Molecular Dissection of the Interaction between the Small G Proteins Rac1 and RhoA and Protein Kinase C-related Kinase 1 (PRK1)
2003; Elsevier BV; Volume: 278; Issue: 50 Linguagem: Inglês
10.1074/jbc.m304313200
ISSN1083-351X
AutoresDarerca Owen, Peter N. Lowe, Daniel Nietlispach, C. Elaine Brosnan, Dimitri Y. Chirgadze, Peter J. Parker, Tom L. Blundell, Helen R. Mott,
Tópico(s)Enzyme Structure and Function
ResumoPRK1 is a serine/threonine kinase that belongs to the protein kinase C superfamily. It can be activated either by members of the Rho family of small G proteins, by proteolysis, or by interaction with lipids. Here we investigate the binding of PRK1 to RhoA and Rac1, two members of the Rho family. We demonstrate that PRK1 binds with a similar affinity to RhoA and Rac1. We present the solution structure of the second HR1 domain from the regulatory N-terminal region of PRK1, and we show that it forms an anti-parallel coiled-coil. In addition, we have used NMR to map the binding contacts of the HR1b domain with Rac1. These are compared with the contacts known to form between HR1a and RhoA. We have used mutagenesis to define the residues in Rac that are important for binding to HR1b. Surprisingly, as well as residues adjacent to Switch I, in Switch II, and in helix α5, it appears that the C-terminal stretch of basic amino acids in Rac is required for a high affinity interaction with HR1b. PRK1 is a serine/threonine kinase that belongs to the protein kinase C superfamily. It can be activated either by members of the Rho family of small G proteins, by proteolysis, or by interaction with lipids. Here we investigate the binding of PRK1 to RhoA and Rac1, two members of the Rho family. We demonstrate that PRK1 binds with a similar affinity to RhoA and Rac1. We present the solution structure of the second HR1 domain from the regulatory N-terminal region of PRK1, and we show that it forms an anti-parallel coiled-coil. In addition, we have used NMR to map the binding contacts of the HR1b domain with Rac1. These are compared with the contacts known to form between HR1a and RhoA. We have used mutagenesis to define the residues in Rac that are important for binding to HR1b. Surprisingly, as well as residues adjacent to Switch I, in Switch II, and in helix α5, it appears that the C-terminal stretch of basic amino acids in Rac is required for a high affinity interaction with HR1b. The Rho family of small GTPases regulates a wide variety of cellular functions including cell growth and differentiation, cell motility and adhesion (via control of the actin cytoskeleton), and cell cycle progression (1.Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5168) Google Scholar, 2.Olson M.F. Ashworth A. Hall A. Science. 1995; 269: 1270-1272Crossref PubMed Scopus (1053) Google Scholar). An understanding, at a molecular level, of how these small G proteins control these cellular events would provide a major advance, potentially leading to therapeutic opportunities in areas such as hypertension (3.Mukai Y. Shimokawa H. Matoba T. Kandabashi T. Satoh S. Hiroki J. Kaibuchi K. Takeshita A. FASEB J. 2001; 15: 1062-1064PubMed Google Scholar), Alzheimer's disease (4.Kawamata T. Taniguchi T. Mukai H. Kitagawa M. Hashimoto T. Maeda K. Ono Y. Tanaka C. J. Neurosci. 