V segment binding site on or near the barbed end of actin (bottom of the blue actin in Figure 3(a)). This actin is shown as the first subunit in the daughter filament in Figure 3(a) (although one group has proposed that the V segment bound actin monomer is incorporated into the mother filament 41 ). The sequence linking V and C varies in length among VCA proteins and does not interact strongly with Arp2/3 complex. 20 NMR spectra are interpreted to show that the C segment forms four turns of amphipathic alphahelix with hydrophobic side-chains and a conserved arginine on one side contacting Arp2/3 complex. 20 The same face of this C-helix can bind intramolecularly to the GTPase binding domain in auto-inhibited WASp. 18 The segment between the C and A varies in length from seven to 18 residues that do not interact with Arp2/3 complex or form a secondary structure, 20 so this segment may be flexible and extended. Cross-linking VCA to Arp3 requires the C-terminal six residues of the A segment of N-WASp (498 EDDDEWED 505), 22 so the A-site on Arp3 is likely to be basic. The conserved W contributes considerable binding energy. 19 NMR spectroscopy showed directly that the conserved W and about six largely acidic residues at the C terminus of the A region interact with Arp2/3 complex. 20 Altogether the CA region bound to Arp2/3 complex might span Å. Taking WASp as an example, the bound C segment with 4.2 turns of alpha-helix (22.7 Å), up to 15 linker residues in an extended conformation (54 Å) and six residues of bound but extended chain in the A segment (21.6 Å) would be 98.3 Å long. N-WASp is slightly longer with two more residues and Scar is slightly shorter with four fewer residues. We used the homology models to search for

11 Homology Models of Arp2/3 Complex 561 conserved sites on Arp2/3 complex where C and A segments might bind. These sites are conserved, since vertebrate WASp can activate Arp2/3 complex from protozoa 13 and fungi (V. Sirotkin & T.D.P., unpublished work). We assumed that VCA binding stabilizes the compact, active conformation of Arp2/3 complex associated with the first actin subunit of the daughter filament (Figure 3). We ignored surfaces believed to be involved in Arp2 Arp3 dimer formation and actin monomer addition (Figure 4). We searched for groups of conserved, solvent-exposed residues on Arp3, Arp2, ARPC1 and ARPC3, especially those on Arp2 and Arp3 that differ in chemical type from the corresponding residues of actin. Arp3 The surface of Arp3 exposed on the backside of Arp2/3 complex has two intriguing sites (that we label A-sites) with potential for binding the acidic residues and tryptophan of A segments (red on Arp3 in Figure 2(c); A-1 and A-2 in Figures 3(b) and 5). Candidate site A-1 consists of a linear array of six conserved basic residues (R329, R333, R334, R337, K340, and R341) that lie along one side of the helix that is extended by the S3 insert, a conserved hydrophobic pocket that could accommodate binding of the conserved W (D. Sept, Washington University, personal communication), as well as a conserved basic residue (K228) at the back of the pocket (Figures 3 and 5). Conserved residues M327 and F328 line the hydrophobic pocket. We note that bound nucleotide will alter this hydrophobic pocket due to a hydrogen bond between K228 and the 2 0 OH of the ribose ring and an interaction between F328 and positions 1 and 2 of the adenine ring. Furthermore, the bound nucleotide creates an additional amino group from position 6 of the adenosine ring that is exposed and could potentially interact with residues in the A sequence, providing communication between the bound nucleotide and VCA. Candidate site A-2 is formed, in part, by residues , which form an insert between the bases of subdomains 1 and 3 that is not found in actin. Conserved residue R161 is next to a conserved hydrophobic pocket composed of residues W153, F379, M383, and L384 that could bind the conserved W in the acidic sequence. Actin has no side-chain corresponding to Arp3 W153, but the other three hydrophobic residues in this pocket correspond to residues I342, I346, and L347 that are conserved in actin. Having A150 in Arp3 (T in C. elegans Arp3) rather than Y144 in actin increases the solvent exposure of the hydrophobic sidechains and along with the insert, creates the pocket-like structure. In crystals of bovine Arp2/3 complex this pocket on Arp3 is occupied by the side-chain of the conserved F302 in the helical insert of ARPC1 from a neighboring complex in the crystal lattice. Other basic residues are conserved on the surface of Arp3, but most are similar in location to conserved basic residues of actin. ARPC3 Candidate site A-3 consists of a group of nine conserved residues (P2, H5, S7, N18, E59, I60, K61, R66, and I116) located on the underside of ARPC3 near the A-1 site on Arp3 (red on ARPC3 in Figure 2(c); A-3 in Figure 5). An additional 24 conserved, exposed residues are relatively evenly spread over the surface of ARPC3 (Figure 2(b)) rather than clustered into a potential binding site. Most of the residues in the Basic Patch 1 noted by Robinson et al. 3 are not conserved by our standards. ARPC1 An extensive patch of 46 conserved residues on ARPC1 is an obvious potential binding site for nucleation promoting factors (cyan on ARPC1 in Figure 2(c)). The cluster of six conserved basic residues might bind the A segment, but crosslinking data 22 favor A sequence binding to Arp3. Alternatively this conserved patch on ARPC1 could be a C-site, but it is.70 Å from the prime A-1 site on Arp3. We therefore reserve this group of conserved residues as a potential mother filament binding site (M-3). Figure 5. Space filling model showing potential binding sites for the A-segment of VCA. Residues are enumerated in Figure 2(c). Blue, basic residues. Red, acidic residues. Purple, uncharged polar residues. Green, non-polar residues. Brown, aromatic residues. Subunits are shaded as in Figure 1. Arp2 A search of the homology models of Arp2 (including subdomains 1 and 2 modeled on skeletal muscle actin) for conserved residues that differ chemically from actin revealed two

