Supporting Information

Soundararajan et al. 10.1073/pnas.0801508105

Methods 22°C when the OD600 nm reached a value of 0.5. Cultures were Protein Purification, Crystallization, and Data Collection. Clones induced for protein expression with 1 mM IPTG and grown at encoding human RGS domain sequences (see Table S4)as 22°C overnight. The next day, the cells were harvested by N-terminally His -tagged fusion proteins with a tobacco-etch centrifugation, washed with ice-cold 150 mM NaCl, and frozen 6 Ϫ virus (TEV) protease cleavage site were individually expressed at 80°C. NMR spectra were acquired at 297 K, using Bruker in E. coli BL21(DE3) or BL21(DE3)-Rosetta cells. Cultures DRX 600 and DMX 750 spectrometers in standard configura- were grown in 1 liter of Terrific Broth medium at 37°C until tion with triple resonance probes equipped with self-shielded OD measurement indicated log-phase growth; the temper- triple-axis gradient coils. Spectra for the resonance and NOE 600 nm assignments were recorded essentially as described in the orig- ature was then reduced to 25°C for 1 h before induction of 15 protein expression by addition of 0.5 mM or 1 mM isopropyl- inal references (see Table S2). N-labeled RGS protein samples at concentrations of 1.0–1.9 mM protein in 90% H O/10% D O ␤-D-thiogalactopyranoside. Protein expression was allowed to 2 2 (pH 6.0 buffer as in Table S4) were used for 3D 15N-separated continue for another4horovernight before cell harvesting by 15 15 NOESY-HSQC, NT1 and NT2 relaxation, and heteronuclear centrifugation and purification by using methods detailed in 15 1 13 15 Table S4.G␣ subunits were purified by using methods similar to N- H NOE experiments. C, N-labeled samples of RGS i proteins at 0.9–1.4 mM concentration in 90% H O/10% D O those in Table S4, but with 300 mM NaCl-containing buffers 2 2 ϩ (pH 6.0 buffer as in Table S4) were used for all H -detected when using Ni2 -NTA column and 150 mM NaCl during S-200 N triple-resonance experiments, 3D CBCA(CO)NNH, CB- gel filtration. The complete details of the materials and methods CANNH, CC(CO)NNH, H(CCCO)NNH, HBHA(CBCA- for the expression and purification of individual RGS proteins CO)NNH, HNCO, HN(CA)CO, and for 3D 13C-separated and RGS domain/G␣ subunit complexes, the reader is asked to i aliphatic-centered- and aromatic-centered NOESY-HSQC spec- refer to the www.sgc.ox.ac.uk/structures/RGS.html web page. tra. Protein samples were then freeze-dried and redissolved in To obtain RGS domain/G␣i complexes, equimolar concentra- 13 ␣ 100% D2O for acquisition of 3D C-separated HMQC-NOESY, tions of RGS protein and G i subunit were mixed and incubated HCCH-COSY, HCCH-TOCSY and 2D NOESY and TOCSY at 4°C for 15 min. The protein mixture was then resolved with an spectra. Data were processed on Silicon Graphics O2 worksta- S-200 gel filtration column preequilibrated with 20 mM Hepes tions using the program XWIN-NMR (version 2.6) of Bruker (pH 7.5), 150 mM NaCl, 5% glycerol, 2 mM DTT, 5 mM MgCl2, Ϫ BioSpin GmbH (Rheinstetten, Germany). 30 ␮M AlCl3, 100 ␮M GDP, and 20 mM NaF (to form AlF4 ). Column fractions of proteins eluting as a complex were analyzed NMR Structure Calculation. Preliminary 3D structures were calcu- by using SDS/PAGE, and appropriate fractions were pooled and lated by using the program CYANA v. 2.0 (20, 21) based on the concentrated to 24–47.5 mg/ml and used for crystallization trials resonance assignments, and NOE peak lists from 13C HMQC- (see Table S4). Crystals were grown at 20°C by using the NOESY, 3D 13C-aliphatic-centered NOESY-HSQC, 3D 13C- sitting-drop method. All crystals were mounted in loops and aromatic-centered NOESY-HSQC, and 3D 15N NOESY-HSQC cryocooled before x-ray diffraction data collection at 100 K. spectra. Dihedral angle restraints were predicted by using the Datasets were collected at the Swiss Light Source (beamline program TALOS (22) (http://spin.niddk.nih.gov/NMRPipe/ SLS-X10). Diffraction data were processed with MOSFLM (14) talos/) and used to aid initial rounds of structure calculations but and the CCP4 suite (15). Structures were solved by molecular were excluded in the final rounds. A precise ensemble of replacement using PHASER (16), and crystallographic models structures showing an obvious RGS domain fold was obtained to were rebuilt by using O (17) or COOT (18). Refinements were which hydrogen bond restraints were added on the basis of the performed by using Refmac5 (19). observed protected amides after sample exchange into D2O. Iterative structure refinement was carried out manually by using NMR Spectroscopy. For NMR structure determinations, RGS10, X-PLOR-NIH v. 2.14 (23); http://nmr.cit.nih.gov/xplor-nih/. RGS14, and RGS18 expression plasmids were separately trans- Structures were validated by using WHATIF http://swift.cm- formed into BL21(DE3)-Rosetta cells and grown at 37°C in bi.kun.nl/WIWWWI and the Protein Structure Validation Suite either [15N]M9 medium or [13C,15N]M9 medium supplemented (PSVS) www-nmr.cabm.rutgers.edu/PSVS. The refined ensem- with 60 ␮g/ml carbenicillin and 30 ␮g/ml chloramphenicol. ble of the lowest energy structures with no NOE violations was These large-scale cultures were grown at 37°C and transferred to submitted to the PDB.

