Structural Basis for Mutual Relief of the Rac Guanine Nucleotide Exchange Factor DOCK2 and Its Partner ELMO1 from Their Autoinhibited Forms

Structural basis for mutual relief of the Rac guanine nucleotide exchange factor DOCK2 and its partner ELMO1 from their autoinhibited forms

Kyoko Hanawa-Suetsugua, Mutsuko Kukimoto-Niinoa,b, Chiemi Mishima-Tsumagaria, Ryogo Akasakaa, Noboru Ohsawaa, Shun-ichi Sekinea,c, Takuhiro Itoa,c, Naoya Tochioa, Seizo Koshibaa, Takanori Kigawaa,d, Takaho Teradaa,b, Mikako Shirouzua, Akihiko Nishikimib,e,f, Takehito Urunoe, Tomoya Katakaig, Tatsuo Kinashig, Daisuke Kohdah, Yoshinori Fukuib,e,f,1, and Shigeyuki Yokoyamaa,b,c,1

aRIKEN Systems and Structural Biology Center, 1-7-22 Suehiro, Tsurumi, Yokohama 230-0045, Japan; bJapan Science and Technology Agency, Core Research for Evolutional Science and Technology, Tokyo 102-0075, Japan; cDepartment of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan; dTokyo Institute of Technology, 4259 Nagatsuta, Midori, Yokohama 226-8502, Japan; eDivision of Immunogenetics, Department of Immunobiology and Neuroscience, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan; fResearch Center for Advanced Immunology, Kyushu University, Fukuoka 812-8582, Japan; gDepartment of Molecular Genetics, Institute of Biomedical Science, Kansai Medical University, Osaka 570-8506, Japan; and hDivision of Structural Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan

Edited by John Kuriyan, University of California, Berkeley, CA, and approved December 31, 2011 (received for review September 6, 2011)

DOCK2, a hematopoietic cell-specific, atypical guanine nucleotide for the Rho-family GTPases. There are 11 mammalian members exchange factor, controls lymphocyte migration through ras-related (DOCK180, DOCK2-11) of the CDM family. The CDM proteins C3 botulinum toxin substrate (Rac) activation. Dedicator of cytokin- share the DOCK-homology regions (DHR)-1 and DHR-2 (also esis 2–engulfment and cell motility protein 1 (DOCK2•ELMO1) com- known as Docker domains), and lack the Dbl homology (DH) plex formation is required for DOCK2-mediated Rac signaling. In this and pleckstrin homology (PH) domains typically present in the study, we identified the N-terminal 177-residue fragment and the mammalian Rho-family GEFs (6, 9, 10). DOCK2 mediates the C-terminal 196-residue fragment of human DOCK2 and ELMO1, re- Rac GEF reaction by means of the DHR-2 domain (6, 10, 11). spectively, as the mutual binding regions, and solved the crystal DOCK2 also interacts with phosphatidylinositol 3,4,5-tripho- structure of their complex at 2.1-Å resolution. The C-terminal Pro- sphate [PtdInsð3;4;5ÞP3] through the DHR-1 domain (7, 12), to rich tail of ELMO1 winds around the Src-homology 3 domain of target the GEF activity to the plasma membrane. Recently, the DOCK2, and an intermolecular five-helix bundle is formed. Overall, crystal structure of the DHR-1 domain of DOCK180 was deter- the entire regions of both DOCK2 and ELMO1 assemble to create a mined (13), as well as those of the DHR-2 domains of DOCK2 rigid structure, which is required for the DOCK2•ELMO1 binding, as and DOCK9 bound with their substrates, Rac1 and cell division revealed by mutagenesis. Intriguingly, the DOCK2•ELMO1 interface cycle 42 (Cdc42), respectively (14, 15). BIOCHEMISTRY hydrophobically buries a residue which, when mutated, reportedly DOCK2, like DOCK180 and DOCK3-5, has a Src-homology relieves DOCK180 from autoinhibition. We demonstrated that the 3 (SH3) domain at its N terminus. The N-terminal 502-residue re- ELMO-interacting region and the DOCK-homology region 2 guanine gion, including the SH3 domain, of DOCK2 interacts with engulf- nucleotide exchange factor domain of DOCK2 associate with each ment and cell motility protein 1 (ELMO1), a mammalian other for the autoinhibition, and that the assembly with ELMO1 homologue of C. elegans CED-12 (16). ELMO1 contains the N- weakens the interaction, relieving DOCK2 from the autoinhibition. terminal ras homolog gene family, member G (RhoG)-binding re- The interactions between the N- and C-terminal regions of ELMO1 gion, the ELMO domain, the PH domain, and the C-terminal se- reportedly cause its autoinhibition, and binding with a DOCK quence with three