Identiﬁcation of Functionally Important Residues of Arp2/3 … · 2019. 12. 19. ·...

Identification of Functionally Important Residues ofArp2/3 Complex by Analysis of Homology Modelsfrom Diverse Species

Christopher C. Beltzner1 and Thomas D. Pollard1,2*

1Department of MolecularCellular and DevelopmentalBiology, Yale University, P.O.Box 208103, New Haven, CT06520-8103 USA

2Department of Cell BiologyYale University, P.O. Box208103, New Haven, CT06520-8103 USA

We constructed homology models from the crystal structure of bovineArp2/3 complex and sequences from six phylogenetically diverse species(Arabidopsis thaliana, Caenorhabditis elegans, Dictyostelium discoideum,Drosophila melanogaster, Saccharomyces cerevisiae, Schizosaccharomycespombe) representing over 800 million years of evolution and used con-served surface residues to search for functionally important structuralelements. The folds of the seven subunits and their core residues are wellconserved, as well as residues at subunit interfaces. Only 45% of solvent-exposed surface residues are conserved and only 15% are identical acrossthe seven species. Arp residues expected to interact with nucleotide andwith the first and second actin subunits in a daughter filament are con-served and similar to actin. Arp residues required to form an Arp dimerdiffer from actin and may contribute to the dissociated state of the Arpsin the unactivated complex. Conserved patches of surface residues guidedus to candidate sites for nucleation promoting factors to interact withArp3, Arp2, and ARPC3. Other conserved residues were used withexperimental constraints to propose how residues on the subunitsARPC1, ARPC2, ARPC4 and ARPC5 might interact with the motherfilament at branch junctions.

q 2003 Elsevier Ltd. All rights reserved.

Keywords: actin; cellular motility; dendritic nucleation; evolution; proteininteractions*Corresponding author

Introduction

Since its initial purification from Acanthamoebaby affinity chromatography on profilin-agarose,1

Arp2/3 complex has been identified in manyother organisms and implicated in the initiation ofnew actin filaments (reviewed by Pollard et al.2).In every case, Arp2/3 complex has been shown tobe composed of two actin-related proteins (Arps),a seven-bladed beta propeller (ARPC1), and fourother subunits (ARPC2–5) with novel folds.3

Homology modeling originally suggested thatArp2 and Arp3 have the surface features requiredto form a dimer and to initiate an actin filamentgrowing in the barbed end direction.4 Highlypurified Arp2/3 complex caps the pointed ends ofactin filaments and has weak nucleating activity5

that is strongly activated by proteins called nuclea-tion promoting factors6 – 10 and by pre-existingfilaments.6,11,12 “Daughter filaments” grow at theirbarbed ends as 708 branches on the “motherfilament”.5,13 Branches formed by the purifiedproteins are identical with those at the leadingedge of migrating eukaryotic cells.14

The cell surface protein ActA of the intracellularbacterium Listeria15 was the first nucleationpromoting factor to be discovered (reviewed byWeaver et al.16). Eukaryotic nucleation promotingfactors related to WASp and Scar have a verprolinhomology (V; also called WH2 for WASp hom-ology) sequence that binds actin monomers and aC-terminal acidic sequence that binds Arp2/3complex. A central sequence (called C) connects

0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.

Supplementary data associated with this article can befound at doi: 10.1016/j.jmb.2003.12.017

E-mail address of the corresponding author:[email protected]

Abbreviations used: Arp, actin-related protein; WASp,Wiskott–Aldrich syndrome protein; HUGO, HumanGenome Organization; Bt, Bos torus; At, Arabidopsisthaliana; Ce, Caenorhabditis elegans; Dd, Dictyosteliumdiscoideum; Dm, Drosophila melanogaster; Sc, Saccharomycescerevisiae; Sp, Schizosaccharomyces pombe.

doi:10.1016/j.jmb.2003.12.017 J. Mol. Biol. (2004) 336, 551–565

the V and A motifs.17 We refer to these regions as“sequences” or “segments” rather than domains,because free VCA polypeptides lack secondarystructure but fold at least partially when they bindthe WASp GTPase binding domain,18 actin19 orArp2/3 complex.20 All three regions of VCA con-tribute to efficient activation of Arp2/3 complex.Less active nucleation promoting factors such asApp1p, Abp1p and cortactin, have an acidicsequence and an actin filament binding site ratherthan an actin monomer binding site,16 but canstabilize actin filament branches.21 These twoclasses of activators may cooperate to activateArp2/3 complex by forming an active ternarycomplex.22 Arp2, Arp3, ARPC1 and ARPC3 can becross-linked to nucleation promoting factors.22,23

A 2.0 A crystal structure of bovine Arp2/3complex3 revealed that the two Arps are physicallyseparated in the inactive complex. Activation wasproposed to involve a conformational change thatorients Arp2 and Arp3 like two subunits along theshort-pitch helix of an actin filament, a confor-mation suitable to initiate polymerization in thebarbed end direction. Binding to a mother filamentand a nucleation promoting factor are bothpostulated to favor the same active conformation.19

Arp2/3 complex contacts three consecutive actinsubunits along one long-pitch helix of the motherfilament in reconstructions of cryo-electron micro-graphs of branches.24 Antibodies to the C terminusof ARPC2 inhibit binding of Arp2/3 complex tothe side of actin filaments and branching.25 Arp2,ARPC1, ARPC2 and ARPC5 can be cross-linked toactin subunits at branch points.26

Investigators have modified individual aminoacids of nucleation promoting factors to probetheir functions,19,20 but modifications of Arp2/3complex have been confined to deletion of wholesubunits. Subcomplexes lacking ARPC3 are stablebut nucleate filaments inefficiently.27 Pentamericsubcomplexes lacking ARPC1 and ARPC5 arestable but have little nucleation activity.27 A dimerof ARPC2/ARPC4 is soluble and binds the side ofactin filaments similar to the whole complex.27

Mutagenesis might advance our understandingof the Arp2/3 complex, but this task is dauntinggiven nearly 2000 residues in the complex. Fortu-nately, actin, nucleation promoting factors andArp2/3 complex from protozoa, fungi and animalscan be used in apparently any combination toreconstitute branching nucleation in biochemicalassays (reviewed by Higgs & Pollard17). Since thisrequires extensive interactions of Arp2/3 complexwith multiple partners, we postulate that selectivepressures have maintained the structure of contactsites since the divergence of protozoa, fungi andanimals between 800 million and 1 billion yearsago. Actin is an extreme example of this trendwith 94% conservation of sequence between fungaland human actins, presumably due to extensivecontacts between polymerized actin subunits andbetween actin and numerous binding proteins.Arp2/3 complex is less conserved, but we

assumed that a survey of conserved surfaceresidues would reveal those with functional sig-nificance. To enable this survey, we constructedhomology models of Arp2/3 complex from sixspecies (a protozoan, a plant, two fungi and twoinvertebrates) for comparison with each other andbovine Arp2/3 complex. We assume that residuesthat interact with partner proteins have less free-dom to vary than those that do not. The limitednumber of conserved residues provided uswith many clues about activation, actin filamentnucleation and branching.