1. Salim S, Dessauer CW (2004) Analysis of the interaction between RGS2 and adenylyl 8. Tesmer JJ, Berman DM, Gilman AG, Sprang SR (1997) Structure of RGS4 bound to cyclase. Methods Enzymol 390:83–99. AlF4-activated G(i alpha1): stabilization of the transition state for GTP hydrolysis. Cell 2. Berman DM, Kozasa T, Gilman AG (1996) The GTPase-activating protein RGS4 stabilizes 89:251–261. the transition state for nucleotide hydrolysis. J Biol Chem 271:27209–27212. 9. Linder ME, Ewald DA, Miller RJ, Gilman AG (1990) Purification and characterization of 3. Popov S, Yu K, Kozasa T, Wilkie TM (1997) The regulators of G protein signaling (RGS) Go alpha and three types of Gi alpha after expression in Escherichia coli. J Biol Chem domains of RGS4, RGS10, and GAIP retain GTPase activating protein activity in vitro. 265:8243–8251. Proc Natl Acad Sci USA 94:7216–7220. 10. Peleg S, Varon D, Ivanina T, Dessauer CW, Dascal N (2002) G(alpha)(i) controls the 4. Kimple RJ, et al. (2004) Guanine nucleotide dissociation inhibitor activity of the triple gating of the G protein-activated K(ϩ) channel, GIRK. Neuron 33:87–99. GoLoco motif protein G18: Alanine-to-aspartate mutation restores function to an 11. Ivanina T, et al. (2004) Galphai1 and Galphai3 differentially interact with, and regulate, inactive second GoLoco motif. Biochem J 378:801–808. the G protein-activated Kϩ channel. J Biol Chem 279:17260–17268. 5. Willard FS, Low AB, McCudden CR, Siderovski DP (2007) Differential G-alpha interac- 12. Rubinstein M, Peleg S, Berlin S, Brass D, Dascal N (2007) Galphai3 primes the G tion capacities of the GoLoco motifs in Rap GTPase activating proteins. Cell Signal protein-activated Kϩ channels for activation by coexpressed Gbetagamma in intact 19:428–438. Xenopus oocytes. J Physiol 581:17–32. 6. Chenna R, et al. (2003) Multiple sequence alignment with the Clustal series of pro- 13. O’Hara CM, Tang L, Taussig R, Todd RD, O’Malley KL (1996) Dopamine D2L receptor grams. Nucleic Acids Res 31:3497–3500. couples to G alpha i2 and G alpha i3 but not G alpha i1, leading to the inhibition of 7. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: Improving the sensitivity of adenylate cyclase in transfected cell lines. J Pharmacol Exp Ther 278:354–360. progressive multiple sequence alignment through sequence weighting, position- 14. Leslie AG (1999) Integration of macromolecular diffraction data. Acta Crystallogr D specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680. 55:1696–1702.

Soundararajan et al. www.pnas.org/cgi/content/short/0801508105 1of16 15. Collaborative Computational Project No 4 (1994) The CCP4 suite: Programs for protein 20. Guntert P, Mumenthaler C, Wuthrich K (1997) Torsion angle dynamics for NMR crystallography. Acta Crystallogr D 50:760–763. structure calculation with the new program DYANA. J Mol Biol 273:283–298. 16. Storoni LC, McCoy AJ, Read RJ (2004) Likelihood-enhanced fast rotation functions. Acta 21. Herrmann T, Guntert P, Wuthrich K (2002) Protein NMR structure determination with Crystallogr D 60:432–438. automated NOE assignment using the new software CANDID and the torsion angle 17. Jones TA, Zou JY, Cowan SW, Kjeldgaard M (1991) Improved methods for building dynamics algorithm DYANA. J Mol Biol 319:209–227. protein models in electron density maps and the location of errors in these models. 22. Cornilescu G, Delaglio F, Bax A (1999) Protein backbone angle restraints from searching Acta Crystallogr A 47 (Pt 2):110–119. a database for chemical shift and sequence homology. J Biomol NMR 13:289–302. 18. Emsley P, Cowtan K (2004) Coot: Model-building tools for molecular graphics. Acta 23. Schwieters CD, Kuszewski JJ, Tjandra N, Clore GM (2003) The Xplor-NIH NMR molecular Crystallogr D 60:2126–2132. structure determination package. J Magn Reson 160:65–73. 19. Murshudov GN, Vagin AA, Lebedev A, Wilson KS, Dodson EJ (1999) Efficient anisotropic 24. Slep KC, et al. (2008) Molecular architecture of G-␣-o and the structural basis for refinement of macromolecular structures using FFT. Acta Crystallogr D 55:247–255. RGS16-mediated deactivation. Proc Natl Acad Sci USA 105:6243–6248.

Soundararajan et al. www.pnas.org/cgi/content/short/0801508105 2of16 RGS Protein: 0 2 4 6 7 8 0 1 2 3 4 6 7 8 1 1 1 1 1 1 2 kDa 188 98 62 49 38 28

17 14

6

Fig. S1. Electrophoresis of purified RGS domains. Proteins were purified from E. coli as described in Methods and resolved by SDS/PAGE and Coomassie blue staining. In some cases, higher-molecular-weight species suggestive of stable oligomers were detected (e.g., RGS2, RGS3), consistent with previous reports of RGS protein dimerization (e.g., ref. 1).

Soundararajan et al. www.pnas.org/cgi/content/short/0801508105 3of16 Immobilized ligand:Gα subunits α. α. . - RED= G GDP BLUE= G GDP AlF4 Gαi1 Analyte:RGS domains at [1 µM] Gαq

RGS1 RGS10 RGS1 RGS10 2500 2000 350 500 ) ) ) 300 400 U 2000 1500 RU 250 300 e(R 1500 200 c 1000 150 200 an

n 1000 100 500 100 500 50 Resonance ( Resonance (RU Resonance (RU) Reso 0 0 0 0 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 Time (s) Time (s) Time (s) Time (s)

RGS2 RGS12 RGS2 RGS12 500 1500 250 500 400 1250 200 400 RU) RU) RU) ( 300 1000 ( 300 e

e( 150 ce ce (RU) c n n 200 750 200 a 100 nan n 500 100 100 so sona

eso 250 50 Re Re R 0 Resonanc 0 0 0 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 Time (s) Time (s) Time (s) Time (s)

RGS3 RGS14 RGS3 RGS14 500 3000 4000 1000 ) 2500 ) 400 U

R 3000 750 2000 300 e( 1500 2000 500 200

1000 nance (RU) o 250 100 1000 sonance (RU esonance (RU) 500 Re Resonanc R Res 0 0 0 0 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 Time (s) Time (s) Time (s) Time (s)

RGS4 RGS16 RGS4 RGS16 200 1500 1500 500 )

1250 U) U 1250 150 U)

400 R R ( 1000 1000

300 ce ( 100

750 750 n nce (R ance

a 200

n 50 500 500 ona on s o

250 250 s 100 Re Res Resonance (RU) 0 0 Re 0 0 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 Time (s) Time (s) Time (s) Time (s)

RGS6 RGS17 RGS6 RGS17 2000 1200 500 300

) 250 U) 1000 U 400 1500 (R (R 200 (RU) 800 e

e 300 150

1000 600 nc

a 200 400 100

500 son 50 200 100 Re Resonance Resonanc Resonance (RU) 0 0 0 0 -50 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 Time (s) Time (s) Time (s) Time (s)

