Supporting Information Hanawa-Suetsugu et al. 10.1073/pnas.1113512109 SI Text DOCK2 DHR-2 fragment (residues 1196–1622) was cloned into SI Results. We examined the intra- and intermolecular interfaces the expression vector pDEST10.1 (Invitrogen), as a fusion with of the Src-homology 3 (SH3) domains. To assess all structures N-terminal His and small ubiquitin-like modifier protease affinity containing an SH3 domain, we collected the Protein Data Bank tags and a TEV protease cleavage site. The DOCK2 DHR-2 frag- (PDB) codes in three different ways. The first was a key word ment was expressed in Sf9 cells, using the Bac-to-Bac Baculovirus search for “SH3” in the PDB. The second was a Protein Structure Expression System (Invitrogen). The gene encoding the human Database Search by DaliLite v. 3, using our dedicator of cytokin- ras-related C3 botulinum toxin substrate 1 (Rac1) (1–177) frag- esis 2 (DOCK2) SH3 domain structure (PDB code 3A98). The ment was cloned into the expression vector pCR2.1 (Invitrogen), last was a homology search in the PDB, using the amino acid as a fusion with an N-terminal His affinity tag and a TEV pro- sequences that are annotated as an SH3 domain in the UniProt tease cleavage site. The T17N mutation was introduced into Rac1 database. Finally, we collected 1,245 PDB codes, and calculated by using a QuikChange site-directed mutagenesis kit (Agilent). the intermolecular and intramolecular interface areas of their The Rac1 (T17N) mutant was synthesized by the E. coli cell-free SH3 domains by PDBePISA (Fig. S5 A and B). system (1, 2). The DOCK2 DHR-2 and Rac1 (T17N) proteins, The intermolecular interface between DOCK2 SH3 and En- after digestion with TEV protease, were separately purified gulfment and cell motility protein 1 (ELMO1) in our structure by ion exchange on a HiTrap Q column and by size-exclusion 2 is approximately 1;450 Å (Fig. S5 A and C), and it is the largest chromatography on a HiLoad 16/60 Superdex 75 pg column. The intermolecular interface (Fig. S5A). The second largest interface DOCK2 DHR-2•Rac1 complex, formed by incubating the pro- 2 (approximately 860 Å ) is that in the p22Phox • p47Phox complex teins together on ice for 1 h, was separated from the uncomplexed structure, which also has the authentic SH3•PXXP interaction proteins by chromatography on a HiLoad 16/60 Superdex 75 pg (Fig. S5D), but it is less than 60% of the size of the DOCK2•- column, preequilibrated with 20 mM Tris•HCl buffer (pH 8.0), ELMO interface. The third is the complex of Cbl-interacting pro- containing 150 mM NaCl, 10% glycerol, and 2 mM DTT. tein of 85 kDa SH3 and ubiquitin. This interface is formed by an 2 approximately 820 Å domain•domain interaction (Fig. S5A). NMR spectroscopy and spectral assignments. All spectra were re- On the other hand, intramolecular interactions of the SH3 corded at 296 K on Bruker Avance 600 and 800 spectrometers domain have been observed with other regions in multidomain equipped with cryoprobes. Resonance assignments were accom- proteins, which may be related to the autoinhibition of the plished using a conventional set of triple-resonance spectra, as protein functions. The SH3 interface in the calcium channel β-2 described previously (4), and were deposited in the Biological 2 subunit is over 1;500 Å (Fig. S5B). The construct lacks the poly- Magnetic Resonance Data Bank (11079). Interproton distance 15 13 proline sequences, and the SH3 domain is buried among the gua- restraints were obtained from N and C edited NOESY spec- nylate kinase domain and two flanking regions. Almost of all the tra, both recorded with a mixing time of 120 ms. All spectra were structures listed in Table S3B include such domain•domain inter- processed using NMRPipe (5), and the programs Kujira (6) and actions. Only neutrophil cytosol factor 1 has the authentic inter- NMRView (7) were employed for optimal visualization and spec- action of the SH3 domain with a polyproline sequence. However, tral analyses. the interacting motif is not a typical PXXP, but a PXXR motif (Fig. S5E). NMR structure calculations. The DOCK2 SH3-ELMO1 peptide fusion complex was determined by the conventional triple-reso- SI Materials and Methods. Protein expression and purification for nance technique (8–12). Automated NOE cross-peak assign- structural determination. The genes encoding the DOCK2 SH3- ments and structure calculations with torsion angle dynamics ELMO1 peptide fusion proteins were obtained by PCR. The were performed using the software package CYANA (13). The DNA fragments, listed in Fig. S1, were cloned into the expression backbone dihedral angle restraints from the TALOS program vector pCR2.1 (Invitrogen), as fusions with an N-terminal His (14) were also