PxxP motifs. The C-terminal region of ELMO1, protein relieves the autoinhibition for ras homolog gene family, including the Pro-rich sequence, binds the SH3-containing region member G binding and membrane localization. In fact, the DOCK2•- of DOCK2, which is required for DOCK2 to activate Rac in vivo (16). Similarly, DOCK180 forms a ternary complex with ELMO1 ELMO1 interface also buries the ELMO1 residues required for the and Rac (17, 18), which is essential for the in vivo catalytic activity autoinhibition within the hydrophobic core of the helix bundle. (17, 19). ELMO1 binds to the N-terminal region (about 200 resi- Therefore, the present complex structure reveals the structural basis dues), including the SH3 domain, of DOCK180 (17, 19). However, by which DOCK2 and ELMO1 mutually relieve their autoinhibition an ELMO1 mutant lacking the Pro-rich sequence retains the ability for the activation of Rac1 for lymphocyte chemotaxis. to bind DOCK180 (20), suggesting that the region flanking the Pro- ∣ ∣ ∣ ∣ rich sequence of ELMO1 also participates in DOCK180 binding. X-ray crystallography NMR protein complex CDM proteins Furthermore, the DOCK180•ELMO1 complex was proposed to immunology function as a bipartite GEF for Rac, in which the DHR-2 domain edicator of cytokinesis 2 (DOCK2), a large protein with a Dmolecular mass of 213 kDa, is specifically expressed in he- Author contributions: M.S., Y.F., and S.Y. designed research; K.H.-S., M.K.-N., C.M.-T., R.A., matopoietic cells (1). DOCK2 plays a critical role in lymphocyte N.O., S.-i.S., T.I., N.T., S.K., T. Kigawa, T.T., A.N., T.U., D.K., and S.Y. performed research; T. Katakai and T. Kinashi contributed new reagents/analytic tools; K.H.-S., M.K.-N., Y.F., migration and activation by regulating the actin cytoskeleton and S.Y. analyzed data; and K.H.-S., M.K.-N., Y.F., and S.Y. wrote the paper. through ras-related C3 botulinum toxin substrate (Rac) activa- The authors declare no conflict of interest. tion (2, 3), and the deletion of DOCK2 enables long-term cardiac This article is a PNAS Direct Submission. allograft survival (4). DOCK2 also controls various immunolo- Freely available online through the PNAS open access option. gical functions, including helper Tcell differentiation (5), neutro- Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, phil chemotaxis (6, 7), and type I interferon induction in plasma- www.pdb.org (PDB ID codes 2RQR, 3A98, 3B13). cytoid dendritic cells (8). DOCK2 belongs to the CDM family 1To whom correspondence may be addressed. E-mail: [email protected] (Caenorhabditis elegans CED-5, mammalian DOCK180, and or [email protected]. Drosophila melanogaster Myoblast city) of the evolutionally con- This article contains supporting information online at www.pnas.org/lookup/suppl/ served, atypical guanine nucleotide exchange factors (GEFs) doi:10.1073/pnas.1113512109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1113512109 PNAS ∣ February 28, 2012 ∣ vol. 109 ∣ no. 9 ∣ 3305–3310 Downloaded by guest on September 30, 2021 of DOCK180 and the PH domain of ELMO1 cooperatively function (20). The crystal structure of the ELMO1 PH domain has recently been determined, and the unusual α-helical N-terminal extension of the PH domain in ELMO1 was found to be essential for DOCK180 binding and DOCK180-mediated Rac signaling (21). In the absence of ELMO1, the N-terminal region, including the SH3 domain, of DOCK180 interacts with the DHR-2 domain, which may facilitate the autoinhibition of the DOCK180 GEF activity (22). ELMO1 binding to the SH3 domain disrupts the SH3•DHR-2 interaction, and allows Rac binding to the DHR-2 domain for the GEF activity (22). This autoinhibition of DOCK180 is relieved by the mutation of Ile31 in the SH3 domain, even though the location of this residue is distant from the PxxP-binding pocket. ELMO1 by itself is also reportedly autoinhibited because of the interaction of the ELMO inhibitory domain (EID), lying between the C-terminal PxxP motif and the PH domain, with the ELMO autoregulatory domain (EAD) residing between the N-terminal RhoG-binding domain and the ELMO domain. Actually, EAD binding to DOCK180, as well as the mutations of Met692 and Glu693 in the EAD, disrupts this intra- Fig. 1. Mutually interactive