RGS7 RGS18 RGS7 RGS18 1000 2000 500 600 500 U) 800 1600 400 U) R (RU) (RU) 300 400 600 e(R e e 1200 ce ( c

n n 300 400 200 800 200 ona 100 ona 200 s s 400 100 Re Re Resonanc Resonanc 0 0 0 0 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 Time (s) Time (s) Time (s) Time (s)

RGS8 RGS20 RGS8 RGS20 2000 800 500 500

) 400 400 U

1500 600 RU) ( (RU) 300 300 e 200 1000 400 200 100

onance 100 500 200 0 s Re Resonanc Resonance (R Resonance (RU) -100 0 0 0 -200 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 Time (s) Time (s) Time (s) Time (s)

Fig. S2. G␣i vs. G␣q selectivity of 14 RGS proteins determined by surface plasmon resonance (SPR). SPR spectroscopic analyses of the binding of indicated RGS proteins (at 1 ␮M final concentration) to immobilized G␣i1-biotin (left side; pink border) or His6-G␣q (right side; cyan border). Proteins were injected over biosensor-immobilized G␣ subunits for 600 seconds (injections start at time ϭ 0). Experiments were conducted with G␣ subunits both in the inactive, GDP-bound Ϫ conformation (red curves) and in the transition state for nucleotide hydrolysis (GDP⅐AlF4 -bound; blue curves). All RGS domains bound to G␣ in the transition state for GTP hydrolysis (i.e., bound to GDP and aluminum tetrafluoride), consistent with their known biochemistry (2, 3). The complete dataset of SPR analyses of G␣ selectivity is summarized in Table S1.

Soundararajan et al. www.pnas.org/cgi/content/short/0801508105 4of16 Surface α ⋅ - [RGS10] (nM) ) : G i1 AlF4 U

1000 R 2000 ( ) 2000 g U 500 Equilibrium analysis: n R i

( 100 1500

1500 d KD=56 nM e n i

c 50 B n Kinetic analysis: 1000 a 1000 30 m n K 5 -1 -1

u =2.2 x 10 M s o 10 a i s 500 r 500 K -2 -1 e 5 b d=1.6 x 10 s i l R 1 i KD=73 nM u

0 0.1 q 0 E

0 0 250 500 750 1000 0 250 500 750 1000

1 Time (s) [RGS10] (nM) S

- )

G Surface α ⋅ : G q AlF4 250 U 250 R R ( )

200 g U [RGS10] (nM) 200 n R i (

150 10000 d e n

i 150 c 1000 B n

100 a 500 m

n µ 100 KD=3 M u o 50 100 i s r e b

50 i 50 l

R 0 i u

-50 q 0 E 0 250 500 750 1000 0.0 2.5 5.0 7.5 10.0 Time (s) [RGS10] (µM)

Surface α ⋅ - [RGS20] (nM) ) : G i1 AlF4 600 U 800 R ( )

g U 450 1000 n R

i 600 ( 500

d e n i c 300 100 B n

400

a 50 m n u

o 150 30 µ i KD=2 M s r 200 e

10 b i l R 0 5 i u

q 0 E

0 0 250 500 750 1000 0 250 500 750 1000

2 Time (s) [RGS20] (nM) S

- ) G Surface α ⋅ : G q AlF4 200 U 250 R R ( )

g U 150 [RGS20] (nM) 200 n R i (

5000 d e n

i 150 c 100 1000 B n

a 500 m n 100

50 u o µ 100 i KD=6 M s r e b

i 50 l R 0 i u

q 0 E 0 250 500 750 1000 0 1000 2000 3000 4000 5000 Time (s) [RGS20] (nM)

Fig. S3. Quantitation of G␣-binding affinities of RGS10 and RGS20 by SPR analyses. SPR spectroscopic analyses were performed as in Fig. S2 by using indicated Ϫ G␣⅐GDP⅐AlF4 surfaces and indicated concentrations of RGS10 (Upper; pink border) and RGS20 (Lower; cyan border). Resultant sensorgrams were used in equilibrium saturation binding analyses (as described in ref. 4) to derive indicated KD values. KD values for G␣q/RGS10, G␣i1/RGS20, and G␣q/RGS20 interactions are likely to be underestimates (i.e., the affinity could be substantially weaker) because of the inability to observe Bmax. Independent analysis of the association Ϫ (Ka) and dissociation (Kd) rates, obtained from sensorgram curves of RGS10 binding to immobilized G␣i1⅐GDP⅐AlF4 (as described in ref . 5), yields an apparent KD ϭ 73 nM (upper right quadrant).

Soundararajan et al. www.pnas.org/cgi/content/short/0801508105 5of16 αV αVI αVII A apo-RGS8 Gαi1/RGS8 RGS6 387 IWQEFLAPGAPSAINLDSHSYEITSQNVKDGGRYTFEDAQ 426 RGS7 384IWQEFLAPGAPSAINLDSKSYDKTTQNVKEPGRYTFEDAQ 323 R7 RGS9 353IYKLFLAPGARRWINIDGKTMDITVKGLKHPHRYVLDAAQ 392 RGS11 354VYEQFLAPGAAHWVNIDSRTMEQTLEGLRQPHRYVLDDAQ 393 RGS17 136IYEDYISILSPKEVSLDSRVREVINRNLLDPNPHMYEDAQ 175 RZ RGS19 142 IYEDYVSILSPKEVSLDSRVREGINKKMQEPSAHTFDDAQ 181 RGS20 314IYEDYISILSPKEVSLDSRVREVINRNMVEPSQHIFDDAQ 353 RGS2 135IYTDFIEKEAPKEINIDFQTKTLIAQNIQEATSGCFTTAQ 174 RGS4 114IYNEFISVQATKEVNLDSCTREETSRNMLEPTITCFDEAQ 153 R4 RGS8 108IFEEFVDVQAPREVNIDFQTREATRKNLQEPSLTCFDQAQ 147 RGS16 117IFEEFICSEAPKEVNIDHETRELTRMNLQTATATCFDAAQ 156 RGS18 138IYEKFIQTDAPKEVNLDFHTKEVITNSITQPTLHSFDAAQ 177

αV αVI αVII apo-RGS14 apo-RGS10 (2I59) Gαi3/RGS10 apo-RGS10 (2DLR) RGS10 85IYMTFLSSKASSQVNVEGQSR-LNEKILEEPHPLMFQKLQ 123 R12 RGS12 768IFSKFLCSKATTPVNIDSQAQ-LADDVLRAPHPDMFKEQQ 807 RGS14 121IYQEFLSSQALSPVNIDRQAW-LGEEVLAEPRPDMFRAQQ 159