Results and Discussion

Construction of homology models anddefinitions of terms

Homology modeling depends on an accuratealignment of model sequences with a reliable tem-plate. Bovine Arp2/3 complex (Figure 1) is a goodtemplate, since its structure is known at highresolution (with the exception of subdomains 1and 2 of Arp2) and, where tested, it has biochemi-cal properties similar to Arp2/3 complex from themodel organisms. Most of the model sequencesare sufficiently similar to the template for reliablealignment, especially elements of secondary struc-ture. Automated sequence alignments based onsecondary structure (carried out by What If) weremost problematic for surface loops and turns withhighly divergent sequences. Gaps in the alignmentoccurred when structural elements differed inlength, large insertions caused uncertainty in thealignment of the surrounding region, or divergencecomplicated alignment. We rebuilt these gaps byhand when the insertions or deletions comprisedsix residues or fewer; other gaps were omittedfrom the model. After rebuilding gaps, the models

Figure 1. Ribbon diagram of the crystal structure ofinactive bovine Arp2/3 complex, PDB 1K8K. Subunitsare named based on the HUGO nomenclature. Themodels of subdomains 1 and 2 of Arp2 (pink) are basedon the uncomplexed actin structure (PDB: 1J6Z).

552 Homology Models of Arp2/3 Complex

were refined to remove steric clashes and improvebond geometries. Refinement was intentionallykept to a minimum.

The crystal structure of bovine Arp2/3 complexlacked electron density for subdomains 1 and 2 ofArp2, so we used sequence alignments betweenthe seven Arp2 sequences and human a-actin forour analysis. For Figures containing Arp2 wemade a homology model of subdomains 1 and 2of bovine Arp2 using uncomplexed rhodamineADP actin (PDB 1J6Z) as the template.

An important consideration when judging thequality of homology models is their intended use.Since our purpose is to locate potential sites ofprotein–protein interactions and to identifyresidues favorable for mutagenesis, the mostimportant aspect is the correct positioning of side-chains. Given that the Ca positions of core residuesof proteins in the same family having .50%sequence identity deviate less than 1 A rms,28 andgiven that the sequences of many of the subunitsof Arp2/3 complex are more than 50% identicalwith the bovine subunits (Table 1), it is highlylikely that core residues are accurately placed inour homology models. This ensures that mostexposed residues are correct as well. (See Guexet al.29 and references within for a more detaileddiscussion of homology models and theirreliability.)

We used the sequence alignments employed formodel building (Supplementary Material, Figures1–7) to analyze the conservation of the proteins.For purposes of comparison, we classify each resi-due as “core”, “subunit interacting”, or “exposed”based on solvent exposure in the bovine crystalstructure. Side-chains of core residues are lessthan 10% exposed to solvent. Subunit interactingresidues have 10% or more of their side-chainsshielded from solvent by interaction with anothersubunit. Side-chains of exposed residues are morethan 10% exposed to solvent. Side-chain solventexposure, expressed as a percentage, was deter-mined by comparing the exposure in the proteinwith the same side-chain conformation in a GLY-XXX-GLY tripeptide, in vacuo. Since hydrogenatoms are not included in the calculation, glycineresidues were classified based on the solventexposure of the alpha carbon. We grouped theamino 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 residuenumbers for bovine Arp2/3 complex and humana-actin throughout.

We use stringent criteria to define phylogeneti-cally “conserved residues” as those with the samechemical type (i.e. acidic, basic, etc) at a particularposition in six of the seven species. Due to gapssome residues are represented in less than sevenspecies. In this case only residues that are chemi-cally equivalent in six of six species are consideredconserved. Residues with five out of six, five out offive, or fewer were classified as not conserved,because revision of the structures could result inan alignment where the residue would no longerbe considered conserved. Conservation of residuesin actin is based on standard sequence alignmentswith the sequences of the major isoform of actinfrom each of the seven species. In the text, we indi-cate conserved residues by capital letters of thesingle letter amino acid code and non-conservedresidues by lower case. Residues identical in allseven species are in bold capital letters. For com-parisons between actin and Arp2 or Arp3, we givethe residue number, identity and conservation. Forexample a comparison of residues in actin andArp2 denoted as 203T-205s refers to a conservedthreonine at position 203 in human a-actin and aserine at position 205 of bovine Arp2 that is notconserved among the seven species. Additionally,we denote substitutions between species by thebovine residue number followed by the residue atthe corresponding location of the otherspecies. For example, substitution of phenylalanine17 in bovine Arp2/3 complex for tyrosine atthe analogous position in Arabidopsis is given asF17Y.

Overall description of models

After rebuilding gaps the Drosophila model has1720 residues, Caenorhabditis elegans has 1700,Dictyostelium has 1677, Saccharomyces cerevisisaehas 1669, S. pombe has 1665 and Arabidopsis has1672, compared with the original bovine structureof 1709 residues. The extended N terminus ofARPC5 is generally incomplete in the models,since the sequences are poorly conserved and varyin length. The Arabidopsis and Dictyostelium modelsboth lack the extended N terminus of ARPC5 thatinteracts with Arp2. The Arabidopsis ARPC5sequence 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 70/80Ce 76/85 76/85 48/64 70/80 54/69 74/85 42/55 62/74Dd 69/79 71/79 41/53 40/58 42/60 67/82 36/55 55/68Sp 60/72 64/75 42/57 48/64 52/67 68/79 26/44 53/67Sc 63/71 66/76 37/53 41/56 46/63 69/78 27/48 52/65At 59/72 61/73 41/53 27/48 46/64 61/77 30/50 47/61

Homology Models of Arp2/3 Complex 553

is not clear if the Arp2 binding region is present.Both fungal models include the N-terminalresidues of ARPC5 that bind Arp2, but lack theresidues connecting this region to the globularpart of the subunit. The models include the shorthelix inserted between blades 6 and 7 of ARPC1but not the connecting residues, because they aredisordered in the crystal structure and are poorlyconserved. A loop consisting of residues 40–50 ofArp3 was omitted from the models, since they aredisordered in the crystal structure and not con-served among the seven species. Other sequencesomitted from the models include several loops ofmore than ten residues: 35 residues in S. cerevisiaeand 14 residues in S. pombe in subdomain 4 ofArp3; 19 residues in S. cerevisiae and 17 residues inS. pombe between the first helix and the first strandof the second a/b domain of ARPC2; 11 residuesat the C terminus of S. cerevisiae. None of theseinserts has significant sequence conservation withknown proteins.