12 562 Homology Models of Arp2/3 Complex candidate sites for binding VCA (red on Arp2 in Figure 2(c); C-1 and C-2 in Figure 3(b)). The C-1 site, predominantly on the back of subdomain 2, consists of I40, I41, R42, K46, I52, L55, M56, L64, S66, N71, and D90. The C-2 site on the front side between subdomains 1 and 3 is composed of residues N26, F27, D346, R349, H352, A359. This analysis of phylogenetically conserved residues provides candidate binding sites for nucleation promoting factors on Arp2/3 complex. The most attractive candidate site is A-1 on subdomain 3 of Arp3 (Figures 2, 3 and 5), the subunit shown by chemical crosslinking to bind the C terminus of VCA. 22,23 We favor this potential binding site over the A-2 hydrophobic pocket between subdomains 1 and 3, because the size, shape and charge appear to be ideal for binding an extended A segment with the conserved W plugged into the hydrophobic pocket. Peptide docking simulations also favor this A-1 site (D. Sept, Washington University, personal communication). VCA peptides are long enough to span between these proposed A sites and a V binding site on actin. Assuming segment A binding to Arp3 and given segment V binding to actin, 42 C must bind somewhere in between at the interface that includes actin, Arp2 and Arp3. The C region binds to both actin and Arp2/3 complex 19 and is required to stimulate actin filament nucleation by Arp2/3 complex. 19 This segment C binding site is poorly defined, because crucial parts of Arp2 are missing from the electron density maps and the conformation of the active complex has not been determined experimentally. Of the potential CA binding sites on Arp2, the C-1 site on subdomain 2 appears to be most favorable for binding the hydrophobic surface of the C helix shown to contact Arp2/3 complex. 20 However the C-1 site is not adjacent to actin in the hypothetical model of the Arps and actin in Figure 3(a). Given the flexibility of subdomains 1 and 2 of Arp2, 43 the active structure may differ from the model and accomodate interaction of the C helix with both actin and either C-1 or C-2. Alternatively, the C region binding site on the Arps may be conserved but similar to actin such as a large group of residues on the back side of Arp2 subdomain 1 (Figure 2(b) and (c); Y72, E75, N76, I78, R80, N81, W82, D83, N119, K122, E125, E129, Y377, G381, and V382) close to Arp3 (subdomain 3 insert) and actin (subdomain 4). The evidence suggests that VCA binds to three separate sites extending from the V-site on the first actin subunit of the daughter filament past a still poorly defined site for the C-helix 20 between actin, Arp3 and Arp2 to the A-1 site on Arp3. These interactions, particularly sandwiching the C-helix among several subunits, would stabilize the association of the active Arp2 Arp3 shortpitch dimer with the first actin subunit of the daughter filament (Figure 3(a)). Potential interactions with the mother actin filament Cryo-EM reconstructions of actin filament branch junctions mediated by Arp2/3 complex 24 suggest the longest dimension of the complex (left side in the standard view, Figure 1) contacts three consecutive subunits along one long-pitch helix of the mother filament. This side of the complex consists of ARPC1, ARPC2, ARPC4 and ARPC5. This concept is supported by a variety of evidence: an antibody to ARPC2 inhibits branching; 25 actin can be chemically crosslinked to ARPC1, ARPC2, and ARPC5 in branch complexes; 26,44 and recombinant ARPC2/ARPC4 dimers bind to actin filaments with affinity similar to the whole complex. 27 Thus we focus on conserved residues on this side of the complex as potential mother filament binding sties. Homology models have three distinct clusters of conserved, solvent-exposed residues on the left side of the complex that could participate in mother filament binding. The M-1 site on ARPC2 consists of 22 conserved residues, seven of which are identical in the seven species (blue residues on ARPC2 in Figures 2(c) and 6). These conserved residues cover a total surface area of 468 Å 2. The M-2 site consists of a cluster of 13 conserved, solvent-exposed residues on ARPC4, largely on the left side of the Arp2/3 complex (blue residues on ARPC4 in Figures 2(c) and 6). This conserved M-2 patch on ARPC4 is extended to the front surface of the complex by three residues and to the back surface by ten more residues. The M-3 site is an extensive patch of 46 conserved residues on ARPC1 that covers 942 Å 2, mainly on the backside of the complex on blades 2, 3 and 4 and on the top surface created by the turns between strands A Figure 6. Model of an actin filament branch mediated by Arp2/3 complex. Actin subunits are shown as gray ribbon diagrams and Arp2/3 complex is a space filling model with the same color code as Figure 1. Blue, residues comprising four conserved sites with potential to interact with the mother filament, M-1 on ARPC2, M-2 on ARPC4, M-4 on ARPC1, M-5 on ARPC5 (residues are enumerated in Figure 2(c)).