Structural Basis of CYRI-B Direct Competition with Scar/WAVE Complex for Rac1

•CYRI-B structure reveals homology to CYFIP of the Scar/WAVE complex•CYRI-B shares a conserved interface with for Rac1 interaction•CYRI proteins form autoinhibited dimers that compete binding•Rac1-CYFIP model shows potential steric clashes RAC1 is major regulator actin dynamics, GTP-bound promoting assembly via complex. CYRI competes interaction in feedback loop regulating dynamics. Here, we reveal nature CYRI-Rac1 interaction, through crystal structures CYRI-B lacking N-terminal helix (CYRI-B?N) and CYRI-B?N:Rac1Q61L complex, providing molecular basis regulation We as having two subdomains - an binding subdomain unique Rac1-effector C-terminal Ratchet undergoes conformational changes induced by binding. Finally, show protein family, CYRI-A can produce hetero- or homodimers, adding additional layer signaling. Regulation cytoskeleton has direct impact on cellular shape, polarity, migration, homeostasis. Actin key effector shaping dynamics at membrane interfaces (Krause Gautreau, 2014Krause M. Gautreau A. Steering cell migration: lamellipodium directional persistence.Nat. Rev. Mol. Cell Biol. 2014; 15: 577-590Crossref PubMed Scopus (291) Google Scholar). Recently, novel Rac1, Fam49B also known (CYFIP-related Rac interactor), been reported bind active oppose activation complex (Fort et al., 2018Fort L. Batista J.M. Thomason P.A. Spence H.J. Whitelaw J.A. Tweedy Greaves J. Martin K.J. Anderson K.I. Brown P. al.Fam49/CYRI interacts locally suppresses protrusions.Nat. 2018; 20: 1159-1171Crossref (15) It was described local inhibitor branched assembly, thought be recruited activating signal-active Rac1-and activity This inhibition buffers polymerization process leading edge Scholar), increases T (Shang 2018Shang W. Jiang Y. Boettcher Ding K. Mollenauer Liu Z. Wen X. C. Hao Zhao S. al.Genome-wide CRISPR screen identifies FAM49B activation.Proc. Natl. Acad. Sci. U S 115: E4051-E4060Crossref (37) Scholar) promote resistance Salmonella infection (Yuki 2019Yuki K.E. Marei H. Fiskin E. Eva M.M. Gopal A.A. Schwartzentruber Majewski Cellier Mandl J.N. Vidal S.M. al.CYRI/FAM49B negatively regulates RAC1-driven cytoskeletal remodelling protects against bacterial infection.Nat. Microbiol. 2019; 4: 1516-1531Crossref CYRI-B, its isoform all possess evolutionarily domain (RBD) termed DUF1394 domain. Based this homology, CYRI's suppression Rac1-mediated proposed competition Nevertheless, structural interplay are not known. The composed five subunits; CYFIP, NCKAP1, Scar/WAVE, ABI, HSPC300. change (Chen 2010Chen Borek D. Padrick S.B. Gomez T.S. Metlagel Ismail A.M. Umetani Billadeau D.D. Otwinowski Rosen M.K. Structure control regulatory WAVE complex.Nature. 2010; 468: 533-538Crossref (275) recruitment plasma interactions charged phospholipids “active” Scholar; Hoeller 2016Hoeller O. Toettcher J.E. Cai Sun Huang Freyre Devreotes P.N. Weiner O.D. Gbeta coupling between oscillators polarity migration.PLoS 2016; 14: e1002381Crossref (24) Veltman 2012Veltman King J.S. Machesky L.M. Insall R.H. SCAR knockouts Dictyostelium: WASP assumes SCAR's position upstream regulators pseudopods.J. 2012; 198: 501-508Crossref (56) Activation releases VCA region Arp2/3, (Davidson Insall, 2011Davidson R. Actin-based motility: reopens old SCARs.Curr. 2011; 21: R66-R68Abstract Full Text PDF (16) While biochemically detected, little about interface. site “A” validated using pulldown experiments A recent cryo-EM fused revealed secondary site, which named “D” 2017Chen B. Chou H.T. Brautigam C.A. Xing Yang Henry Doolittle L.K. Walz T. GTPase activates distinct sites.Elife. 