included in the calculations, with allowed ranges affinity tag and a tobacco etch virus (TEV) protease cleavage site. of Æ30°. The final structure calculations with CYANA were The 13C∕15N-labeled fusion proteins were synthesized by the started from 100 conformers with random torsion angle values. Escherichia coli cell-free protein expression system (1–3), and The 20 conformers with the lowest final CYANA target function were purified using a chelating column, as described previously values were selected for the final structure set. The structures (4). The purified proteins were concentrated to 0.7–1.0 mM in were validated using PROCHECK-NMR (15, 16). The structural 20 mM Tris-d11-HCl buffer (pH 7.0), containing 100 mM NaCl, statistics of the DOCK2 SH3-ELMO1 peptide fusion protein are 1 mM dithiothreitol-d10, 10% D2O, and 0.02% NaN3. summarized in Table S1. Figures were generated with the MOL- The expression plasmids for the human DOCK2 (residues 1– MOL (17) and PyMol (18) (http://www.pymol.org) programs. 177) and ELMO1 (residues 532–727) fragments were constructed in a similar manner. The selenomethionine-labeled proteins were Identification of the ELMO1-interacting region of DOCK2 and the synthesized using the large-scale dialysis mode of the E. coli cell- DOCK2-interacting region of ELMO1. In our search for DOCK2 free reaction (1, 2). After digestion with TEV protease, the pro- and ELMO1 regions that are suitable for crystallographic studies, tein complex was purified by ion exchange on a HiTrap Q column we expressed a series of N-terminal fragments of DOCK2 (resi- and by size-exclusion chromatography on a HiLoad 16/60 Super- dues 1–160, 1–177, 1–190, 9–160, 9–177, 9–190, 21–160, 21–177, dex 75 pg column, preequilibrated with 20 mM Tris•HCl buffer and 21–190) and C-terminal fragments of ELMO1 (residues 532– (pH 8.0), containing 150 mM NaCl and 2 mM DTT. All columns 717, 541–717, 550–717, 532–727, 541–727, and 550–727), using were purchased prepacked from GE Healthcare. the small-scale dialysis mode of the E. coli cell-free reaction The boundaries for the DOCK2 DOCK-homology region 2 (1, 2). We found that fragments 1–177 and 1–190 of DOCK2 (DHR-2) domain were determined by expressing various DOCK2 and fragments 532–717, 532–727, and 541–727 of ELMO1 were DHR-2 fragments, using the small-scale dialysis mode of the highly expressed and produced soluble DOCK2•ELMOl com- E. coli cell-free reaction (1, 2). The gene encoding the human plexes. Among them, only the protein complex between frag- Hanawa-Suetsugu et al. www.pnas.org/cgi/doi/10.1073/pnas.1113512109 1of9 ments 1–177 of DOCK2 and 532–727 of ELMO1 produced dif- fore, the present DOCK2 DHR-2 domain•Rac1 structure is fraction quality crystals. suitable for generating a structural model of the DOCK2•EL- MO1•Rac1 ternary complex (Fig. 5 B and C). Analytical ultracentrifugation. All experiments were performed at 4 °C with an An-50 Ti rotor using Beckman Optima XL-I analy- Calculations of SH3 intermolecular and intramolecular interfaces. To tical ultracentrifuge. The sample buffer was 20 mM Tris•HCl assess all structures containing an SH3 domain, we collected the buffer (pH 8.0), containing 150 mM NaCl and 5 mM β-mercap- PDB codes in three different ways. The first was a key word toethanol. The solvent density and the protein partial specific vo- search for “SH3” in the PDB (http://www.pdbj.org/). The second lume (υ) were estimated using the program Sednterp (http://www. was a Protein Structure Database Search by DaliLite v. 3 (http:// jphilo.mailway.com/). Sedimentation equilibrium experiments ekhidna.biocenter.helsinki.fi/dali_server/), using our DOCK2 were performed with protein concentrations of 0.80, 0.40, and SH3 domain structure (PDB code 3A98). The last was a homol- 0.20 mg∕mL. Data were obtained at 10,000, 12,000, and ogy search in the PDB, using the amino acid sequences that are 14,000 rpm. The equilibrium data were fitted using the manufac- annotated as an SH3 domain in the UniProt database (http:// turer’s software. www.uniprot.org/uniprot/). Finally, we collected 1,245 PDB codes, and calculated the intermolecular and intramolecular in- Crystallization and data collection. The DOCK2•ELMOl complex terface areas of their SH3 domains by PDBePISA (http:// crystals for structure analysis were grown by the hanging-drop www.ebi.ac.uk/msd-srv/prot_int/pistart.html)(Fig.S5A and B). vapor-diffusion method, by mixing the protein solution with an We listed the PDB codes of the proteins with intermolecular in- 2 equal volume of reservoir solution, containing 100 mM diammo- terfaces greater than 800 Å and intramolecular interfaces larger 2 nium hydrogen citrate and 12% PEG 3350. The crystals were than 1;000 Å .