General features

Of 562 core residues, 80.0% are conserved; 20.5%of the conserved core residues are polar (chargedand uncharged), 63.8% are non-polar and 15.7%are aromatic. Among the core residues the conser-vation of each chemical type is roughly the samewith the non-polar residues being the highest at83.0% and the uncharged polar residues being thelowest at 67.8%. Of the conserved charged polarresidues (acidic and basic) 86.7% are identical. Ofthe conserved aromatic residues in the cores only43.5% are identical and overall only 33.5% of theconserved core residues are identical. This demon-strates how poorly Arp2/3 complex toleratesconservative substitutions of charged polarresidues in the protein cores.

Residues comprising subunit interfaces are moreconserved than residues on the free surfaces. Ofthe 335 residues at subunit interfaces 69.6% areconserved; 17.2% of the conserved residues areacidic, 22.7% are basic, 15.5% are uncharged polar,30.9% are non-polar, and 13.7% are aromatic. Theconservation of uncharged polar residues is lowerat 57.1% than the other chemical types, whichvary from 69.9 to 73.6%. Conserved basic,uncharged polar and non-polar residues are

equally tolerant to conservative mutations with41.5%, 36.1% and 36.1% of the conserved residuesbeing identical. Conserved acidic and aromaticresidues are less tolerant of conservative changeswith 60.0% and 56.3% of the conserved residuesbeing identical. The complementary surfaces ofsubunit interfaces tend to be equally conserved,while other parts of the subunits have diverged tovarious degrees (Table 2). For example, at the inter-face of Arp3 and ARPC3, 85% of the ARPC3residues and 80% of the Arp3 residues areconserved, while overall only 54% of total ARPC3residues and 75% of total Arp3 residues are con-served. The subunit interfaces of ARPC1 andARPC5 are the least conserved. These two subunitsdepend on each other to bind to the complex.27

Exposed surface residues

Of the 818 solvent-exposed residues in bovineArp2/3 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%) arearomatic. Less than half (44.6%) of these residuesare conserved. Of the conserved exposed residues16.4% are acidic, 21.9% are basic, 21.4% areuncharged polar, 34.0% are non-polar, and 6.3%are aromatic. Of the exposed residues, aromaticresidues are more conserved (59.0%) than otherchemical types, which vary from 40.5% to 46.5%.Conservative substitutions are frequent amongconserved exposed uncharged polar residues withonly 14 of the 79 conserved residues being identi-cal. Overall only 33.2% of the conserved exposedresidues (15% of the total surface residues) areidentical across the seven species. The followingsections explore the functional significance ofconserved surface residues.

Nucleotide binding to Arp2 and Arp3

The original crystal structure of bovine Arp2/3complex lacked bound nucleotide but new struc-tures have ADP or ATP bound to both Arps(R. Littlefield, B. Nolen & T.D.P., unpublishedresults). Hydrolyzable ATP bound to the Arps isrequired for nucleation of branches.30,31 Thesestudies agree that the affinity of Arp3 for ATP isin 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 80/45Arp2 87/53ARPC1 54/29 45/18ARPC2 76/28 74/20ARPC3 85/23ARPC4 88/44 56/13 71/35 53/8ARPC5 45/0 53/11


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 crystalstructure. Conserved residues are the same chemical type in at least six of the seven species. Exposed residues have10% or more of their side-chain surface area exposed to solvent. (a) Three views of a space filling model of bovineArp2/3 complex with subunits colored as in Figure 1. (b) Conserved exposed residues are colored blue and subunitsare outlined. (c) Conserved exposed residues colored according to proposed functions. Residues identical in all sevenspecies are bold in this legend. Orange, nucleotide binding by Arp2 and Arp3. Dark blue, potential mother filamentbinding 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 promotingfactors. 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 andbetween 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 K337Arp2 D12 T15 F17 K19 Q141 D161 V163 R214 K217 E218 S307 Y310 K351Arp3 D11 T14 Y16 K18 Q144 D172 V174 k225 K228 E229 S325 F328 R374


Figure 3 (legend opposite)


affinity of Arp2 for ATP. The Kd is in the low nano-molar range according to LeClainche et al.30 and inthe low micromolar range according to Dayelet al.31 The affinity of actin is 0.12 nM for Ca-ATPand 1.2 nM for Mg-ATP.32

To explore nucleotide binding by Arp2 and Arp3we made a structure-based alignment of the bovineArps with rabbit skeletal muscle a-actin (PDB1ATN) and compared the residues known toparticipate in nucleotide binding to actin. Of theresidues present in the recently determined ATPand ADP structures, all are correctly predicted bythis method (orange in Figure 2(c)) (R. Littlefield& T.D.P., unpublished results). For subdomains 1and 2 of Arp2 we used sequence alignmentsbetween bovine Arp2 and human a-actin. Theseresidues are shown in italics (Table 3).

Most residues making side-chain interactionswith ATP are conserved among actin, Arp2 andArp3. Four residues (D12, K19, D158, and K214)are identical among the three proteins across thesampled species. Of the Arp2 residues included inthe bovine crystal structure, the only substitutionbetween the seven species is S307T in C. elegans;otherwise all the residues are identical among theseven species. The only substitution that occursbetween actin and residues in the bovine crystalstructure of Arp2 is T304-S307. In actin, T304makes a side-chain hydrogen bond to the carbonyloxygen of G157. This contact is not maintained byS307 in bovine Arp2; however, G160 is located atthe same position as G157 in actin and thehydroxyl group is oriented in the proper direction.This hydrogen bond may simply be weakened inArp2. Of the Arp2 residues missing from thebovine crystal structure, there are two substitutionsbetween the species; F17Y in Arabidopsis and K351Rin S. pombe. This region has two substitutionsbetween Arp2 and actin, one conservative and onenon-conservative. The conservative substitution isS15-T15. In actin, the side-chain of this residuemakes a hydrogen bond to an oxygen on the beta-phosphate. The non-conservative substitution isL17-F17. In actin the side-chain of this residue isnear the alpha and beta phosphate groups.

