to facilitate crystallization of the wrc we genetically deleted the c - terminal proline - rich region and sh3 domain of abi2 , and replaced the proline - rich region of wave1 with an 18-residue linker .
the resulting miniwrc is inactive toward the arp2/3 complex but can be stimulated by rac1-gmppnp .
crystals of miniwrc contained one complex in the asymmetric unit and diffracted to 2.3  at a synchrotron light source .
phases were obtained by multiple isomorphous replacement with anomalous scattering ( miras ) using preparations containing selenomethionine - labeled sra1 and nap1 ( supplementary table 1 ) .
miniwrc has an elongated shape with approximate dimensions of 200  110  80  ( fig .
two - dimensional class averages from electron micrographs of negatively stained miniwrc and full - length wrc are indistinguishable , and of similar dimensions as the crystal structure ( supplementary fig .
the structure of miniwrc is thus likely a faithful representation of the structured elements of the wrc .
miniwrc can be delineated into two subcomplexes : an sra1:nap1 dimer and a wave1:abi2:hspc300 trimer ( fig .
1 ) . sra1 and nap1 have homologous structures ( see below ) and interact extensively to create an elongated pseudo - symmetric dimer , which forms a platform for the trimer ( supplementary fig .
the n - terminal helix of sra1 links to the rest of the complex through a flexible sequence that lacks electron density ( residues 23 - 56 ) , and contacts an adjacent molecule in the crystal lattice ( supplementary fig .
3 ) . the trimer contacts the sra1:nap1 dimer in a tripartite manner . a long four - helix bundle created by a helix from hspc300 ( residues 14 - 68 ) , two helices from abi2 (
residues 1 - 39 and 43 - 112 ) and a helix from wave1 ( residues 26 - 81 ) contacts sra1 extensively and is aligned roughly parallel to the long axis of the dimer ( supplementary figs . 4 and 5 ) .
the most extensive contacts are made by hspc300 , which is sandwiched between sra1 and abi2:wave1 across the entire length of its helix .
the  homeo - domain homologous region of abi2 ( residues 112 - 155 ) adopts an extended conformation running around the rim of a large cavity on nap1 ( supplementary fig . 6 ) .
the c - terminus of wave1 , including the v- and the c - regions , forms an irregular , loosely - packed chain that lies against a concave surface of sra1 adjacent to the long side of the four - helix bundle . as detailed below , interactions of elements in the c
- terminus of wave1 with sra1 and each other are central to regulation of wrc activity .
the structure reveals that sra1 and nap1 have the same domain organization ; their coordinates can be superimposed with a root mean square deviation ( r.m.s.d . ) of 6.9  for 681 c atoms with dali z - score 17.9 ( supplementary fig .
thus , they belong to the same protein family despite their low sequence identity ( 13% ) .
homology between sra1 and nap1 is also supported by hhpred , which showed additional human members of this family that are similar in size : kiaa1033/swip and strumpellin , with similarity extending over their entire lengths . when analyzed pair wise , swip is more similar to sra1 and strumpellin is more similar to nap1 .
we and others recently reported that swip and strumpellin form a pentameric complex ( shrc , for wash regulatory complex ) containing the proteins ccdc53 and fam21 , and another wasp family member , wash . within the shrc
hhpred and biochemical analyses suggest that ccd53 , and the n - termini of wash and fam21 are structurally and/or functionally similar to hspc300 and the n - termini of wave and abi , respectively .
these many similarities , coupled with the similar overall shape of the wrc and shrc , suggest that the shrc is analogously organized as a large swip : strumpellin platform bound to a helical bundle of wash : ccdc53:fam21 , with the wash vca sequestered by a similar mechanism .
we validated the structural organization observed in the crystal by replacing wild type wave2 in hela cells with mutants targeting the trimer interface . mutating the wave - hspc300 interface ( i50d / l54dwave2 ) or the wave - hspc300/abi interface ( l40d / f51dwave2 ) appreciably decreased co - immunoprecipitation of wave2 with the four other components of the wrc ( supplementary fig .
the wave1 vca is bound by a conserved surface of sra1 and residues 82 - 184 of wave1 , which form five helices ( 2-6 ) and a series of intervening loops .
this element of wave1 traces a meandering path across a concave surface of sra1 , and we refer to it as the  meander region  ( fig .
contacts between the meander region and sra1 bury over 2,100  ( ~56% of the total wave1-sra1 interface ; supplementary fig .