regions of DOCK2 and ELMO1. (A) Domain or- molecular inhibitory interaction, resulting in RhoG binding and ganizations of DOCK2 and ELMO1. Known protein interaction and functional membrane localization (23). Consequently, DOCK2 and ELMO1 regions are indicated. Blue and red bars indicate the DOCK2 and ELMO1 mutually relieve their autoinhibitions by an unknown interaction regions included in the NMR construct. Green and yellow bars indicate the mode involving the DOCK2 N-terminal and ELMO1 C-terminal regions used for the crystal structure determination. ELMO1-interacting re- regions, a scenario that is far more complicated than simple gion (EIR) and DOCK2-interacting region (DIR) indicate the ELMO1-interact- SH3•PxxP binding. ing region and the DOCK2-interacting region, respectively. (B) Overviews of In the present study, we identified the regions required for the the crystal structure of the DOCK2•ELMOl complex. Ribbon representations • interaction between DOCK2 and ELMO1, and determined their of the DOCK2 ELMOl complex. DOCK2 is colored green and ELMO1 is yellow. The six proline residues in the ELMO1 Pro-rich tail are colored red. The two complex structure. The SH3 domain of DOCK2 binds to the views are related by a 90° rotation about the vertical axis. C-terminal sequence of ELMO1 much more extensively than other SH3 domains, which only bind the PxxP motif. Moreover, SH3 domain resembles those of other SH3 domains, and consists α the -helical region following the SH3 domain forms an intermo- of five β-strands packed in two β-sheets, which form a β-barrel- lecular five-helix bundle with both the N- and C-terminal exten- like structure (Fig. S2D). The ELMO1 peptide binds to the sions of the PH domain of ELMO1. Thus, the entire region is DOCK2 SH3 domain as a polyproline II helix in a class II orien- assembled into a rigid structural unit. The residues required for tation (27, 28). Tyr44 of DOCK2 is buried in the center of the the respective autoinhibitions (i.e., Ile31 of DOCK2 and Met692 interface and hydrophobically interacts with Pro714 and Pro717 of ELMO1) are buried in the hydrophobic core of the intermo- of ELMO1. These analyses revealed that, among the three can- lecular assembly, thus revealing the structural basis of the mutual didates, P714-x-x-P717 serves as the PxxP motif. On the other autoinhibition relief. Further mutagenesis demonstrated that this hand, the N-terminal seven residues of the ELMO1 peptide unique assembly mode is important for ELMO1 to relieve the (residues 697–703) were poorly defined, probably because of high autoinhibition of DOCK2, so it can participate in Rac activation mobility, although they were necessary for the structure determi- in lymphocyte chemotaxis. nation (Figs. S1 and S2C). This observation prompted us to exam- Results ine whether any additional interactions, besides the SH3 domain– NMR Analysis of the DOCK2 SH3 Domain Interaction with ELMO1 Pep- PxxP motif interaction, also occur between DOCK2 and ELMO1. tides. The C terminus of ELMO1 contains six Pro residues located close together (residues 707, 710, 711, 712, 714, and 717), con- Crystal Structure of the Complex of the DOCK2 and ELMO1 Interacting stituting three potential PxxP motifs, which are the potential SH3 Domains. First, we determined the ELMO1-interacting region of domain binding sites. To define the interaction between the DOCK2 and the DOCK2-interacting region of ELMO1 that are DOCK2 SH3 domain and the ELMO1 Pro-rich sequence, we first suitable for crystallographic studies (see SI Text). We generated – generated various fusion constructs for cell-free protein synthesis longer fragments of DOCK2 (residues 1 177; the SH3 domain – (24–26) and obtained one sample suitable for structure determi- and the flanking region) and ELMO1 (residues 532 727; includ- nation by NMR spectroscopy (Fig. S1). The fusion construct ing the PH domain and the Pro-rich region) by cell-free protein includes a 26-residue ELMO1 peptide (residues 697–722) fused synthesis (Fig. S3). The DOCK2 fragment precipitated during to the N terminus of the DOCK2 SH3 domain (residues 8–70) the synthesis, but it was prepared as a soluble protein complex (Fig. 1A and Fig. S2A). For comparison, we also analyzed the by cell-free