BCαB αB

αA αA αVI

SIII SIII αVII αVII

V α α3 V α3 α SII SII

Fig. S4. Structure-based primary sequence alignment of the ␣V, ␣VI, and ␣VII regions of R4-, R7-, R12-, and RZ-subfamily RGS domains and differential G␣ switch III region interactions observed within R4- versus R12-subfamily RGS protein/G␣i complexes. (A) A multiple sequence alignment of human RGS proteins, from the ␣V helix through to the ␣VII helix, was constructed by using ClustalW (6, 7). Conserved amino acids identified by ClustalW are boxed. Selected R4, R7, and RZ subfamily RGS proteins (Upper) and R12 subfamily RGS proteins (Lower) are presented. Filled circles denote conserved residues forming the RGS domain hydrophobic core (as first defined in ref. 8), and open circles highlight conserved residues making direct contacts with G␣ in the RGS4/G␣i1 crystal structure (8), PDB ID 1AGR; Upper) or RGS10/G␣i3 crystal structure (this study, PDB ID 2IHB; Lower). ␣-Helical secondary structure, as defined by PyMOL, is denoted with solid lines for apo-RGS structures and dotted lines for RGS domain/G␣ complex structures. The coloring scheme is consistent with that of Fig. 1: namely, RGS8 in green [PDB IDs 2IHD (apo), 2ODE (complex)]; RGS10 in red [PDB IDs 2I59 (apo), 2IHB (complex)] or purple [PDB ID 2DLR ([apo)]; and RGS14 in blue [PDB ID 2JNU (apo)]. The disordered residues of the RGS10/G␣i3 crystal structure are indicated by open squares. Primary sequences in the alignment are all human, and SwissProt accession numbers are presented in parentheses: RGS2 (P41220), RGS4 (P49798), RGS6 (P49758), RGS7 (P49802), RGS8 (P57771), RGS9 (O75916), RGS10 (O43665), RGS11 (O94810), RGS12 (O14924), RGS16 (O15492), RGS17 (Q9UGC6), RGS19 (P49795), and RGS20 (O76081). (B) The structures of RGS1/G␣i1 (PDB ID 2GTP) and RGS8/G␣i3 (PDB ID 2ODE) are superimposed, with RGS1 in wheat and RGS8 in gray-blue. The all-helical domains of G␣i1 and G␣i3 are shown in dark blue and cyan, and their respective Ras-like domains are colored in salmon and brick red; the switch regions of both G␣ subunits are colored orange. The terminal amine of Arg-143[RGS1] is 3.0 Å from the backbone carbonyl of Glu-236 in switch III of G␣i1; similarly, the terminal amine of Arg-128[RGS8] is 3.2 Å from Glu-236 in switch III of G␣i3.(C) The structure of RGS10 (green) in complex with G␣i3 (PDB ID 2IHB) reveals that Glu-236[G␣i3] makes no contacts with the RGS domain of this R12 subfamily member. The all-helical domain, Ras-like domain, and switch regions of G␣i3 are colored in dark blue, brick red, and orange, respectively.

Soundararajan et al. www.pnas.org/cgi/content/short/0801508105 6of16 A RGS1 72 SLEKLLANQTGQNVFGSFLKSEFSEENIEFWLACEDYKKTE---SDLLPCKAEEIYKAFV 128 RGS2 83 AFDELLASKYGLAAFRAFLKSEFCEENIEFWLACEDFKKTK--SPQKLSSKARKIYTDFI 140 RGS3 1073 SLEKLLVHKYGLAVFQAFLRTEFSEENLEFWLACEDFKKV--KSQSKMASKAKKIFAEYI 1130 RGS4 62 SLENLISHECGLAAFKAFLKSEYSEENIDFWISCEEYKKI--KSPSKLSPKAKKIYNEFI 119 RGS5 64 SLDKLLQNNYGLASFKSFLKSEFSEENLEFWIACEDYKKI--KSPAKMAEKAKQIYEEFI 121 R4 RGS8 56 SFDVLLSHKYGVAAFRAFLKTEFSEENLEFWLACEEFKKT--RSTAKLVSKAHRIFEEFV 113 RGS16 65 SFDLLLSSKNGVAAFHAFLKTEFSEENLEFWLACEEFKKI--RSATKLASRAHQIFEEFI 122 RGS18 86 SFDKLLSHRDGLEAFTRFLKTEFSEENIEFWIACEDFKKS--KGPQQIHLKAKAIYEKFI 143 RGS21 21 NMDTLLANQAGLDAFRIFLKSEFSEENVEFWLACEDFKKTKN--ADKIASKAKMIYSEFI 78 RGS13 34 SFENLMATKYGPVVYAAYLKMEHSDENIQFWMACETYKKIA--SRWSRISRAKKLYKIYI 91 RGS6 336 SFDEILKDQVGRDQFLRFLESEFSSENLRFWLAVQDLKKQ---PLQDVAKRVEEIWQEFL 392 RGS7 333 GMDEALKDPVGREQFLKFLESEFSSENLRFWLAVEDLKKR---PIKEVPSRVQEIWQEFL 389 R7 RGS9 302 NFSELIRDPKGRQSFQYFLKKEFSGENLGFWEACEDLKYG---DQSKVKEKAEEIYKLFL 358 RGS11 303 SFRELLEDPVGRAHFMDFLGKEFSGENLSFWEACEELRYG---AQAQVPTLVDAVYEQFL 359 RGS10 33 SLENLLEDPEGVKRFREFLKKEFSEENVLFWLACEDFKKM--QDKTQMQEKAKEIYMTFL 90 R12 RGS12 715 SFERLLQDPVGVRYFSDFLRKEFSEENILFWQACEYFNHVPAHDKKELSYRAREIFSKFL 745 RGS14 67 SFERLLQDPLGLAYFTEFLKKEFSAENVTFWKACERFQQIPASDTQQLAQEARNIYQEFL 126