8) , as is its contact surface on sra1 , suggesting its interactions and irregular structure are conserved .
the v- and c - regions of the vca lie on the surface of sra1 and form two amphipathic helices ( residues 500 - 514 and 531 - 543 , respectively ) that also pack against 2 and 6 of wave1 , respectively ( supplementary fig .
the a - region of the vca ( residues 545 - 559 ) is likely disordered , as it is not observed in the electron density . during actin filament nucleation ,
the v - region recruits an initial actin monomer to the nascent filament , while the c- and a - regions contribute binding energy and induce activating conformational changes in the arp2/3 complex .
the structure and complementary experimental data below indicate that sequestration of both the v- and the c - regions by sra1 and the meander region of wave1 underlies vca inhibition within the wrc .
inhibition of the v - region involves a combination of contacts to actin - binding residues and induction of structure that is incompatible with actin binding ( fig .
2b ) . in the complex of the v - region of wave2 with actin ,
residues equivalent to 497 - 507 of wave1 form a helix that inserts into the cleft between actin subdomains 1 and 3 .
residues equivalent to 508 - 516 are extended , and the ile509 and arg512 equivalents contact actin . in the structure of miniwrc ,
the entire v - region is helical , and the side chains of leu 501 , leu 502 , ile 505 , ile 509 and arg 512 are buried in the sra1 interface , making the v - region inaccessible to actin .
sequestration of the v - region is an important contributor to wrc inhibition , as mutating v - helix contact residues leu 841 , phe 844 and trp 845 of sra1 constitutively activates miniwrc toward the arp2/3 complex ( l841a / f844a / w845asra1-miniwrc , fig .
the c - helix is also critical for activation of arp2/3 complex , as mutations of val 531 , leu 535 or arg 538 in the wave1 vca reduce activity toward arp2/3 complex by at least 50% . in the miniwrc
, the c - helix buries its hydrophobic face in the sra1-6 interface ( fig .
intermolecularly , val 531 , ala 532 , leu 535 and ile 539 of wave1 make van der waals contacts with sra1 .
intramolecularly , val 531 , ile 534 , leu 535 , arg 538 and val 541 of the c - helix pack against 6 .
hence , the structure shows that the wrc sequesters c - helix residues important for activation of the arp2/3 complex , resulting in inhibition .
this mechanism is also supported by mutagenesis : perturbing contacts of the c - helix with either sra1 ( l697d / y704dsra1-miniwrc , fig .
additionally , replacement of wild type wave2 with equivalent levels of the analogous 6 mutant ( w160e / k161dwave2 ) in hela cells does not alter the integrity of the complex ( supplementary fig .
4 ) , but causes a dramatic redistribution of actin , with loss of stress fibers and assembly of filaments at the cell periphery , again consistent with constitutive activation of the wrc ( fig .
interactions of the vca , and perhaps the structure of the entire meander - vca element , appear to be highly cooperative , as perturbation of either the v- or c - region contacts produces wrc activity near that of the isolated vca ( figs .
rac plays an important role in controlling actin polymerization and lamellipodia formation through activation of the wrc in vivo .
rac1 binds to recombinant wrc and activates it in vitro at micromolar concentrations under optimized assay conditions without disrupting the integrity of the complex ( supplementary fig .
12 ) . by analogy to the activation of wasp by cdc42 , we reasoned that removing the vca from the wrc would increase its affinity for rac1 .
indeed , in pull down assays immobilized gst - rac1:gmppnp ( a gtp analog ) bound a vca - deleted wrc ( wrc ) to a greater extent than miniwrc ( supplementary fig .