Arp3 has several conservative differences fromactin among residues that participate in nucleotidebinding: S15-T14, R211-k225, T304-S325, Y307-F328, and K337-R374. In actin, R211 forms a hydro-gen bond with the side-chain of E215, whichhydrogen bonds to the 20-OH of the ribose ring. Inbovine Arp3 k225 makes a hydrogen bond with

the hydroxyl group of Y16. Y307 interacts withpositions 1 and 2 of the adenine ring. K337 is nearposition 7 of the adenine ring, but does not form ahydrogen bond. In bovine Arp3 R374 is orientedtoward the adenine ring and does not make anyhydrogen bonds. The contributions of S15 andT304 to nucleotide binding in actin are describedabove. T14 of Arp3 does not maintain the hydro-gen bond of actin S15. S325 of Arp3 does maintainthe hydrogen bond of actin T304. Position 225 ofArp3 varies between the species, where Arabidopsishas a K225R substitution, and both fungi have aK225E substitution. L17-Y16 is a non-conservativesubstitution between actin and Arp3; however,S. cerevisiae Arp3 has L like actin, presumably areverse substitution. The remaining positions areconserved and the same as actin, except for substi-tutions of Q144N and V174A in Arabidopsis andE229Q in S. cerevisiae.

The lack of nucleotide in the bovine crystal struc-ture and biochemical data showed that both Arpshave a lower affinity for nucleotide than actin,although one group reports a significantly loweraffinity only for Arp3. Our study does not reveal abasis for a lower affinity of Arp2 for nucleotide,because most residues in direct contact withnucleotide are identical in the seven species ofArp2 and identical with actin. Interactions withother subunits or other features of Arp2 may favorthe “open” conformation captured in the crystalslacking nucleotide.

Our study does reveal a potential basis for loweraffinity of Arp3 for nucleotide. Five conservativedifferences and one non-conservative differencebetween Arp3 and actin are maintained acrossmost of the seven species sampled. While most ofthese substitutions are conservative, they changethe structure of the nucleotide-binding cleft andcould contribute to lower affinity. Only oneposition in Arp3, 225, is not conserved among thespecies studied. The charge reversal mutations, Rto E, found in the fungal species suggest that eitherthis residue has little effect on nucleotide bindingor that fungal Arp3 has even lower affinity fornucleotide. The insert from 154–161 in Arp3between subdomains 1 and 3 may influence thetransition from the open to closed conformationsand therefore affect nucleotide affinity. Only 154T,155S and 161R are conserved in this insert and thelength of the insert varies, with Arabidopsis havingthree fewer residues and Dictyostelium having oneless residue.

Figure 3. Ribbon diagrams of models of an Arp2–Arp3 short-pitch filament dimer. (a) Two stereo views of theArp2–Arp3 short-pitch filament dimer with ARPC3 and the first subunit of the daughter actin filament. The moleculeswere aligned on the Holmes actin filament model with Arp2/3 complex subunits colored as in Figure 1 and actin inblue. The arrowhead indicates a steric clash between subdomain 2 of Arp2 and ARPC3. The arrow indicates a stericclash between subdomain 3 of Arp3 and subdomain 4 of actin. (b) Two stereo views of the Arp2 and Arp3 withARPC3 from the structure of inactive complex shown in Figure 1. Green, conserved residues with potential to bindthe C-segment (C-1, C-2) and the A-segment (A-1, A-2, A-3) of VCA nucleation promoting factors (enumerated inFigure 2(c)).


Potential contacts between the Arps and withthe daughter actin filament

Actin filaments are polar owing to the commonorientation of the asymmetrical subunits along thedouble helical polymer. The ends are designatedas barbed and pointed from the arrowhead-likepattern created by bound myosin heads.33 Sub-domains 1 and 3 of actin are exposed at the barbedend.34 In solution the rate-limiting step for pureactin monomers to initiate a new filament is theformation of a nucleus consisting of an actin trimer.Both possible actin dimers are unstable, but theshort-pitch dimer is thought to be more stablethan the long-pitch dimer and on the pathway totrimers.35

We used the actin filament model proposed byHolmes34 to construct a model of Arp3 (associatedwith ARPC3), Arp2 and the first subunit of thedaughter filament (Figure 3(a)). We positionedArp2 and Arp3 by creating a least-squares fit ofthe Ca positions of each Arp and actin and notedthe residues in the Arps corresponding to theactin residues predicted to make contacts in theHolmes model. We used standard sequence align-ments with actin for the parts of Arp2 not in themodel. In this simple model subdomain 2 of Arp2clashes with ARPC3, so in reality some rearrange-ment is required. This model assumes a confor-mational change in the inactive complex thatrearranges Arp2 and Arp3 to adopt orientationssimilar to two subunits along the short-pitch helixof an actin filament.3,24 Arp2 is at the barbed endof the short-pitch Arp2–Arp3 dimer, because thatis the arrangement of the Arps in the crystal struc-ture and ARPC3 blocks the pointed end of Arp3.In this model, Arp3 contacts Arp2 and the firstactin subunit in the daughter filament, while Arp2contacts Arp 3 and the first two actin subunits inthe daughter filament. The daughter filamentnucleus is completed adding an actin monomer tothe Arp2–Arp3 dimer. This arrangement is favoredover formation of a long-pitch dimer by Arp2 andArp3, which would require massive rearrangementof the complex. Another model of activationinvolving the incorporation of one of the Arpsinto the mother filament12 is not supported byelectron microscopy24 or observations of branchingin real time by fluorescence microscopy.36 – 38

We used this model to identify potential contactsbetween the Arps (called R sites, green inFigures 2(c), 4(a) and (b)) and between the Arpsand the first two actin subunits of the daughterfilament (called D sites, green in Figures 2(c),4(c)–(e)). Most surface residues of Arp2 and Arp3that are proposed to make R site contacts in theshort-pitch dimer in the activated complex are con-served among the Arps, but only 58% of these keyresidues are of the same chemical type as the corre-sponding residues of actin. Six Arp2 residues andfive Arp3 residues conserved among the Arps arethe same chemical type as actin (blue in Figure4(a) and (b)). Three Arp2 residues and five Arp3

residues conserved among the Arps differ inchemical type from the corresponding residues inactin (red in Figure 4(a) and (b)). Three Arp2residues and five Arp3 residues are not conserved(green in Figure 4(a) and (b)). We propose thatthese differences in the Arp2–Arp3 dimer interfacerelative to actin favor the dissociated, inactiveconformation of the Arps in spite of their closeproximity in the complex. In the absence of nuclea-tion promoting factors, this inactive, dissociatedconformation is captured in the crystals.