RGS1 129 HSDAAKQINIDFRTRESTAKKIKAPTPTCFDEAQKVIYTLMEKDSYPRFLKSDIYLNLL 187 RGS2 141 EKEAPKEINIDFQTKTLIAQNIQEATSGCFT-T-A-Q-K-RMVRYASALAMIESNTNPSKYLPDRKFMLPEGSMEFFYSQADNLPC 199 RGS3 1131 AIQACKEVNLDSYTREHTKDNLQSVTRGCFD-L-A-QMKQRSIAFMGFLLMAEVKQDHSDYCPRPFMLDRKSSDALGYSLGDHLKI 1189 RGS4 120 SVQATKEVNLDSCTREETSRNMLEPTITCFD-E-A-Q-K-K-I-F-N-L-M-E-K-D-S-Y-R-R-F-L-K-S-R-F-Y-L-D-L-V 178 RGS5 122 QTEAPKEVNIDHFTKDITMKNLVEPSLSSFD-M-A-Q-K-R-I-H-A-L-M-E-K-D-S-L-P-R-F-V-R-S-E-F-Y-Q-E-L-I 180 R4 RGS8 114 DVQAPREVNIDFQTREATRKNLQEPSLTCFD-Q-A-Q-G-K-V-H-S-L-M-E-KMDCSKYGP-R-F-L-R-SLKAMAYLPD-L-L 172 RGS16 123 CSEAPKEVNIDHETHELTRMNLQTATATCFD-A-A-Q-G-K-T-R-TMLWMNETKLDTSRYSP-R-F-L-K-SLPSADYHRPDVLGA 181 RGS18 144 QTDAPKEVNLDFHTKEVITNSITQPTLHSFD-A-A-Q-S-R-V-Y-Q-L-M-E-QMDCSRYT-R-F-L-K-SLDAIAYFLPD-L-M 202 RGS21 79 EADAPKEINIDFGTRDLISKNIAEPTLKCFDMEEATQTKLLIFYFCSLQMIANKMDCSEFSPKREFKLTKFSFEKILYIKHKGLSV 137 RGS13 93 QPQSPREINIDSSTRETIIRNIQEPTETCFE-E-A-Q-K-I-V-Y-M-H-M-E-R-D-S-Y-P-R-F-L-K-S-E-M-Y-Q-K-L-L 150 RGS6 393 APGAPSAINLDSHSYEITSQNVKDGGRYTFE-D-A-Q-E-H-I-Y-K-L-MK-S-D-S-Y-A-R-F-L-R-S-N-A-Y-Q-D-L-L 451 RGS7 390 APGAPSAINLDSKSYDKTTQNVKEPGRYTFE-D-A-Q-E-H-I-Y-K-L-MK-S-D-S-Y-P-R-F-I-R-S-S-A-Y-Q-E-L-L 488 R7 RGS9 359 APGARRWINIDGKTMDITVKGLKHPHRYVLD-A-A-Q-T-H-I-Y-M-L-MK-K-D-S-Y-A-R-Y-L-K-S-P-I-Y-K-D-M-L 417 RGS11 360 APGAAHWVNIDSRTMEQTLEGLRQPHRYVLD-D-A-Q-L-H-I-Y-M-L-MK-K-D-S-Y-P-R-F-L-K-S-D-M-Y-K-A-L-L 418 RGS10 91 SSKASSQVNVEGQS-RLNEKILEEPHPLMFQ-K-L-Q-D-Q-I-F-N-L-MK-Y-D-S-Y-S-R-F-L-K-S-D-L-F-L-K-H-K 148 R12 RGS12 746 CSKATTPVNIDSQA-QLADDVLRAPHPDMFK-E-Q-Q-L-Q-I-F-N-L-MK-F-D-S-Y-T-R-F-L-K-S-P-L-Y-Q-E-C-I 832 RGS14 127 SSQALSPVNIDRQA-WLGEEVLAEPRPDMFR-A-Q-Q-L-Q-I-F-N-L-MK-F-D-S-Y-A-R-F-V-K-S-P-L-Y-R-E-C-L 184

B Analyte: wildtype RGS2 Analyte: RGS2(C106S,N184D) Surface: - Gαi1.GDP.AlF4 Gαi1.GDP

Fig. S5. Multiple sequence alignment of the RGS domains of R4-, R7-, and R12-subfamily RGS proteins. A multiple sequence alignment of the RGS domains of indicated human RGS proteins was constructed by using ClustalW (6, 7). Conserved amino-acids identified by ClustalW are boxed. Highlighted in red are the residues of RGS2 (cysteine 106, asparagine 184, and glutamate 191) considered primary determinants of its unique selectivity for G␣q (Fig. 2). Highlighted in blue (conserved glutamate in R4-subfamily members) and yellow (conserved lysine in for R7- and R12-subfamily members) is the position in which a charge reversal causing electrostatic repulsion is considered important to the G␣i selectivity of the R7- and R12-subfamily members. Primary sequences in the alignment are all human, and SwissProt accession numbers are presented in parentheses: RGS1 (Q08116), RGS2 (P41220), RGS3 (P49796), RGS4 (P49798), RGS5 (O15539), RGS6 (P49758), RGS7 (P49802), RGS8 (P57771), RGS9 (O75916), RGS10 (O43665), RGS11 (O94810), RGS12 (O14924), RGS13 (O14921), RGS14 (O43566), RGS16 (O15492), RGS18 (Q9NS28), RGS19 (P49795), RGS20 (O76081), and RGS21 (Q2M5E4). (B) Enhanced binding of RGS2 to immobilized, transition-state G␣i1 upon conversion of cysteine 106 to serine and asparagine 184 to aspartate, as measured by SPR analysis performed as in Fig. S2. Wild-type RGS2 (Left) or double point mutant RGS2 (Right) was injected for 600 s at a concentration of 3 ␮M over immobilized, biotinylated G␣i1 of the indicated nucleotide state, followed by a 300-s dissociation phase.

Soundararajan et al. www.pnas.org/cgi/content/short/0801508105 7of16 A ) x

a 100 m 80 % (

s i 60 s

y 200 nM Gα

l i1 o

r 40

d + 50 nM RGS1 y

h 20 + 50 nM RGS16 P

T 0 G 0 100 200 300 400 Time (s) B ) x

a 100 m 80 % (

s i 60 s

y 200 nM Gα

l i3 o

r 40

d + 50 nM RGS8 y

h 20 + 50 nM RGS10 P

T 0 G 0 100 200 300 400 Time (s)

Fig. S6. RGS1, -8, -10, and -16 are potent GTPase-accelerating proteins for G␣i substrates. Single-turnover GTP hydrolysis assays were conducted by using 32 32 [␥- P]GTP-bound G␣i1 (A)orG␣i3 (B). G␣ subunits were bound to [␥- P]GTP in the absence of Mg2ϩ. Single-turnover reactions with 200 nM G␣⅐GTP were initiated by adding Mg2ϩ-containing buffer with either 50 nM indicated RGS protein or a buffer control. Inorganic phosphate produced by GTP hydrolysis was measured by activated charcoal filtration and liquid scintillation count- ing. Data were fit to single exponential functions by using GraphPad PRISM. Substoichiometric amounts of these RGS proteins markedly accelerated GTP hydrolysis by their cognate G␣-binding partners; rate constants (sϪ1; 95% CI in parentheses) were obtained as follows: G␣i1 alone, 0.009 (0.0087 to 0.0095); ϩ RGS1, 0.2 (0.15 to 0.29); ϩ RGS16, 0.2 (0.16 to 0.26) (A); G␣i3 alone, 0.010 (0.0096 to 0.011); ϩ RGS8, 0.13 (0.11 to 0.16); ϩ RGS10, 0.14 (0.10 to 0.17) (B).