13 ) . using equilibrium dialysis we measured a dissociation constant ( kd ) of 1 - 2 m for wrc .
miniwrc bound rac with lower affinity , having kd of 7 - 10 m ( fig .
3b ) had affinity similar to that of wrc , consistent with notion that sequestration of the vca motif competes with rac1 binding .
these results imply that rac1 binds to the body of the wrc competitively with the vca ( either directly or indirectly ) , leading to activation of the complex . to better define the rac1 interaction surface , we searched for wrc mutants with defects in rac1 binding
/ y704d , l841a / f844a / w845a or e250k / q399a of sra1 did not perturb the binding of wrc to rac1 ( data not shown )
. however , sra1 mutations c179r , r190d , m632d and e434k / f626a , severely impaired binding of wrc to rac1 without altering the integrity of the recombinant complex ( fig .
these highly conserved sra1 residues are located in a patch adjacent to 4-6 of the wave1 meander region ( fig . 3c and supplementary fig .
furthermore , truncation of 6 ( 154wave1-wrc ) also decreases rac1 binding ( fig . 3c and supplementary fig .
13 ) , suggesting that the helix or adjacent parts of the meander may contact the bound gtpase directly or stabilize its interaction sites on sra1 .
these data implicate a rac1 binding site on the wrc involving an sra1 surface and perhaps part of the meander region of wave1 .
interactions of rac1 could then trigger conformational changes in the meander region and/or its contact site on sra1 . since the meander appears to cooperatively stabilize the v- and c - regions of wave1 ( see above ) , these perturbations could drive wrc activation by causing release of the vca .
several groups report that phosphorylation of the strictly conserved tyr 150 of wave2 ( tyr 151 in wave1 and wave3 ) by the abl kinase is important for wrc - mediated actin assembly and lamellipodia formation .
additionally , phosphorylation of wave tyr 125 by src or thr 138 by cdk5 , alters cellular actin dynamics .
tyr 151 of wave1 is located in the 5-6 loop of the meander region , and is buried in a hydrophobic pocket formed by sra1 and wave1 ( fig . 3c and supplementary fig 9 ) .
phosphorylation of tyr 151 would thus disrupt the contacts between the meander region and sra1 , leading to destabilization of the c - helix of the vca motif and wrc activation . to test this idea , we reconstituted a miniwrc containing a phospho - mimicking y151e mutation in vitro , and generated the analogous full - length wrc in hela cells using a knock down / reexpression strategy .
consistent with the structural predictions , the mutant complexes displayed high actin assembly activity both in vivo and in vitro ( figs .
mutation of the y151-binding pocket in sra1 ( f686esra1-miniwrc ) equivalently activates the wrc ( fig .
3d ) . tyr 125 of wave1 is also strictly conserved from animals to plants ( supplementary fig .
this residue is located in 3 and its side chain packs against gln 685 and makes a hydrogen bond to asp 689 of sra1 ( fig 3c and supplementary fig . 9 ) .
phosphorylation of tyr 125 should disrupt the contact with asp 689 , and could destabilize the meander region of wave1 , leading to release of the vca .
consistent with this , replacement of wave2 with a y124d mutant in cells increased lamellipodia formation ( fig .
makes intramolecular contacts with 4 and 5 ; its hydroxyl group is part of a network of hydrogen bonds that span between these secondary elements ( fig . 3c and supplementary fig .
phosphorylation of thr 138 may thus also perturb the structure of the meander region , again contributing to activation of the wrc .
together , the data suggest that , analogous to rac1 activation , phosphorylation could destabilize the meander region and/or its interactions with sra1 , leading to release of the vca and activation of the wrc .
the wrc is typically clustered to high density at its sites of action in cells .
this is believed to be necessary for spatially restricted actin assembly during , for example , polarized cell movement .
clustering is mediated by the combined actions of phosphoinositide lipids and rac , as well as various sh3 containing proteins .
the polybasic region of wave2 ( equivalent to residues 172 - 184 of wave1 ) can bind phosphoinositide lipids in vitro , and is essential for membrane recruitment of the wrc and formation of lamellipodia in cells .
surface electrostatic calculations show that the face containing the wave1:abi2:hspc300 four - helix bundle is negatively charged ( fig .