Crystallography, electron microscopy and bio-chemistry all suggest that elongation, in the barbedend direction, from an Arp2–Arp3 dimer is themost plausible mechanism for Arp2/3 complex toinitiate a daughter filament. The Arp residueswith potential to contact actin are highly conservedand identical with the corresponding residues ofactin. Eleven residues at the barbed end of Arp2that are predicted to interact with the first subunitin the actin filament (D1 site) are conserved andidentical with actin (blue in Figure 4(c)). Eight resi-dues at the barbed end of Arp3 expected to contactactin (D2 site) are conserved and identical withactin (blue in Figure 4(d)). Three Arp2 D1 residues(red in Figure 4(c)) and two Arp3 D2 residues (redin Figure 4(d)) are conserved among the Arps, butdiffer in chemical type from actin. One D1 residueof Arp2 and one D2 residue of Arp3 are notconserved (green in Figure 4(c) and (d)).

Given the low tendency of the Arps to dimerize,we propose that interactions of the first subunit inthe daughter filament with the D1 and D2 sites ofthe two Arps stabilize the active conformation.This provides a mechanism for the actin monomerbound to nucleation promoting factors tocontribute to efficient activation of Arp2/3complex.

Arp2 residues forming the D3 site proposed tointeract with the pointed end of the second subunitin the daughter filament diverge more from actinthan those that contact the first subunit. Five Arp2D3 residues are conserved and identical with actin(blue in Figure 4(e)). One D3 residue is conservedamong the Arp2 proteins but differs chemicallyfrom actin (red in Figure 4(e)) and four D3 residuesare not conserved among Arp2 proteins (green inFigure 4(e)). Consequently, the first actin subunitof the daughter filament may contribute morethan Arp2 to binding the second daughter subunit.

Conserved inserts in the Arps predicted tointeract with the daughter filament

Both Arps have conserved inserts relative toactin, particularly a prominent insert correspond-ing to actin residues 322–325 (see Figure 2 ofRobinson et al.3). These inserts extend an alpha-helix at the base of subdomain 3 and the followingextended chain that leads to subdomain 1. TheseS3 inserts are predicted to interact with thefirst and second actin subunits of the daughterfilament.


The Arp2 S3 insert consisting of residues 325–335 extends the helix by four residues (Figure 4(e)).All of the inserted residues are conserved across thesampled species except for 326, where both fungihave F rather than L. This short insert contactsARPC5, but leaves the conserved, solvent-exposedresidues 328R, 329V, 330L and 331K (pink in Figure4(e)) to interact with the second actin subunit with-out steric interference. This interaction may stabilizethe base of the daughter filament.

The Arp3 S3 insert consisting of residues 347 to358 extends the helix by two turns and the follow-

ing chain by about 15 A (Figure 4(d)). This insertincludes nine conserved residues (344D, 346R,347L, 350S, 351E, 353L, 354S, 356G, and 362P (pinkin Figure 4(d))) but varies in length, with bothfungi having two fewer residues. In the currentmodel of nucleation this large insert mustrearrange to allow binding of the first actin subunitof the daughter filament (arrow in Figure 3(a)). Theabsence of density for residues 354–359 from theelectron density map indicates some flexibility. Inparticular, conserved residues 347L, 350S, 351E,353L, 354S, 356G, and 362P may have a critical

Figure 4. Space filling models ofArp2 and Arp3 with residues pre-dicted to participate in interactionsbetween Arp2, Arp3 and actinmonomers. Arp2 and Arp3structures were aligned withhuman a-actin (PDB: 1ATN) toidentify residues corresponding tothose mediating interactionsbetween actin monomers in theHolmes actin filament model. Blue,conserved Arp residues of thesame chemical type as actin. Red,residues conserved among theseven species of Arp2 or Arp3 butdiffering in chemical type fromactin. Green, residues not con-served among the seven species ofArp2 or Arp3. Pink, conservedresidues in the S3 inserts. Cyan:non-conserved residues in the S3inserts. (a) Back view of Arp2 withR-1 residues predicted to interactwith Arp3 in a short-pitch helix.Residues conserved among Arp2proteins and of the same chemicaltype as actin (196R-200R, 197G-201G, 201Tp-205N (presidue 201 is Vin bovine and T or S in the otherspecies), 267I-271I, 269M-273V,270E-274E). Residues conservedamong Arp2 proteins but differingin 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 thesame chemical type as actin (110L-117L, 111N-118N, 171L-186I, 270E-291D, 285C-307C). Residues conserved amongArp3 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 Arp2with D-1 residues predicted to interact with the first actin subunit of the daughter filament. Residues conservedamong 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 proteinsbut 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 inchemical type from actin (166Y-181A, 324T-362P). Position 289I-311v is not conserved. (e) Bottom view of Arp2 withD-3 residues predicted to interact with the second actin subunit of the daughter filament. Residues conserved amongArp2 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.


role in structural rearrangements in this region, sincethey are conserved and present a possible steric clashwith 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” withinteresting species-specific variation. Although itwas beyond the resolution of their fiber diffractiondata, Holmes et al.34 postulated that residues 266F,267I, 268G, 269M swing out from the surface ofthe monomer to insert between two subunits inthe adjacent long-pitch helix of a filament. Thesehydrophobic interactions along the axis of thefilament were suggested to stabilize the polymer.Mutagenesis confirmed the importance of theseresidues for polymerization.39 Assuming that theHolmes mechanism applies to Arp2/3 complex,the hydrophobic plug of Arp3 would interact withArp2 and the hydrophobic plug of Arp2 wouldinteract with Arp3 and the first actin subunit ofthe daughter filament. Residues 287–290 of Arp3correspond to the actin hydrophobic plug. Thethree metazoan Arp3 molecules have a consensussequence FxNP, while Arabidopsis, Dictyostelium,S. cerevisiae, and S. pombe have a consensussequence IaSS. The hydrophobic plugs of Arp2,residues 270–273, have consensus sequences ofLINV in the three metazoans and Lvdv inArabidopsis, Dictyostelium, S. cerevisiae, andS. pombe. Residue 266 of actin is also variable,being F in bovine, C. elegans, Dictyostelium, andDrosophila, M in Arabidopsis, V in S. cerevisiae andA in S. pombe. An additional position in Arp3 119,which corresponds to position 112 in actin, is pro-posed to interact with Arp2 and varies betweenspecies in a similar pattern: T in metazoans; A inDictyostelium; and P in Arabidopsis, S. cerevisiae,and S. pombe.