coexpression with the ELMO1 fragment. The purified DOCK2 SH3 domain alone and compared its chemical shifts for complex between the DOCK2 and ELMOl fragments (hereafter the backbone 15N and 1HN atoms with those in the fusion sample. referred to as the DOCK2•ELMOl complex) was stable and The residues located in the arginine and threonine pair (RT) monomeric with 1∶1 stoichiometry, as determined by analytical loop, the n-Src loop, β4, and the helix-like loop between β4 ultracentrifugation (Fig. S4A). and β5 exhibited large chemical shift changes (Fig. S2B). We determined the crystal structure of the DOCK2•ELMOl We determined the solution structure of the DOCK2 SH3– complex (Table S2). The asymmetric unit contained two virtually ELMO1 peptide fusion complex (Table S1). The rmsd from the identical DOCK2•ELMOl complexes. Overall, DOCK2 and mean structure was 0.78 Å for the backbone atoms and 1.19 Å for ELMO1 form a large, rigid complex involving the entire regions all heavy (non-hydrogen) atoms in the well-ordered region (resi- of the two protein fragments. The DOCK2 structure is composed dues 704–722 for the ELMO1 peptide and residues 9–68 for the of the N-terminal SH3 domain (residues 1–69) and a three-helix DOCK2 SH3 domain) (Fig. S2C). The topology of the DOCK2 bundle domain (residues 81–167; Dα1–3). The electron density

3306 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1113512109 Hanawa-Suetsugu et al. Downloaded by guest on September 30, 2021 of the 11-residue region connecting the two domains was not tail. In contrast, the DOCK2 SH3 domain binds the N-terminal observed. The ELMO1 structure is composed of the PH domain half (Asp700–Ile713), mostly through hydrophobic interactions. (residues 560–674), its N- and C-terminal extended helices Eα1 Second, the DOCK2 SH3 domain also interacts with Eα1 and and Eα3 (residues 532–558 and 681–697, respectively), and the C- Eα3 of ELMO1, through its RT loop (Fig. 2B). Among the RT- terminal Pro-rich tail (residues 700–727) (Fig. 1B and Fig. S3). loop residues, Tyr17 stacks its side chain between those of Pro532 The DOCK2 SH3 domain and the ELMO1 Pro-rich tail interact of Eα1 and Pro710 in the ELMO1 tail, whereas Ile31 and Gly32 α α extensively with each other. Furthermore, Dα1–3 of DOCK2 and hydrophobically interact with Ile533 of E 1, Leu698 of E 3, and Eα1 and Eα3 of ELMO1 form an intermolecular five-helix bun- Leu701 and Ile706 of the tail. In addition, Asn18 hydrogen bonds 2 α dle (Fig. 1B). A surface area of approximately 2;370 Å is buried with Pro532 and Elu535 of E 1, whereas Gln30 hydrogen bonds α in the interface between DOCK2 and ELMO1. with Arg697 of E 3. Within the SH3 domain, Ile31, corresponding to Ile32, which is reportedly required for the autoinhibition of The SH3 Domain of DOCK2 and the Pro-Rich Tail of ELMO1. The overall DOCK180 (21), is located at the center of the interactions fold of the DOCK2 SH3 domain and the binding manner of the (Fig. 2B). α PxxP-motif (Pro714–Pro717) are consistent with those defined Third, DOCK2 interacts with the boundary between E 3 and by our NMR analysis. However, the SH3 domain reinforces the the ELMO1 tail. Lys67, at the C terminus of the SH3 domain, hydrogen bonds with Leu699 of Eα3 and Asp700 in the tail. interaction with the regions flanking the PxxP motif of ELMO1 Arg128 and Ser129 of Dα1 form hydrogen bonds with Leu699 (Fig. 2A). The overall intermolecular binding mode of the DOCK2 2 of Eα3 and Glu702 in the tail. The residues involved in these in- SH3 domain with ELMOl, burying approximately 1;450 Å ,isby teractions in the three regions are well conserved in the CDM far the most extensive among the SH3 domain complexes charac- family (Fig. S3). terized to date (SI Text and Fig. S5). The interactions occur in three – regions. First, the 28-residue Pro-rich tail (Asp700 Asn727) winds Five-Helix Bundle. The intermolecular five-helix bundle consists 1;310 2 around the SH3 domain, burying approximately Å .The of Dα1–3 of DOCK2 and Eα1 and Eα3 of ELMO1 (Fig. 3 and DOCK2 SH3 domain forms extensive hydrophobic interactions Fig. S4 B and C). In the DOCK2 three-helix bundle domain, Dα2 and several hydrophilic interactions with the ELMO1 tail. At the is arranged in an antiparallel manner relative