2017; 6: e29795Crossref (52) mutagenesis studies most potent lamellipodia cells Schaks 2018Schaks Singh S.P. Kage F. Klünemann Steffen Blankenfeldt Stradal T.E. Rottner Distinct sites rac have non-redundant functions vivo.Curr. 28: 3674-3684 e3676Abstract (30) Importantly, atomic level release remains unresolved. present mouse (residues 26–324 hereafter CYRI-B?N) human CYRI-B?N constitutively mutant Rac1Q61L.GppNHp. first provides details dictating specificity module. these structures, define interacting provide clues how directly Furthermore, leads homo- heterodimerize. Dimerization uses same binding, us propose autoinhibition mechanism CYRI. low sequence identity (?18% humans) subunit As there no data CYRI, set out solve structure. Despite sharing 80% identity, significant differences solubility were observed, latter showing better solubility. We, therefore, focused our crystallographic efforts CYRI-B. Initial screens up full-length although crystals obtained, they diffracted poorly resisted optimization. To obtain capable diffracting high resolution, residues (1–25) involved truncated, but entire 26–324) retained. solved 2.37Å resolution (Table 1), experimental phasing, replacement successful.Table 1Crystallographic Table StatisticsCYRI-B?N (PDB: 7AJL)CYRI-B?N: Complex 7AJK)Wavelength (Å)0.970.97Resolution range41.74–2.37 (2.45–2.37)55.45–3.10 (3.21–3.10)Space groupP 1 21 1P 62 2 2Unit cell44.73 166.68 45.1390.0 112.3 90.081.87 81.87 355.8790.0 90.0 120.0Total reflections76389914,916Unique reflections24,72313,492Multiplicity23.0 (21.5)10.6 (11.3)Completeness (%)99.94 (100.00)97.82 (99.77)Mean I/sigma(I)25.0 (3.5)18.7 (3.6)Wilson B-factor62.5488.66R-merge7.2 (86.4)9.2 (66.2)R-meas7.5 (90.8)9.6 (68.1)CC1/20.97 (0.97)0.99 (0.92)Reflections used refinement24,71013,490Reflections R-free1,242672R-work23.924.7R-free27.229.2No. non-hydrogen atoms4,7293,603Macromolecules4,5683,558Solvent16112RMS(bonds)0.0130.013RMS(angles)1.541.54Ramachandran favored (%)95.994.7Ramachandran allowed (%)4.15.1Ramachandran outliers (%)0.00.2Rotamer (%)7.26.0Clashscore15.614.1Average B-factor84.586.9Macromolecules84.987.0Solvent74.265.5 Open table new tab each monomer consists 12 alpha helices packed into bundles running perpendicular other L-shape fold (Figure 1A). asymmetric unit contains molecules “slotting” compact dimer 1B). produces large contact area 1524Å2, yet relatively few bonding contacts (Figures 1C, 1D) monomers. Briefly, R161 hydrogen bond A192, R165 M166 binds Q324, I168 R320, N169 S321 Q324. With such density monomer, suggests despite dimerization may weak. Using DALI server (http://ekhidna2.biocenter.helsinki.fi/dali/), compared PDB entries. showed only reasonable (RMSD <5Å protein) CYFIP1 1E). Remarkably, given divergence sequence, overlay RMSD 2.7Å C? atoms, 1.9Å 26–214 greatest corresponds helical bundle where alignment 215–324 gives 5.1Å atoms due insertion antiparallel ?-hairpin alignment, module comprising 1F). runs size exclusion chromatography S3A). Thus test if observed occur solution it heterodimerize CYRI-A, assays, weak reproducible MBP-CYRI-A HA-CYRI-B S1A Figure 4B). Since CYRI-B:CYRI-A heterodimer available validate interface, mutated based homodimer Both single double mutation arginine reduce 50% S1B). investigate heterodimerization could context, performed proximity ligation assay (PLA). positive signal strongly wild-type significantly reduced RRDD mutant, confirming possibility S2B S2C).To determine whether overlaid 3P8C) S3B). resulting minimal suggesting feature containing proteins. In line observation, higher-order oligomers (Pipathsouk 2019Pipathsouk Brunetti R.M. Town J.P. Breuer Pellett Marchuk Tran N.T. Krummel M.F. Stamou self-organization templates lamellipodial formation.bioRxiv. https://doi.org/10.1101/836585Crossref (0) understand domains general specifically, determined Rac1Q61L. Attempts co-crystallize Rac1Q61L.GppNHp successful, likely affinity (measured dissociation constant (Kd) 25?M – Figures S3C S3D). overcome challenge, 10-residue linker (GSAGSAGSAG) CYRI-B?N's C-terminus identified successfully produced 3.1Å 2A, 1). one copy Rac1Q61L molecule trans from symmetry mate S3E S3F). Examination CRYI-B?N primarily mediated extensive switch-I 25–39) along adjacent ?