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 func- tion (20). The crystal structure of the ELMO1 PH domain has re- cently 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 signal- ing (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 do- main, even though the location of this residue is distant from the PxxP-binding pocket. ELMO1 by itself is also reportedly auto- inhibited because of the interaction of the ELMO inhibitory do- main (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.