to Dα1 and Dα3. center of the interface, the side-chain imino group of Trp44 of Almost all of the interactions between the three helices are hy- DOCK2 hydrogen bonds with the main-chain carbonyl group of drophobic, involving a large number of aliphatic side chains of Ile, Lys715, within the PxxP motif of ELMO1. The DOCK2 SH3 do- Val, and Leu residues and those of Trp98 and Met120 (Fig. S4B). main forms six hydrogen bonds that interact with the C-terminal Similarly, the ELMO1 Eα1 and Eα3 helices are arranged in an half (Asn719–Asn727), following the PxxP motif, of the ELMO1 antiparallel manner and form multiple hydrophobic interactions involving the side chains of Ile and Leu residues (Fig. S4C). On the other hand, Dα1 and Dα2 of DOCK2 and Eα1 and Eα3of ELMO1 form the interface between the DOCK2 and ELMO1 2 helix bundles, burying approximately 930 Å (Fig. 3). The interface involves not only a number of hydrophobic interactions, but also several hydrophilic interactions. The residues Leu95, Trp96, BIOCHEMISTRY Trp102, Val107, Phe114, Met121, and Met125 of DOCK2 hydrophobically interact with Ile544, Leu547, Ile548, Leu681, Leu689, Met692, Leu696, and Leu699 of ELMO1. Met692 of ELMO1, which is required for ELMO autoinhibition (23), is buried at the center of the five-helix bundle. In addition, the Glu693 side chain of ELMO1 hydrogen bonds with the Lys103 side chain of DOCK2. Similarly, the Tyr106 side chain of DOCK2 hydrogen bonds with the Asp685 side chain of ELMO1, and the Arg128

Fig. 2. DOCK2•ELMO1 interactions around the SH3 domain. (A) Interface between the DOCK2 SH3 domain and the ELMO1 Pro-rich tail. The six proline residues (Pro707, Pro710, Pro711, Pro712, Pro714, and Pro717) in the ELMO1 Pro-rich tail are colored red, and the labels of the two Pro residues forming the PxxP motif are underlined. Hydrogen bonds are indicated by dashed lines. The DOCK2 and ELMO1 residues are labeled in black and red, respectively. (B) Interface between the DOCK2•ELMO1 five-helix bundle and the DOCK2 Fig. 3. DOCK2•ELMO1 five-helix bundle formation. Interface within the SH3 domain. The DOCK2 and ELMO1 residues are labeled in black and red, DOCK2•ELMO1 five-helix bundle, showing the interactions between the respectively. DOCK2 and ELMO1 helices. Hydrogen bonds are indicated by dashed lines.

Hanawa-Suetsugu et al. PNAS ∣ February 28, 2012 ∣ vol. 109 ∣ no. 9 ∣ 3307 Downloaded by guest on September 30, 2021 side chain of DOCK2 hydrogen bonds with the Leu699 main- chain carbonyl group of ELMO1. The hydrophobic residues within the three helices of DOCK2 are highly conserved among the CDM family members (Fig. S3A). Actually, the mutations of such hydrophobic residues, L96D/W99E and I132D/L133D in DOCK180 (L95/W98 and L131/L132, respectively, in DOCK2), reportedly disrupt the ability to bind ELMO1 (21). Therefore, DOCK180 forms a three-helix bundle similar to that of DOCK2, and uses it to interact with ELMO1. On the other hand, nearly all of the hydrophobic interactions between the two helices are conserved in the ELMO family (Fig. S3B). The DOCK2 residues on the interface with ELMO1 are well conserved with those of DOCK180, whereas three residues, Trp96, Met121, Met125, are not conserved (Fig. S3A). As for ELMO1, the present interaction mode with DOCK2 (Fig. 3) agrees well with the results of a previous mutational study, which showed that Leu547 and Ile548 of ELMO1 are required for DOCK180 binding (21). There- fore, DOCK180 is also likely to form a five-helix bundle, as observed in the present DOCK2•ELMO1 complex. In addition, the C terminus of Eα1 of ELMO1 forms hydrophobic interactions with the Eα2 helix within the PH domain (Fig. S4D). These hydrophobic residues are strictly conserved in the ELMO family (Fig. S3B). The Eα1•Eα2 interactions fix the arrangement of the PH domain relative to the five-helix bundle (Fig. 1B). The core structure of the PH domain of ELMO1 in the DOCK2•ELMOl complex is essentially the same as that of the ELMO1 fragment consisting of the PH domain and the Eα1 helix (21). However, Eα1 significantly bends, at the cen- tral Pro542, and interacts with Eα3 to form the five-helix bundle with DOCK2, whereas Eα1 was straight in the previous structure, due to crystal packing (21) (Fig. S4E).