4a ) , while much of the face of the complex adjacent to the polybasic region is positively charged ( fig .
this polar distribution suggests that when the wrc is recruited to the plasma membrane , the side covered by the four - helix bundle is exposed to the cytoplasm , while the opposite side contacts the membrane . in this orientation rac
4c ) , and its c - terminal isoprene group , the polybasic region of wave and the basic surface of the sra1/nap1 dimer could all be directed toward the plasma membrane .
the meander region and the vca motif of wave would face the cytoplasm , making them accessible to other regulators ( e.g. kinases ) , and to the arp2/3 complex and actin .
this organization would allow simultaneous phosphoinositide and rac binding , cooperatively recruiting the wrc to membranes and enhancing allosteric activation .
self - association of the wrc at membranes , and consequent enhanced activity , could be mediated by inter - complex binding of the n - terminal helix of sra1 with the wave / abi / hspc300 trimer , as observed in the crystal lattice ( supplementary fig .
sra1 was recently reported to support translation inhibition through simultaneous binding to the translational regulator , fmrp , and the translation initiation factor , eif4e .
however , the putative mode of eif4e binding is incompatible with the wrc structure ( fig . 1b , supplementary fig .
thus , eif4e may bind to isolated sra1 , but not the wrc , consistent with the finding that eif4e co - immunoprecipitates with sra1 but not wave .
these observations suggest that sra1 may partition between the wrc , which regulates actin dynamics , and a free ( or alternatively complexed ) state , which regulates translation .
protocadherin-10 which binds the wrc , and fmrp  are implicated in autism and other mental disorders , suggesting that an appropriate balance of these pathways or their joint action may be needed for proper neuronal development and function .
future studies of the intact wrc and its separate components will reveal how this system coordinates multiple processes in normal and abnormal cellular function .
sra1 , nap1 , wave1 , abi2 and hspc300 were overexpressed separately , partially purified , assembled into an sra1:nap1 dimer and a wave1/abi2/hspc300 trimer , respectively , and then assembled into the miniwrc pentamer .
crystals of miniwrc were obtained by hanging - drop vapor - diffusion at 4 c .
all the data sets were collected at the id-19 beamline ( aps , chicago ) and processed with the hkl3000 suit and ccp4 suites .
experimental phases were determined from se - miras data collected on samples containing either semet - sra1 or semet - nap1 , and analyzed using shelxd .
the atomic model of the complex was built using buccaneer and coot , and refined using refmac 5 .
equilibrium dialysis was done at room temperature and protein concentrations were determined using deep purple gel staining ( ge healthcare ) .
actin polymerization and gst - rac1 pull - down assays were performed as described previously .
hela cells were grown directly on coverslips , fixed in 4% paraformaldehyde , and prepared for immunofluorescence as described .
 members of the wiskott - aldrich syndrome protein ( wasp ) family control cytoskeletal dynamics by promoting actin filament nucleation by the arp2/3 complex . 
 the wasp relative , wave , regulates lamellipodia formation within a 400 kda , hetero - pentameric wave regulatory complex ( wrc ) . 
 the wrc is inactive toward the arp2/3 complex , but can be stimulated by the rac gtpase , kinases and phosphatidylinositols . 
 we report the 2.3  crystal structure of the wrc and complementary mechanistic analyses . 
 the structure shows that the activity - bearing vca motif of wave is sequestered by a combination of intramolecular and intermolecular contacts within the wrc . 
 rac and kinases appear to destabilize a wrc element that is necessary for vca sequestration , suggesting how these signals stimulate wrc activity toward the arp2/3 complex . 
 spatial proximity of the rac binding site and a large basic surface of the wrc suggests how the gtpase and phospholipids could cooperatively recruit the complex to membranes . 
Overall structure of the WRC
Mechanism of WRC inhibition
Activation by Rac
Activation by phosphorylation
Discussion
Methods summary
Supplementary Material