-strand contribution switch-II 2B), 1097Å2. effectors, complexes; Rac1: Arfaptin 1I4T), pPhox67 1E96) PRK 2RMK) 2C, 2D, 2E). Although utilizes made Q162, A159 L160, Q162 (Lapouge 2000Lapouge Smith Walker Gamblin S.J. Smerdon Rittinger TPR p67phox Rac.GTP.Mol. Cell. 2000; 899-907Abstract (254) fewer than both make (Modha 2008Modha Campbell L.J. Nietlispach Buhecha H.R. Owen Mott polybasic required PRK1.J. Chem. 2008; 283: 1492-1500Abstract (44) Tarricone 2001Tarricone Xiao Justin N.N. Arfaptin-mediated cross-talk Arf signalling pathways.Nature. 2001; 411: 215-219Crossref (199) CYRI-B?N. results different conformation when bound 2F). Thus, unique, explains Rac1. Binding combination polar hydrophobic interactions. 3A 3B ). R160 residue forms salt bridge side chain D38 main-chain carbonyl group F37. Q153 R64 S41 N26 respectively. M147 N52 S157 peptide N39 explaining modest P29S clinically important (Hodis 2012Hodis Watson I.R. Kryukov G.V. Arold S.T. Imielinski Theurillat Nickerson Auclair Li Place al.A landscape driver mutations melanoma.Cell. 150: 251-263Abstract (1609) Krauthammer 2012Krauthammer Kong Ha B.H. Evans Bacchiocchi McCusker Cheng Davis M.J. Goh G. Choi al.Exome sequencing recurrent somatic melanoma.Nat. Genet. 44: 1006-1014Crossref (773) allows undergo spontaneous nucleotide exchange absence GEF, producing shift (Davis 2012Davis Holman E.C. Halaban Schlessinger Boggon T.J. RAC1P29S spontaneously cancer-associated GTPase.Proc. 110: 912-917Crossref (84) dual Q61L effectors CRIB 2019Whitelaw Lilla Paul N.R. Fort Zanivan CYRI/Fam49 represent class interactors.Communicative Integr. 12: 112-118Crossref (4) P29 located 3A), speculate serine result bonds, perhaps increase Previously shown another report indicates P150 bonds mutating aspartic acid would introduce repulsive charge-charge thus disrupt formation S4A). Rac1's distance nearest 4.4Å, reversal charge will S4A S4B). case S4C S4D). close making contacts, clashes, inhibiting 3C). further rationally introduced alanine arginine, 3D). Following experiments, resulted 3E 3F). conclude effector-binding identify specifically Overlaying dimer, see 4A). raises autoinhibit regulate functionality. assays repeated presence increasing levels seen 4B, 4C, 4D, homo heterodimers disrupted addition Interestingly, following swap 5A, Video S1). clash Q2 Y305 5A). domain-swapped occupy shifted 5Å, allowing without might act alternative 5C). When overlaying 5D), observed. implies either specific captured occur. However, failed induce solution, swapping crystallization artifact. Nonetheless, does Therefore, assuming evolved fragment retained activity, acquired defining features, distinguishing pure destabilization intramolecular common Rho family complexes proteins, formins (Kühn 2015Kühn Erdmann Block Schwenkmezger Geyer FMNL2-Cdc42 yields insights filopodia formation.Nat. Commun. 2015; 7088Crossref (39) IRSp53 (Kast 2014Kast D.J. Disanza Boczkowska Madasu Scita Svitkina Dominguez Mechanism combinatorial Cdc42 downstream effectors.Nat. Struct. 413-422Crossref (38) alpha-helical 215–324) 26–215), subdomains. 5A), upon destabilizes RBD clashes. precise biological role Rac1-induced elusive. question field analogy interact while Cdc42. less related GTPases RhoA RND1 6A). Together comparison 4JS0) selectivity becomes apparent. Residues A27, G30 lysine Cdc42; 6B–6E). breaking whereas W56 phenylalanine Cdc42, reduces extent Mutation S41A serves selectivity, mimics disrupts ?30% 3D, E). Sequence more distantly members 6A) demonstrates even greater variation together similarity extrapolate similar mode. tempting reason variations explain selective For example, A27K (a variation) 6B), mimicking cdc42, prevent summary, module, alignments evidence specificity. S4). them isoforms exception M147N; however, amino acid's 3B). (R159D, R160D R160D, R161D CYRI-B) comparable reductions involve limited sought use template interaction. ?4-?6 meander WAVE1) previously 7A 7B). All docked Of (R190, C179, E434, F626, M632), (R190 CYFIP) CYFIP1/2 CYRI-A/B coupled 6A, S5A, S5B). nine (Q68, N154, S157, R160, R161, N185, S188, L189 A192) chemical properties. supports informed Ra