1998; 18: 7402-7410Crossref PubMed Google Scholar), and cancer. Small G proteins act as molecular switches, being active in the GTP-bound form and inactive in the GDP-bound form. The interaction of the G protein (in its GTP-bound conformation) with an effector protein triggers a downstream signaling cascade. Effectors for Rho include at least 11 proteins: diacylglycerol kinase, phospholipase D, phosphatidylinositol phosphate 5-kinase, kinectin, rhotekin, rhophilin, p140 Diaphanous, myosin-binding subunit, citron, Rho kinase, and PRK 1The abbreviations used are: PRKprotein kinase C-related kinasePAKp21-activated kinaseGAPGTPase activator proteinGSTglutathione S-transferaseSPAscintillation proximity assayNMRnuclear magnetic resonanceNOEnuclear Overhauser effectr.m.s.d.root mean square deviationGMPPNPguanosine 5′-[β,γimido]triphosphate.1The abbreviations used are: PRKprotein kinase C-related kinasePAKp21-activated kinaseGAPGTPase activator proteinGSTglutathione S-transferaseSPAscintillation proximity assayNMRnuclear magnetic resonanceNOEnuclear Overhauser effectr.m.s.d.root mean square deviationGMPPNPguanosine 5′-[β,γimido]triphosphate. (or PKN). Within this group of effector proteins there are at least two Rho-binding motifs defined by sequence homology: REM (or class 1 Rho-binding motif) include the PRKs, rhophilin and rhotekin, whereas RKH (Rho effector homology 2 or class 2 Rho-binding motif) include the Rho kinases and kinectin (5.Bishop A.L. Hall A. Biochem. J. 2000; 348: 241-255Crossref PubMed Scopus (1655) Google Scholar). protein kinase C-related kinase p21-activated kinase GTPase activator protein glutathione S-transferase scintillation proximity assay nuclear magnetic resonance nuclear Overhauser effect root mean square deviation guanosine 5′-[β,γimido]triphosphate. protein kinase C-related kinase p21-activated kinase GTPase activator protein glutathione S-transferase scintillation proximity assay nuclear magnetic resonance nuclear Overhauser effect root mean square deviation guanosine 5′-[β,γimido]triphosphate. The importance of the PRK effector family for the biological functions of the Rho family of small G proteins is indicated by the interactions that have been described for PRK1 and -2. Many proteins that interact with PRK1/2 are involved with the cytoskeletal network, e.g. α-actinin (6.Mukai H. Toshimori M. Shibata H. Takanaga H. Kitagawa M. Miyahara M. Shimakawa M. Ono Y. J. Biol. Chem. 1997; 272: 4740-4746Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) and vimentin (7.Matsuzawa K. Kosako H. Inagaki N. Shibata H. Mukai H. Ono Y. Amano M. Kaibuchi K. Matsuura Y. Azuma I. Inagaki M. Biochem. Biophys. Res. Commun. 1997; 234: 621-625Crossref PubMed Scopus (64) Google Scholar). As a major function of the Rho family proteins is known to be the control of the actin cytoskeleton, finding interactions between cytoskeletal proteins and Rho effectors was to be expected. PRKs are also implicated in the control of transcription factors (8.Kitagawa M. Mukai H. Takahashi M. Ono Y. Biochem. Biophys. Res. Commun. 1998; 252: 561-565Crossref PubMed Scopus (13) Google Scholar, 9.Takanaga H. Mukai H. Shibata H. Toshimori M. Ono Y. Exp. Cell Res. 1998; 241: 363-372Crossref PubMed Scopus (30) Google Scholar), mitogenesis (10.Gao Q.S. Kumar A. Srinivasan S. Singh L. Mukai H. Ono Y. Wazer D.E. Band V. J. Biol. Chem. 2000; 275: 14824-14830Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), and cell cycle regulation (11.Misaki K. Mukai H. Yoshinaga C. Oishi K. Isagawa T. Takahashi M. Ohsumi K. Kishimoto T. Ono Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 125-129Crossref PubMed Scopus (24) Google Scholar). PRK1 and -2 have also been shown to play a role in apoptosis, being activated by caspase 3 (12.Takahashi M. Mukai