Characterization of DOCK2 and DOCK180 Mutants. On the basis of the DOCK2•ELMO1 interactions described above, we mutated several ELMO1-interacting residues in the full-length DOCK2 protein. First, six putative hydrogen-bonding residues in the SH3 domain (Fig. 2 A and B) were changed to Ala (Fig. 4A). Although the Q30A and S62A mutations had no effect, the W4A, E39A, W44A, and R46A mutations significantly reduced the ELMO1 binding. In particular, the Ala mutation of Trp44, which is located Fig. 4. The SH3–PxxP interaction is essential for ELMO1-binding in DOCK2. in the center of the SH3•PxxP interface (Fig. 2A), had the most (A–C) Interaction of the ELMO1 binding residues with DOCK2. HEK293T cell profound effect on ELMO1 binding (Fig. 4A). Therefore, the lysates expressing HA-tagged DOCK2 (WT and mutants) were incubated with hydrogen bonds of Trp44 with the PxxP motif, and those of Trp4, GST-tagged ELMO1. The DOCK2-HA in the pull-downs and total cell lysates was detected by immunoblotting with an anti-HA antibody. The results were Glu39, and Arg46 with the C-terminal region downstream of the quantified by densitometry and are expressed as the ratio of ELMO1 bound PxxP motif, are important for ELMO1 binding. On the other hand, to total DOCK2 after normalization to the WT value, with an arbitrary value the point mutations of Trp102 and Tyr106 of DOCK2 (Fig. 3) to of 1. The results indicate mean SD of three independent experiments. Glu (Y106E and W102E) substantially reduced ELMO1 binding (D) Chemotactic response of BW5147α−β− cells expressing DOCK2 (WT or mu- (Fig. 4B). Similarly, the R128A mutation nearly completely abol- tant). BW5147α−β− cells (−) stably expressing HA-tagged DOCK2 (clones 4B3 ished the ELMO1 binding, whereas the S129A mutation did not and 4C2) or HA-tagged DOCK2 W44A (clones 2B9 and 3G10) were allowed to affect it (Fig. 4A). Arg128 is located at the C terminus of the Dα2 migrate on stromal cells prepared from mouse lymph nodes. The migration speed was calculated by analyzing at least 148 cells per clone. **P < 0.01.(E) helix, in the vicinity of the SH3 domain, and interacts with the main − − α The effect of the W44A mutation on Rac activation. BW5147α β cells (−) chain at the border between the E 3 helix and the Pro-rich tail of expressing wild-type DOCK2 (clone 4C2) or the W44A mutant (clone 3G10) ELMO1 (Figs. 2B and 3). These results indicate that both binding were stimulated with CXCL12 (400 ng∕mL) before the assay. Data are repre- interfaces, the SH3 domain and the helix bundle, are important for sentative of three independent experiments. (F) The effect of the W44A mu- the DOCK2•ELMO1 complex formation. tation on ELMO1-mediated relief of DOCK2 autoinhibition. The GFP-tagged In contrast to the marked reduction in ELMO1 binding by the DOCK2 N-terminal fragment (Met1–Glu200), with or without the W44A DOCK2 W44A mutation (Fig. 4A), the point mutations of the mutation, and the FLAG-tagged DOCK2 C-terminal fragment (Ser1195– corresponding Trp residue of DOCK180 (W45A and W45K) re- Met1828) were expressed in HEK293T cells in the presence or absence of the portedly had minimal effects on ELMO1 binding (21, 22). Con- HA-tagged ELMO1. TCL, total cell lysate. The association between the N- and sequently, we confirmed the observed difference by mutating the C-terminal fragments was analyzed by reciprocal immunoprecipitations with the relevant antibodies. Data are representative of two independent experi- corresponding Trp residues to Lys in DOCK2 and DOCK180. ments. The W44K mutation almost completely abolished the ability of DOCK2 to bind to ELMO1, whereas the DOCK180 W45K mu- purpose, we stably expressed wild-type DOCK2 and the W44A tation retained it (Fig. 4C). Therefore, these mutagenesis results mutant in BW5147α−β− cells, which lack the endogenous expres- indicated that DOCK2 and DOCK180 differ in their require- sion of DOCK2 (2). As compared with the nontransfected cells, ments for the SH3•PxxP interaction in ELMO1 binding. This the expression of wild-type DOCK2 in BW5147α−β− cells signif- finding led us to examine the functional significance of the icantly increased the migration speed (Fig. 4D). However, the ex- SH3•PxxP interaction in DOCK2-mediated chemotaxis. For this pression of the W44A mutant failed to restore the motility in two