The actin plug is more hydrophobic than theplugs of Arp2 and Arp3 and the Arp plugs fromArabidopsis, Dictyostelium, S. cerevisiae and S. pombeare even less hydrophobic than the three metazoanArps. Experimental mutations that reduce thehydrophobicity of the yeast actin plug cause acold-sensitive growth defect and compromiseactin filament stability.39 We propose that the polarcharacter in the plugs of the Arps in non-metazoanspecies is a second factor (besides the non-complementary interfaces) that contributes to alow affinity of Arp2 and Arp3 in the inactivecomplex. Accordingly, S. pombe Arp2/3 complex istenfold less efficient than bovine Arp2/3 complex innucleating actin filaments in the presence of saturat-ing amounts of nucleation promoting factors(V. Sirotkin & T.D.P., unpublished observations).

Potential binding sites for nucleationpromoting factors

Much has been learned about interactions ofVCA-type nucleation promoting factors with

Arp2/3 complex, but their binding sites on thecomplex are still unknown. A yeast two-hybridassay identified ARPC3 as the first potential con-tact of Scar-VCA.40 VCA has also been chemicallycross-linked to ARPC3, ARPC1, Arp2 andArp3,22,23 but the crosslinked residues have notbeen identified.

Spectroscopic, calorimetric and hydrodynamicevidence suggest that free VCA is unstructured,19

but the C segment can fold into short alpha-helicesbound intramolecularly to either the WASp GBD(GTPase binding domain)18 or intermolecularly toArp2/3 complex.20 The C segment contributesenergetically to binding of the V segment to actinmonomers and to binding of the A segment toArp2/3 complex,19 so the C segment may interactwith both actin and Arp2/3 complex in theactivated complex with a bound actin monomer(modeled in Figure 3(a)). The following paragraphsdiscuss what is known about the parts of VCA.

Human profilin-I and thymosin-b4 compete withVCA for binding to actin monomers,19 suggestingthat their binding sites at least partially overlap.This would place the V segment binding site on ornear the barbed end of actin (bottom of the blueactin in Figure 3(a)). This actin is shown as thefirst subunit in the daughter filament in Figure3(a) (although one group has proposed that the Vsegment bound actin monomer is incorporatedinto the mother filament41). The sequence linkingV and C varies in length among VCA proteins anddoes not interact strongly with Arp2/3 complex.20

NMR spectra are interpreted to show that the Csegment forms four turns of amphipathic alpha-helix with hydrophobic side-chains and a con-served arginine on one side contacting Arp2/3complex.20 The same face of this C-helix can bindintramolecularly to the GTPase binding domain inauto-inhibited WASp.18

The segment between the C and A varies inlength from seven to 18 residues that do not inter-act with Arp2/3 complex or form a secondarystructure,20 so this segment may be flexible andextended.

Cross-linking VCA to Arp3 requires theC-terminal six residues of the A segment ofN-WASp (498 EDDDEWED 505),22 so the A-site onArp3 is likely to be basic. The conserved Wcontributes considerable binding energy.19NMRspectroscopy showed directly that the conservedW and about six largely acidic residues at theC terminus of the A region interact with Arp2/3complex.20

Altogether the CA region bound to Arp2/3 com-plex might span 80–100 A. Taking WASp as anexample, the bound C segment with 4.2 turns ofalpha-helix (22.7 A), up to 15 linker residues in anextended conformation (54 A) and six residues ofbound but extended chain in the A segment(21.6 A) would be 98.3 A long. N-WASp is slightlylonger with two more residues and Scar is slightlyshorter with four fewer residues.

We used the homology models to search for


conserved sites on Arp2/3 complex where C and Asegments might bind. These sites are conserved,since vertebrate WASp can activate Arp2/3 com-plex from protozoa13 and fungi (V. Sirotkin &T.D.P., unpublished work). We assumed that VCAbinding stabilizes the compact, active confor-mation of Arp2/3 complex associated with thefirst actin subunit of the daughter filament(Figure 3). We ignored surfaces believed to beinvolved in Arp2–Arp3 dimer formation andactin monomer addition (Figure 4). We searchedfor groups of conserved, solvent-exposed residueson Arp3, Arp2, ARPC1 and ARPC3, especiallythose on Arp2 and Arp3 that differ in chemicaltype from the corresponding residues of actin.

Arp3

The surface of Arp3 exposed on the backside ofArp2/3 complex has two intriguing sites (that welabel A-sites) with potential for binding the acidicresidues and tryptophan of A segments (red onArp3 in Figure 2(c); A-1 and A-2 in Figures 3(b)and 5). Candidate site A-1 consists of a lineararray of six conserved basic residues (R329, R333,R334, R337, K340, and R341) that lie along oneside of the helix that is extended by the S3 insert,a conserved hydrophobic pocket that could accom-modate binding of the conserved W (D. Sept,Washington University, personal communication),as well as a conserved basic residue (K228) at theback of the pocket (Figures 3 and 5). Conservedresidues M327 and F328 line the hydrophobicpocket. We note that bound nucleotide will alterthis hydrophobic pocket due to a hydrogen bondbetween K228 and the 20OH of the ribose ring andan interaction between F328 and positions 1 and 2

of the adenine ring. Furthermore, the boundnucleotide creates an additional amino groupfrom position 6 of the adenosine ring that isexposed and could potentially interact with resi-dues in the A sequence, providing communicationbetween the bound nucleotide and VCA. Candi-date site A-2 is formed, in part, by residues 154–161, which form an insert between the bases ofsubdomains 1 and 3 that is not found in actin.Conserved residue R161 is next to a conservedhydrophobic pocket composed of residues W153,F379, M383, and L384 that could bind the con-served W in the acidic sequence. Actin has noside-chain corresponding to Arp3 W153, but theother three hydrophobic residues in this pocketcorrespond to residues I342, I346, and L347 thatare conserved in actin. Having A150 in Arp3 (T inC. elegans Arp3) rather than Y144 in actin increasesthe solvent exposure of the hydrophobic side-chains and along with the 154–161 insert, createsthe pocket-like structure. In crystals of bovineArp2/3 complex this pocket on Arp3 is occupiedby the side-chain of the conserved F302 in thehelical insert of ARPC1 from a neighboringcomplex in the crystal lattice. Other basic residuesare conserved on the surface of Arp3, but most aresimilar in location to conserved basic residues ofactin.