H. Toshimori M. Mlyamoto M. Ono Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11566-11571Crossref PubMed Scopus (119) Google Scholar) and being involved in the regulation of Akt (13.Koh H. Lee K.H. Kim D. Kim S. Kim J.W. Chung J. J. Biol. Chem. 2000; 275: 34451-34458Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Recently, PRKs have been shown to be involved in keratinocyte cell-cell adhesion with increased PRK activity promoting cell-cell adhesion (14.Calautti E. Grossi M. Mammucai C. Aoyama Y. Purro M. Ono J.L. Dotto G. J. Cell Biol. 2002; 156: 137-148Crossref PubMed Scopus (144) Google Scholar). The activity of PRKs in so many processes intimately involved in disease progression makes dissection of their regulation of widespread interest. PRK1 and -2 are highly related serine/threonine kinases that have a catalytic domain homologous to those of the protein kinase C family in their C termini and a unique regulatory domain in their N termini (Fig. 1) (15.Mukai H. Ono Y. Biochem. Biophys. Res. Commun. 1994; 199: 897-904Crossref PubMed Scopus (140) Google Scholar, 16.Palmer R.H. Ridden J. Parker P.J. Eur. J. Biochem. 1995; 227: 344-351Crossref PubMed Scopus (121) Google Scholar). The N terminus of the protein was found to contain a pseudo-substrate sequence that acts as an auto-inhibitory region (17.Kitagawa M. Shibata H. Toshimori M. Mukai H. Ono Y. Biochem. Biophys. Res. Commun. 1996; 220: 963-968Crossref PubMed Scopus (39) Google Scholar). This regulatory region also contains three leucine zipper type motifs (HR1 repeats a-c). The first of these repeats, HR1a, is now known to incorporate the inhibitory pseudo-substrate site (17.Kitagawa M. Shibata H. Toshimori M. Mukai H. Ono Y. Biochem. Biophys. Res. Commun. 1996; 220: 963-968Crossref PubMed Scopus (39) Google Scholar). PRK1/2 kinase activity is enhanced by binding of the small GTP-binding proteins Rho and Rac in a GTP-dependent manner (18.Amano M. Mukai H. Ono Y. Chihara K. Matsui T. Hamajima Y. Okawa K. Iwamatsu A. Kaibuchi K. Science. 1996; 271: 648-650Crossref PubMed Scopus (394) Google Scholar, 19.Lu Y. Settleman J. 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A. 1998; 95: 11566-11571Crossref PubMed Scopus (119) Google Scholar, 24.Cryns V.L. Byun Y. Rana A. Mellor H. Lustig K.D. Ghanem L. Parker P.J. Kirschner M.W. Yuan J.Y. J. Biol. Chem. 1997; 272: 29449-29453Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Progress has been made recently in the dissection of the regulatory mechanisms employed by the Rho family effector molecules PAK (25.Lei M. Lu W.G. Meng W.Y. Parrini M.C. Eck M.J. Mayer B.J. Harrison S.C. Cell. 2000; 102: 387-397Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar) and Wiskott-Aldrich syndrome protein (26.Kim A.S. Kakalis L.T. Abdul-Manan M. Liu G.A. Rosen M.K. Nature. 2000; 404: 151-158Crossref PubMed Scopus (610) Google Scholar). It appears that many of the Rho family effectors are regulated in a similar manner, being maintained in a closed, inactive conformation via inhibitory intramolecular contacts. These inhibitory contacts can then be relieved by interactions with e.g. other proteins, fatty acids, and phosphoinositides. The involvement of small G proteins in the control of PRK1/2 was first defined when PRK1 was found to be a target for Rho (18.Amano M. Mukai H. Ono Y. Chihara K. Matsui T. Hamajima Y. Okawa K. Iwamatsu A. Kaibuchi K. Science. 1996; 271: 648-650Crossref PubMed Scopus (394) Google Scholar, 21.Watanabe G. Saito Y. Madaule P. Ishizaki T. Fujisawa K. Morii N. Mukai H. Ono Y. Kakizuka A. Narumiya S. Science. 