3308 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1113512109 Hanawa-Suetsugu et al. Downloaded by guest on September 30, 2021 independent clones, although its expression level was comparable to that of the wild-type DOCK2 (Fig. 4D). Consistent with this finding, chemokine-induced Rac activation was readily detected in BW5147α−β− cells expressing the wild-type DOCK2, but not the W44A mutant (Fig. 4E). Collectively, these results indicate that the SH3•PxxP interaction is required for DOCK2-mediated Rac activation. The SH3 domain of DOCK180 has been implicated in its autoinhibition by binding to the catalytic DHR-2 domain and thus blocking Rac access to it (22). Toexplore the mechanism by which the SH3•PxxP interaction regulates the DOCK2-mediated Rac activation, we transiently expressed the DOCK2 N-terminal fragment (Met1–Glu200), with or without the W44A mutation, and the C-terminal fragment (Ser1195–Met1828), including the DHR-2 domain, in HEK293Tcells. Regardless of the presence of the W44A mutation, the N-terminal fragment of DOCK2 asso- ciated with the C-terminal fragment, suggesting that a similar autoinhibitory mechanism occurs in DOCK2 (Fig. 4F). However, upon the coexpression of ELMO1, this association was disrupted in the case of the wild-type DOCK2, but not the W44A mutant (Fig. 4F). These results suggest that the SH3•PxxP interaction is required to relieve the autoinhibition of DOCK2 and to allow Rac access to the catalytic DHR-2 domain. Discussion DOCK2 is a key regulator of the immune system. In this study, we determined the crystal structure of the complex of the DOCK2 and ELMO1 interacting regions, which revealed a large, rigid assembly. Three α-helices of DOCK2 and two α-helices of ELMO1 form a helix bundle. The SH3 domain of DOCK2 interacts with the long Pro-rich tail and the two α-helices of ELMO1 in an unu- sually extensive manner. Together, these structural elements, as well as the PH domain of ELMO1, accrete to form a rigid assembly. A mutational analysis demonstrated that the entire assembly of these two regions is required for the full-length DOCK2 and

ELMO1 proteins to form a complex and activate Rac in vivo. BIOCHEMISTRY The SH3 domain of DOCK180 reportedly interacts with the catalytic DHR-2 domain, which may sterically block Rac access to the DHR-2 domain and thereby cause the autoinhibition of Fig. 5. Hypothetical model of the DOCK2•ELMO1•Rac1 ternary complex. (A) DOCK180 (22). Actually, in the present study, we observed the The schematic model of the mutual relief of DOCK2 and ELMO1 from their binding of the N-terminal ELMO-interacting region with the C- autoinhibited forms for Rac activation. EIR, ELMO1-interacting region; DIR, terminal DHR-2 containing region of DOCK2 and its reduction DOCK2-interacting region. (B) Side view, showing the membrane attachment by the assembly with ELMO (Fig. 4F). Therefore, a similar me- of the DOCK2•ELMO1•Rac1 ternary complex. The membrane is shown as a chanism underlies the autoinhibition of the DOCK2 GEF activ- light yellow bar. The membrane attachment site of Rac1 at the C terminus is ity. In the case of DOCK180, the mutation of the conserved Ile depicted by a red dashed line. (C) Top view from the membrane side. The residue within the SH3 domain (I32K) disrupts the SH3•DHR-2 present structure of the DOCK2•ELMO1 complex (green and yellow) is over- interaction, and relieves DOCK180 of the autoinhibition (22). laid onto the structures of the DOCK180 DHR-1 domain (orange, PDB code 3L4C) (13), the dimeric DOCK2 DHR-2 domain•Rac1 complex (blue and red, This Ile residue corresponds to Ile31 in DOCK2, within the RT PDB code 3B13), and importin subunit β-1 (ARM, armadillo-like repeat) (gray, loop of the SH3 domain. Ile31 is not close to the PxxP-binding PDB code 2QNA) (30). The two views are related by a 90° rotation about the site, but is buried at the center of the hydrophobic interface with horizontal axis. the Eα1 and Eα3 helices and the following peptide segment of ELMO1 (Fig. 2B). lar autoinhibitory interactions. Thus, the structural basis by which On the other hand, the ELMO1 protein also has an autoinhi- DOCK2 and ELMO1 mutually relieve their autoinhibited forms bitory mechanism (23). The EID binds to the EAD in the auto- has now been revealed (Fig. 5A). inhibited form, and EAD binding with DOCK180 relieves The PH domain of ELMO1 is firmly fixed, relative to the five- ELMO1 of its autoinhibition. Therefore, DOCK2/DOCK180 and helix bundle, in the rigid DOCK2•ELMO1 complex. The ELMO1 mutually relieve their autoinhibited forms (Fig. 5A). The ELMO1 PH domain reportedly cooperates with DOCK180 to double mutation of Met692 and Glu693 to Ala within the EAD activate Rac (20). The mutations of the PH domain residues, reportedly disrupts the interaction between the EID and the EAD. such as G559A and W665A, in ELMO1 abrogate the formation Correspondingly, Met692 is buried at the center of the hydrophobic core of the intermolecular five-helix bundle, whereas the of the functional ternary complex with DOCK180 and nucleo- Glu693 side chain interacts with the Lys103 side chain of DOCK2, tide-free Rac, although they do not affect the DOCK180 binding • as revealed by the present structure. As described above, the (20). In the present DOCK2 ELMO1 structure, Gly559 and hydrophobic core of the five-helix bundle also buries Ile31 of Trp665 of ELMO1 are involved in intramolecular interactions DOCK2, which is the putative key residue for the DOCK2 auto- between the Eα1 and Eα2 helices (Fig. S4D). Therefore, their inhibition. Therefore, the tight assembly between the ELMO1- mutations are expected to impair the proper positioning of the interacting region of DOCK2 and the DOCK2-interacting region ELMO1 PH domain relative to the other portions of the of ELMO1 deprives these regions of their respective intramolecu- DOCK•ELMO complex. Thus, the fixed arrangement of the