Soundararajan et al. www.pnas.org/cgi/content/short/0801508105 8of16 Fig. S7. Most RGS domains do not change conformation upon G␣ binding. Structures of RGS domains (green) from select RGS protein/G␣ complexes and their corresponding apo-RGS domain structures (light gray) were superimposed by using PyMOL. RGS4 and RGS10 are the only RGS proteins in this group with obvious differences between unbound and G␣i-bound states (B and E). For each image, the protein structures presented, their respective PDB IDs, and their r.m.s.d. values for backbone superposition are as follows: RGS1/G␣i1 (2GTP), apo-RGS1 (2BV1), 0.5 Å (A); RGS4/G␣i1 (1AGR), apo-RGS4 (1EZT), 1.9 Å (B); RGS8/G␣i3 (2ODE), apo-RGS8 (2IHD), 0.4 Å (C); RGS9/G␣t/i1 (1FQK), apo-RGS9 (1FQI), 0.9 Å (D); RGS10/G␣i3 (2IHB), apo-RGS10 (2I59), 1.8 Å (E); and RGS16/G␣i1 (2IK8), apo-RGS16 (2BT2), 0.5Å(F). The NMR structure of RGS7 (PDB ID 2D9J) was also superimposed with the crystal structure of the RGS9/G␣t/i1 complex (PDB ID 1FQK), yielding a backbone r.m.s.d. of 1.5 Å (data not shown).

Soundararajan et al. www.pnas.org/cgi/content/short/0801508105 9of16 Fig. S8. The structural architecture of the RGS1/G␣i1 and RGS16/G␣i1 com- plexes does not contain interactions with the all-helical domain of G␣i1. Orientation of structures follows that of Fig. 3. The Ras-like domain of G␣i1 is colored in salmon, the all-helical domain of G␣i1 is colored in light blue, and 2ϩ Ϫ switch regions are highlighted in orange. GDP, Mg and AlF4 are shown in magenta, yellow, and cyan, respectively.

Soundararajan et al. www.pnas.org/cgi/content/short/0801508105 10 of 16 Ϫ Fig. S9. G␣i3-specific amino acid residues are predominantly located on the retral surface of G␣. The structure of G␣i3⅐GDP⅐AlF4 , from the RGS8/G␣i3 complex (PDB ID 2ODE), was rendered by using PyMOL to show the anterior (A) and posterior (B) surfaces of G␣i3. Amino acid differences between human G␣i1 (SwissProt accession no. P63096) and human G␣i3 (SwissProt accession no. P08754) were discerned by using ClustalW (refs. 6 and 7 and data not presented). Residues that are different between G␣i3 and G␣i1 are drawn in the ‘‘stick’’ format and colored based on whether the amino acid substitution is conservative (green) or Ϫ 2ϩ nonconservative (red). Switch regions are depicted in gray-blue. GDP is shown in magenta, the AlF4 ion is cyan, and Mg ion is depicted as a yellow sphere. G␣i1 and G␣i3 are 93% identical at the amino acid level and have very similar rates of nucleotide binding and hydrolysis (9). Despite these similarities, G␣i1 and G␣i3 do have some differential biological properties, such as the G␤␥-independent gating of GIRK/Kir3 potassium channels. Binding of G␣ to Kir3 channels causes an attenuation of basal current (10) but primes the channels for agonist-mediated activation; this effect is highly selective to G␣i3 over G␣i1, as determined by both in vivo and in vitro assays (11, 12), suggesting that structural determinants unique to G␣i3 enable specific molecular interactions with the potassium channel. Similarly, studies of D2 dopamine receptor coupling to G␣i subunits suggest that the D2L isoform is selective for G␣i3 over G␣i1 (13), whereas the D2S isoform is selective for G␣i1 over G␣i3.

Soundararajan et al. www.pnas.org/cgi/content/short/0801508105 11 of 16 Table S1. G␣ subunit selectivity of R4, R7, RZ, and R12 subfamily RGS proteins Immobilized ligand

G␣i1 G␣q

Ϫ Ϫ RGS protein subfamily Analyte GDP GDP⅐AlF4 GDP GDP⅐AlF4

Ϫ G␤1␥2 ϩϩϩ Ϫ ϩϩϩ Ϫ R4 RGS1 Ϫ ϩϩϩ Ϫ ϩϩϩ R4 RGS2 ϪϪϩ* ϩϩϩ R4 RGS3 Ϫ ϩϩϩ Ϫ ϩϩϩ R4 RGS4 Ϫ ϩϩϩ Ϫ ϩϩϩ R7 RGS6 Ϫ ϩϩϩ Ϫ Ϫ R7 RGS7 Ϫ ϩϩϩ Ϫ Ϫ R4 RGS8 Ϫ ϩϩϩ Ϫ ϩϩϩ R12 RGS10 Ϫ ϩϩϩ Ϫ ϩ R12 RGS12 Ϫ ϩϩϩ Ϫ Ϫ R12 RGS14 Ϫ ϩϩϩ Ϫ Ϫ R4 RGS16 Ϫ ϩϩϩ Ϫ ϩϩϩ RZ RGS17 Ϫ ϩϩϩ Ϫ ϩϩϩ R4 RGS18 Ϫ ϩϩϩ Ϫ ϩϩϩ RZ RGS20 Ϫ ϩϩϩ Ϫ ϩ

The selectivity of RGS proteins for immobilized G␣i1 and G␣q was probed by using SPR as described in Fig. S2 and Materials and Methods. All RGS domains were tested at 1 ␮M, and the control protein G␤1␥2 was tested at 100 nM. Representative binding curves from these experiments are presented in Fig. S2. The observed strength of binding is represented as ЉϪЉ no binding, ЉϩЉ weak binding, or ЉϩϩϩЉ strong binding. *Although appreciable binding of RGS2 to G␣q⅐GDP was observed, this binding was irreversible (e.g., Fig. S2) and most likely reflects nonspecific electrostatic interactions with the biosensor surface. Poor discrimination of the nucleotide state of G␣q was also observed in Ni-NTA bead precipitation assays [Heximer SP, et al. (1997) Proc Natl Acad Sci USA 94:14389–14393]. More recently, however, a Ϫ flow-based assay for the G␣q/RGS protein interaction has shown that RGS2 is highly selective for the GDP⅐AlF4 -loaded form of G␣q [Gu S, et al. (2007) J Biol Chem 282:33064–33075].