ARPC3

Candidate site A-3 consists of a group of nineconserved residues (P2, H5, S7, N18, E59, I60, K61,R66, and I116) located on the underside of ARPC3near the A-1 site on Arp3 (red on ARPC3 inFigure 2(c); A-3 in Figure 5). An additional 24 con-served, exposed residues are relatively evenlyspread 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 byRobinson et al.3 are not conserved by ourstandards.

ARPC1

An extensive patch of 46 conserved residues onARPC1 is an obvious potential binding site fornucleation promoting factors (cyan on ARPC1 inFigure 2(c)). The cluster of six conserved basic resi-dues might bind the A segment, but crosslinkingdata22 favor A sequence binding to Arp3. Alterna-tively this conserved patch on ARPC1 could be aC-site, but it is .70 A from the prime A-1 site onArp3. We therefore reserve this group of conservedresidues as a potential mother filament binding site(M-3).

Arp2

A search of the homology models of Arp2(including subdomains 1 and 2 modeled onskeletal muscle actin) for conserved residues thatdiffer chemically from actin revealed two

Figure 5. Space filling model showing potentialbinding sites for the A-segment of VCA. Residues areenumerated 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.


candidate sites for binding VCA (red on Arp2 inFigure 2(c); C-1 and C-2 in Figure 3(b)). The C-1site, 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 thefront side between subdomains 1 and 3 is com-posed of residues N26, F27, D346, R349, H352,A359.

This analysis of phylogenetically conservedresidues provides candidate binding sites fornucleation promoting factors on Arp2/3 complex.The most attractive candidate site is A-1 on sub-domain 3 of Arp3 (Figures 2, 3 and 5), the subunitshown by chemical crosslinking to bind theC terminus of VCA.22,23 We favor this potentialbinding site over the A-2 hydrophobic pocketbetween subdomains 1 and 3, because the size,shape and charge appear to be ideal for bindingan extended A segment with the conserved Wplugged into the hydrophobic pocket. Peptidedocking simulations also favor this A-1 site(D. Sept, Washington University, personal com-munication). VCA peptides are long enough tospan between these proposed A sites and a Vbinding site on actin.

Assuming segment A binding to Arp3 and givensegment V binding to actin,42 C must bind some-where in between at the interface that includesactin, Arp2 and Arp3. The C region binds to bothactin and Arp2/3 complex19 and is required tostimulate actin filament nucleation by Arp2/3complex.19 This segment C binding site is poorlydefined, because crucial parts of Arp2 are missingfrom the electron density maps and the confor-mation of the active complex has not been deter-mined experimentally. Of the potential CAbinding sites on Arp2, the C-1 site on subdomain2 appears to be most favorable for binding thehydrophobic surface of the C helix shown to con-tact Arp2/3 complex.20 However the C-1 site isnot adjacent to actin in the hypothetical model ofthe Arps and actin in Figure 3(a). Given theflexibility of subdomains 1 and 2 of Arp2,43 theactive structure may differ from the model andaccomodate interaction of the C helix with bothactin and either C-1 or C-2. Alternatively, the Cregion binding site on the Arps may be conservedbut similar to actin such as a large group ofresidues 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) andactin (subdomain 4).

The evidence suggests that VCA binds to threeseparate sites extending from the V-site on thefirst actin subunit of the daughter filament past astill poorly defined site for the C-helix20 betweenactin, Arp3 and Arp2 to the A-1 site on Arp3.These interactions, particularly sandwiching theC-helix among several subunits, would stabilizethe association of the active Arp2–Arp3 short-pitch dimer with the first actin subunit of thedaughter filament (Figure 3(a)).

Potential interactions with the motheractin filament

Cryo-EM reconstructions of actin filamentbranch junctions mediated by Arp2/3 complex24

suggest the longest dimension of the complex (leftside in the standard view, Figure 1) contacts threeconsecutive subunits along one long-pitch helix ofthe mother filament. This side of the complex con-sists of ARPC1, ARPC2, ARPC4 and ARPC5. Thisconcept is supported by a variety of evidence: anantibody to ARPC2 inhibits branching;25 actin canbe chemically crosslinked to ARPC1, ARPC2, andARPC5 in branch complexes;26,44 and recombinantARPC2/ARPC4 dimers bind to actin filamentswith affinity similar to the whole complex.27 Thuswe focus on conserved residues on this side of thecomplex as potential mother filament binding sties.

Homology models have three distinct clusters ofconserved, solvent-exposed residues on the leftside of the complex that could participate inmother filament binding. The M-1 site on ARPC2consists of 22 conserved residues, seven of whichare identical in the seven species (blue residues onARPC2 in Figures 2(c) and 6). These conservedresidues cover a total surface area of 468 A2. TheM-2 site consists of a cluster of 13 conserved, sol-vent-exposed residues on ARPC4, largely on theleft side of the Arp2/3 complex (blue residues onARPC4 in Figures 2(c) and 6). This conserved M-2patch on ARPC4 is extended to the front surfaceof the complex by three residues and to the backsurface by ten more residues. The M-3 site is anextensive patch of 46 conserved residues onARPC1 that covers 942 A2, mainly on the backsideof the complex on blades 2, 3 and 4 and on the topsurface created by the turns between strands A

Figure 6. Model of an actin filament branch mediatedby Arp2/3 complex. Actin subunits are shown as grayribbon diagrams and Arp2/3 complex is a space fillingmodel with the same color code as Figure 1. Blue,residues comprising four conserved sites with potentialto interact with the mother filament, M-1 on ARPC2,M-2 on ARPC4, M-4 on ARPC1, M-5 on ARPC5 (residuesare enumerated in Figure 2(c)).


and B of these blades (cyan residues on ARPC2 inFigure 2(c)).