Hanawa-Suetsugu et al. PNAS ∣ February 28, 2012 ∣ vol. 109 ∣ no. 9 ∣ 3309 Downloaded by guest on September 30, 2021 ELMO1 PH domain may be important for functional ternary ordered C-terminal region (residues 168–177), and thus is not complex formation with DOCK and Rac. involved in ELMO1 binding. In the typical mammalian RhoGEFs, such as Trio and Dbs, the Our observation that DOCK2•ELMO1 complex formation DH and PH domains act as a “cassette” and coordinately bind is essential for the DOCK2 function makes it an attractive ther- their cognate Rho GTPases (Rac1 and Cdc42) for efficient acti- apeutic target for immunologic disorders, such as autoimmune – vation (29). By analogy to the DH PH domains, the PH domain diseases and graft rejection, caused by tissue infiltration by lym- of ELMO1 may facilitate Rac1 binding by the DHR-2 domain. A phocytes. Although the mechanism by which DOCK180 main- • structural model of the DOCK180 Rac complex has been pro- tains the SH3 domain-independent, tight binding of ELMO1 posed (13), and thus we integrated the present DOCK2•ELMO1 remains elusive, the observed difference raises the possibility that structure into a similar model of the DOCK2•Rac1 complex ELMO1 binding might be specifically disrupted by small com- (Fig. 5 B and C, and Table S3). In this preliminary model, the two DOCK2•ELMO1 complexes might interact with two Rac1 pounds that bind to the interface in DOCK2, but not DOCK180. molecules on the flat surface of the plasma membrane. The In this context, our structure will provide important information PH domain of ELMO1 may be arranged similarly to that in for the development of an inhibitor that selectively disrupts the Rac1•Trio DH–PH cassette (Protein Data Bank code 2NZ8). DOCK2•ELMO1 complex formation. The ELMO1-interacting residues of DOCK2 are also highly conserved in DOCK180. In fact, the present structure indicates Materials and Methods The protein samples for structure determination were produced by the cell- that their overall interaction modes may be similar to each other. free synthesis method. The solution structure of the DOCK2 SH3–ELMO1 fu- Nevertheless, we found an interesting difference between sion protein [Protein Data Bank (PDB) code 2RQR] was determined by NMR. DOCK2 and DOCK180. The mutation of Trp44, within the SH3 The crystal structures of the DOCK2•ELMOl (PDB code 3A98) and DOCK2- domain of DOCK2, abolished the ELMO1 binding ability, but DHR2•Rac1 (PDB code 3B13) complexes were determined by the single-wa- the corresponding mutation of Trp45 of DOCK180 did not affect velength anomalous dispersion and molecular replacement methods, respec- it (Fig. 4C). Phe63, within the SH3 domain of DOCK2, forms tively. Detailed experimental procedures are provided in the SI Text. hydrophobic interactions with the ELMO1 PxxP-tail, but it is replaced with Tyr in DOCK180. Similarly, in the binding interface ACKNOWLEDGMENTS. We thank Y. Tomo, M. Aoki, E. Seki, M. Ikari, K. Hanada, in the five-helix bundle, Trp96, Met121, and Met125 of DOCK2 T. Matsuda, N. Matsuda, Y. Motoda, Y. Fujikura, T. Harada, S. Watanabe, and are replaced with Arg, Ile, and Ile, respectively, in DOCK180 T. Nagira for preparing proteins for NMR experiments. We also thank (Fig. S3A). These amino acid replacements may alter the ELMO- H. Niwa, K. Katsura, S. Tojo, M. Ueno, K. Honda, M. Goto, M. Inoue, M. Aoki, binding affinity between DOCK2 and DOCK180. Alternatively, A. Sakamoto, M. Toyama, Y. Terazawa, T. Fujimoto, M. Wakiyama, A. Shima- an additional region of DOCK180, including Gly171, whose mu- da, and Y. Fujii for technical assistance. We are grateful to Dr. Osamu Ohara (Kazusa DNA Research Institute, Japan) for the DOCK2 clone (KIAA0209). This tation reduces ELMO1 binding (22), may contribute to the tigh- work was supported by the RIKEN Structural Genomics/Proteomics Initiative, ter binding of ELMOl to DOCK180, as compared to DOCK2. the National Project on Protein Structural and Functional Analyses, and by In the current DOCK2•ELMOl complex structure, the corre- the Targeted Proteins Research Program, the Ministry of Education, Culture, sponding Gly residue of DOCK2 (Gly172) is located within a dis- Sports, Science, and Technology of Japan.

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3310 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1113512109 Hanawa-Suetsugu et al. Downloaded by guest on September 30, 2021