Soundararajan et al. www.pnas.org/cgi/content/short/0801508105 12 of 16 Table S2. Database identifiers of apo- and G␣-complexed RGS domain structures RGS protein Method of structure subfamily PDB ID code* Description determination† Reference‡

R4 2BV1 RGS1 X-ray crystallography This study R4 2AF0 RGS2 X-ray crystallography This study R4 None RGS3 NMR (BMRB no. 15178) This study R4 2OJ4 RGS3 X-ray crystallography Unpublished R4 1EZT, 1EZY RGS4 NMR (BMRB no. 4386) 10852703 R4 2CRP RGS5 NMR Unpublished R4 2IHD RGS8 X-ray crystallography This study R4 2BT2 RGS16 X-ray crystallography This study R4 3C7L RGS16 X-ray crystallography (24) R4 2OWI, 2JM5 RGS18 NMR (BMRB no. 7106) 16964532 R4 2DLV RGS18 NMR Unpublished R7 2ES0 RGS6 X-ray crystallography This study R7 2A72 RGS7 X-ray crystallography This study R7 2D9J RGS7 NMR Unpublished R7 1FQI RGS9 X-ray crystallography 11234020 RZ 1ZV4 RGS17 X-ray crystallography This study RZ 1CMZ RGS19 NMR (BMRB# 4407) 10452897 RZ none RGS20 NMR (BMRB# 5872) 14872136 R12 2I59 RGS10 NMR (BMRB# 7272) 17180548 R12 2DLR RGS10 NMR Unpublished R12 2JNU RGS14 NMR (BMRB no. 15128) This study RA 1DK8 Axin X-ray crystallography 10811618 GEF 1HTJ PDZ-RhoGEF X-ray crystallography 11470431 GEF 1IAP p115-RhoGEF X-ray crystallography 11524686 Ϫ R4 2GTP RGS1 bound to G␣i1⅐GDP⅐AlF4 X-ray crystallography This study Ϫ R4 1AGR RGS4 bound to G␣i1⅐GDP⅐AlF4 X-ray crystallography 9108480 Ϫ R4 2ODE RGS8 bound to G␣i3⅐GDP⅐AlF4 X-ray crystallography This study Ϫ R4 2IK8 RGS16 bound to G␣i1⅐GDP⅐AlF4 X-ray crystallography This study Ϫ R4 3C7K RGS16 bound to G␣o⅐GDP⅐AlF4 X-ray crystallography (24) Ϫ R7 1FQK RGS9 bound to G␣t/i1⅐GDP⅐AlF4 X-ray crystallography 11234020 Ϫ R7 1FQJ RGS9 bound to PDE␥ and G␣t/i1⅐GDP⅐AlF4 X-ray crystallography 11234020 Ϫ R12 2IHB RGS10 bound to G␣i3⅐GDP⅐AlF4 X-ray crystallography This study Ϫ GEF 1SHZ p115-RhoGEF bound to G␣13/i1⅐GDP⅐AlF4 X-ray crystallography 15665872 Ϫ GRK 2BCJ GRK2 bound to G␣q⅐GDP⅐AlF4 X-ray crystallography 16339447

*Accessible at the Protein Data Bank, www.pdb.org. †Assigned chemical shifts are accessible at the Biological Magnetic Resonance Data Bank (BMRB), www.bmrb.wisc.edu. ‡Published references are listed as PMID numbers accessible at PubMed, www.pubmed.org.“Unpublished” denotes structures determined by other investigators and freely available in the Protein Data Bank but without an accompanying published article.

Soundararajan et al. www.pnas.org/cgi/content/short/0801508105 13 of 16 Soundararajan tal. et www.pnas.org/cgi/content/short/0801508105

Table S3. X-ray data collection and refinement statistics

Data collection RGS1/G␣i1 RGS16/G␣i1 RGS8/G␣i3 RGS10/G␣i3 PDB ID 2GTP 2IK8 2ODE 2IHB Space group P212121 P21212P21212P43212 Cell parameters: a, b, c,Å;␣ , ␤, ␥, ° 90.5, 102.9, 128.8; 90, 90, 90 82.7, 104.3, 124.0; 90, 90, 90 112.8, 130.2, 68.5; 90, 90, 90 100.6, 100.6, 145.4; 90, 90, 90 Rmax 2.55 (2.55–2.64) 2.7 (2.7–2.8) 1.9 (1.9–1.95) 2.7 (2.7–2.75) Unique reflections 39,406 (3,908) 29,438 (2,882) 80,222 (7,904) 20,815 (851) Completeness, % 98.5 (99.0) 99.3 (99.2) 100 (100) 97.2 (80.2) Redundancy 3.5 (3.4) 4.7 (4.7) 14.6 (14.6) 8.9 (4.5) Rsym * 0.100 (0.633) 0.133 (0.814) 0.094 (0.000) 0.110 (0.400) I/␴(I) 12.6 (2.0) 11.1 (2.1) 29.4 (2.7) 17.9 (2.6) Reflections in refinement (Rfree set) 37,274 (1,976) 27,900 (1,491) 76,139 (4,021) 19,430 (1,040) † ‡ Rwork /Rfree , % 22.8/27.6 23.5/29.1 18.0/21.1 20.9/3.6 Total no. of atoms 7,209 6,814 7,749 3,451 Average B-factor, Å2 34 64 36 58 R.m.s. deviations Bond distances, Å 0.011 0.008 0.014 0.009 Bond angles, ° 1.27 1.06 1.37 1.07 Ramachandran Favorable, % 97.7 96.4 98.8 97.9 Allowed, % 99.9 100.0 98.8 99.8

Values in parentheses refer to the highest-resolution shell. *Rsym ϭ ¥hkl¥iI Ϫ͗Ii͘/¥hkl¥iIi, where Ii is the intensity of a given measurement and the sums are over all measurements and reflections. † Rwork ϭ ¥F(obs) Ϫ F(calc)/¥F(obs) for 95% of the reflection data used in refinement. ‡ Rfree ϭ ¥F(obs) Ϫ F(calc)/¥F(obs) for the remaining 5%. 4o 16 of 14 Soundararajan

Table S4. Cloning, expression, and purification of RGS proteins and RGS domain/G␣i complexes Residues Tag/ Crystallization

tal. et Protein (PDB ID) GenBank gi no. Vector/antibiotic cloned Purification columns Buffer exchange Conc conditions