In the crystal of the bovine Arp2/3 complex ahelix between blades 6 and 7 of ARPC1 contactsArp3 in a neighboring complex of the crystallattice. Robinson et al.3 speculated that this helixmight bind actin in a similar manner, making thishelix candidate site M-4 to link the complex to amother filament (Figures 2(c) and 6). Only tworesidues of this 13-residue helix are conserved:A298, which has a substitution of L in S. pombe,and F302, which is identical in all seven species.

We searched for a model using conserved sur-face residues to contact the mother filament thatmet the following experimental constraints. (1) Anangle of 708 between the mother and daughterfilaments.5 (2) The barbed ends of both motherand daughter filaments on the acute side of thebranch. (3) The mother and daughter filaments inthe same plane, but with the branch junctionslewed around the mother filament, much like amyosin head, rather than being attached to theside.24 (4) The C terminus of ARPC2 contacting themother filament, since antibodies to this sequenceblock branching.25 (5) ARPC1, ARPC2, and ARPC5close to actin at branch junctions, since all threesubunits can be chemically crosslinked to actin.44

(6) Arp2/3 complex in contact with three subunitsalong one long-pitch helix of the mother filament.24

We assumed that the conformation of Arp2/3 com-plex in the branch junction is similar to the activeconformation postulated by Robinson et al.3 withthe two Arps aligned like two subunits along theshort pitch of an actin filament helix and initiatingthe daughter filament. Thus we aligned thedaughter filament with Arp2. Both nucleationpromoting factors and mother filaments favor thisconformation and their binding is coupledthermodynamically.19 We also assumed that theactive conformation postulated by Robinson et al.3

would not significantly perturb the structure of anARPC1-ARPC2-ARPC4-ARPC5 tetramer andtherefore used the original bovine crystal structureand not a model of the active conformation.

We aligned the branch according to these criteriaand then searched manually for orientations thatoptimize contacts between conserved patches onthe complex and the mother filament (Figure 6).Requiring that the C terminus of ARPC2 be closeto the mother filament precluded contact of thelarge patch of conserved residues on ARPC1 withthe mother filament. The insert between blades 6and 7 of ARPC1 needs to be rotated approximately1808 (around the short axis) to interact with anactin subunit in the mother filament similar to thecontact with Arp3 in the crystal. The residuesflanking this helix are disordered in the crystal, sothe backbone may be sufficiently flexible to accom-modate this rotation. This model does not bring thelarge patch of conserved residues on ARPC1 or theconserved residues on the backside of ARPC4 intocontact with the mother filament. The model doesavoid a steric clash of ARPC5 with a mother

filament and brings into contact with the motherfilament conserved residues L46, S85, and K87(M-5). A total of 868 A2 of conserved residues onthe Arp2/3 complex surface contact the motherfilament in this model. The model has the Arp2/3complex and daughter filament rotated about 308around the long axis of the mother filamentrelative to the 2D projection in the reconstructionof electron micrographs.24

Conclusion

The surface residues conserved in Arp2/3 com-plex over the past 800 million years are attractivecandidates for mediating interactions of the com-plex with actin, actin filaments and nucleationpromoting factors. The identification of hypotheti-cal interaction sites will provide a guide for thechemical, mutagenic and structural studies thatare required to confirm these interactions.

Materials and Methods

The following sequences were used for the construc-tion of homology models (sequences are in the order ofArp2, Arp3, ARPC1-5): At, AAD31071, BAB01081,AAD20675, AAO63372, NP_564364, N/A, NP_567216; Ce,AAF36012, P53489, CAB54510, AAL32261, NP_499667,NP_498020, NP491099; Dd, S48844, AAC99776,AAC99777, AAC99778, AAC99779, AAC99780,AAC99781; Dm, AAF50488, P45888, CAB38634, Q9VIM5,NP_573193, NP_608996, NP_608693; Sc, NP_012599,CAA96460, P38328, P53731, NP_013474, P33204, P40518;Sp, A41790, Q9UUJ1, T45528, NP_593903, T39690, T39069,T37856. An Arabidopsis ARPC4 sequence was assembledby blasting with 10–20 amino acid pieces of bovineARPC4 against the published Arabidopsis genome nucleo-tide sequence. This assembled sequence is 90% similar toa predicted protein (AAO37935) of the same size in Oryzasativa. Initial searches in the annotated protein sequenceyielded a protein product containing a putative kinesindomain and an incomplete ARPC4 domain. This sequencemost likely represents an incorrect annotation.

Homology models were built using the program WhatIf45 running on a LINUX platform. Briefly, PDB 1K8Kwas entered into the soup with GETMOL. The secondarystructure profile was obtained with SOUPRF. GETSEQentered the sequence to be modeled. ALIPRF made theinitial alignment of the sequence to the template. Theinitial alignment was edited by hand. The final sequencealignment to be modeled was entered with GETPIR. Theinitial model was then built with the slow option ofBLDPIR. Unmodeled regions were rebuilt with FILGAP.Models were regularized with an energy minimizationoption, REFI. Surface areas and relative solvent exposurewere calculated with SETACC and VACACC. Alignedsequences were entered into an Excel spreadsheet andmacros were written to assign chemical types, determinethe conservation of residues at each position, to assessside-chain solvent exposure data and to assign residueclasses.

Model visualization and least-squares fits of moleculeswere performed using Swiss-PdbViewer (SPDBV).46 Sur-face representations used in Figures were also made


with SPDBV. Coordinates for homology models and thebranch junction model (Figure 6) and scripts for viewingconserved residues (Figure 2b) with SPDBV areavailable as Supplementary Material.

Acknowledgements

This work was funded by NIH research grantsGM26338 and GM066311 to T.D.P. We thank DavidSept for sharing his work on A segment bindingto Arp2/3 complex. We thank Jeffrey Kuhn forhelp with Excel macros and the staff of the YaleCenter for Structural Biology for help withcomputational methods.

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Edited by B. Honig

(Received 18 July 2003; received in revised form2 December 2003; accepted 4 December 2003)

Supplementary Material comprising 16 Figuresis available on Science Direct


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