RGS1 (2BV1) 21361447 pLIC-SGC1 Amp S50 - A192 Ni NTA affinity purification, GF buffer: 50 mM Tris (pH 8.0), No/23 1:1 ratio of RGS1 and www.pnas.org/cgi/content/short/0801508105 S-75 16/60 GF column, and Ni 0.5M NaCl, 0.5 mM TCEP 4.1 M sodium formate NTA for tag removal and 3% glycerol equilibrated with 4.1 M sodium formate RGS2 (2AF0) 4506517 pNIC28-Bsa4 Kan K71 - T211 Ni NTA affinity purification Yes/30 2:1 mix of RGS2 and and S-200 16/60 GF column 2.0 M ammonium sulphate, 0.2 M NaCl, 0.1 M cacodylate pH 6.5 RGS6 (2ES0) 31742476 pNIC28-Bsa4 Kan P325 - Q470 Ni NTA affinity purification, No/ 30 1:2 mix of RGS6 and S-75 16/60 GF column, and Ni 7% PEG-10K, 0.1 M NTA for tag removal ammonium acetate, 0.1 M Bis-Tris pH 5.5 RGS7 (2A72) 21361149 pNIC28-Bsa4 Kan K320 - S463 Ni NTA affinity purification, No/ 62 1:1 ratio of RGS7 to S-200 16/60 GF column, and Ni reservoir containing NTA for tag removal 25% PEG-3350, 0.2 M ammonium acetate, 0.1 M Bis-Tris pH 5.5 RGS8 (2IHD) 16117777 pNIC28-Bsa4 Kan L60 - S191 Ni NTA affinity purification Yes/ 10 1:1 ratio of RGS8 to and S-75 16/60 GF column reservoir containing 0.2 M NaI, 20% PEG-3350, 10% ethylene glycol RGS10 (2I59) 52694755 pLIC-SGC1 Amp Q30 - L165 Ni NTA affinity purification, Ni 20 mM phosphate (pH 6.0), 50 No/ 17 NMR NTA for tag removal mM NaCl, 1 mM DTT, 0.02% sodium azide RGS14 (2JNU) 21361304 pLIC-SGC1 Amp T56 – T207 Ni NTA affinity purification, Ni 20 mM phosphate (pH 6.0), 50 No/ NMR NTA for tag removal mM NaCl, 1 mM DTT, 0.02% 20.6 sodium azide RGS16 (2BT2) 34452690 pLIC- SGC1 Amp R53 - T190 Ni NTA affinity purification 10 mM Borax (pH 9.0), 0.5 M Yes/ 1:2 ratio of RGS16 to and S-200 16/60 GF column NaCl, 1 mM DTT 15.9 reservoir solution containing 26% PEG-3350, 0.1 M ammonium acetate, 0.1 M Bis-Tris pH 5.5 RGS17 (1ZV4) 21361405 pLIC- SGC1 Amp N72 - S206 Ni NTA affinity purification 10 mM Borax (pH 9.0), 0.5 M Yes/ 1:1 ratio of RGS17 and S-75 16/60 GF column NaCl, 1 mM DTT 20.3 and1MNasuccinate pH 8.2, 1% PEG-2000 MME, 0.1 M Hepes pH 7.0, 0.01 M MgCl2 and seeding RGS18 (2OWI) 18640750 pLIC-SGC1 Amp V75 – E223 Ni NTA affinity purification, Ni 20 mM phosphate (pH 6.0), 50 No/ 20 NMR 5o 16 of 15 NTA for tag removal mM NaCl, 1 mM DTT, 0.02% sodium azide Soundararajan tal. et www.pnas.org/cgi/content/short/0801508105

Table S4. Continued Residues Tag/ Crystallization Protein (PDB ID) GenBank gi no. Vector/antibiotic cloned Purification columns Buffer exchange Conc conditions

Ϫ RGS1/G␣i1 21361447, 33946324 pLIC-SGC1 /pProEXHT S50 -A192/ Ni NTA affinity and GF GF buffer containing AlF4 No/ 24 1:1 ratio of G␣i1/RGS1 complex (both Amp) R32 - F354 columns for individual and precipitant (2GTP) components followed by GF containing 0.2 M NaNO3, 0.1 M BT Prop pH 6.5, 20% PEG-3350, 10% ethylene glycol Ϫ RGS8/G␣i3 16117777, 5729850 pNIC28-Bsa4 L63 - S198/ Ni NTA affinity and S-200 GF GF buffer containing AlF4 No/ 2:1 ratio of G␣i3/RGS8 complex /pProEXHT (Kan/Amp) A31 - Y354 columns for individual 47.5 and precipitant (2ODE) components followed by GF containing 0.2 M (NH4)2SO4, 0.1 M Bis-Tris pH 5.5, 25% PEG-3350 Ϫ RGS10/G␣i3 52694755, 5729850 pLIC-SGC1/pProEXHT S10 - E160/ Ni NTA affinity and S-200 GF GF buffer containing AlF4 No/ 34 1:1 ratio of complex (both Amp) A31 - Y354 columns for individual G␣i3/RGS10 and (2IHB) components followed by GF precipitant containing 0.2 M (NH4)2SO4, 0.1 M Bis-Tris pH 6.5, 25% PEG-3350 Ϫ RGS16/G␣i1 34452690, 33946324 pLIC-SGC1/pProEXHT R53 - T190/ Ni NTA affinity and GF GF buffer containing AlF4 No/ 24 1:1 ratio of complex (both Amp) R32 - F354 columns for individual G␣i1/RGS16 and (2IK8) components followed by GF precipitant containing 0.2 M (NH4)2SO4, 0.1 M Bis-Tris pH 6.5, 25% PEG-3350

PDB ID and GenBank gi numbers are derived from www.pdb.org and www.ncbi.nlm.nih.gov, respectively. Vector/antibiotic, Vector into which protein fragments were cloned and the antibiotic resistance of that vector (Amp, ampicillin; Kan, kanamycin); residues cloned, start and end residues of the polypeptide cloned; purification column, chromatography steps used for protein (and complex) purification; GF, gel filtration chromatography; Ni NTA, nickel–nitriloacetic acid; buffer exchange, the buffer into which the respective protein(s) was exchanged for stability and concentration purposes or for NMR measurements; Tag/Conc, presence or absence of hexahistidine tag in the final protein from which structure was determined and the final concentration of protein in mg/ml. Full information for each protein and complex can be obtained at www.sgc.ox.ac.uk/structures/MM. 6o 16 of 16