1996; 271: 645-648Crossref PubMed Scopus (347) Google Scholar). The minimal region required for Rho binding was found to be residues 33-111 (Fig. 1), which contains the first HR1 repeat, HR1a, and the pseudo-substrate site (27.Shibata H. Mukai H. Inagaki Y. Homma Y. Kimura K. Kaibuchi K. Narumiya S. Ono Y. FEBS Lett. 1996; 385: 221-224Crossref PubMed Scopus (58) Google Scholar). Rho was subsequently found to bind to both isolated HR1a and HR1b domains but to HR1a in a more GTP-dependent manner, whereas no interaction was detected for HR1c (28.Flynn P. Mellor H. Palmer R. Panayotou G. Parker P.J. J. Biol. Chem. 1998; 273: 2698-2705Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). G protein-interacting proteins are classically defined as effectors if they bind selectively to the GTP-bound (active) form of the G protein. These results therefore suggested that the HR1a repeat was the important Rho-binding site as it provided discrimination in favor of the active form of Rho. Both PRK1 and -2 have been demonstrated recently (19.Lu Y. Settleman J. Genes Dev. 1999; 13: 1168-1180Crossref PubMed Scopus (112) Google Scholar, 20.Vincent S. Settleman J. Mol. Cell. Biol. 1997; 17: 2247-2256Crossref PubMed Scopus (172) Google Scholar) to be targets not only for Rho but also for Rac. With the Rho family of small G proteins occupying such a central role in a diverse set of cellular processes, many of which are important and altered in disease progression, much effort has been expended over the last few years to elucidate the interactions of these proteins. Progress has been made in determining the structures of Rho family G proteins in complex with various effector proteins (29.Abdul-Manan N. Aghazadeh B. Liu G.A. Majumdar A. Ouerfelli O. Siminovitch K.A. Rosen M.K. Nature. 1999; 399: 379-383Crossref PubMed Scopus (277) Google Scholar, 30.Lapouge K. Smith S.J.M. Walker P.A. Gamblin S.J. Smerdon S.J. Rittinger K. Mol. Cell. 2000; 6: 899-907Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 31.Mott H.R. Owen D. Nietlispach D. Lowe P.N. Manser E. Lim L. Laue E.D. Nature. 1999; 399: 384-388Crossref PubMed Scopus (149) Google Scholar, 32.Morreale A. Venkatesan M. Mott H.R. Owen D. Nietlispach D. Lowe P.N. Laue E.D. Nat. Struct. 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Kaibuchi K. Hakoshima T. Mol. Cell. 1999; 4: 793-803Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). This structure describes the fold of the HR1a domain as an antiparallel coiled-coil domain termed the ACC finger domain. The ACC finger domain describes a new category of G protein binding domains quite distinct from those found in other known Rho family effectors. It also differs from the ubiquitin fold Ras binding domains of Raf (37.Nassar M. Horn G. Herrmann C. Scherer A. McCormick F. Wittinghofer A. Nature. 1995; 375: 554-560Crossref PubMed Scopus (555) Google Scholar, 38.Emerson S.D. Madison V.S. Palermo R.E. Waugh D.S. Scheffler J.E. Tsao K.L. Kiefer S.E. Liu S.P. Fry D.C. Biochemistry. 1995; 34: 6911-6918Crossref PubMed Scopus (95) Google Scholar) and Ral GEF (39.Geyer M. Herrmann C. Wohlgemuth S. Wittinghofer A. Kalbitzer H.R. Nat. Struct. Biol. 1997; 4: 694-699Crossref PubMed Scopus (100) Google Scholar, 40.Huang L. Weng X.W. Hofer F. Martin G.S. Kim S.H. Nat. Struct. Biol. 1997; 4: 609-615Crossref PubMed Scopus (66) Google Scholar) and from the Ran binding domain of importin (41.Vetter I.R. Arndt A. Kutay U. Gorlich D. Wittinghofer A. Cell. 1999; 97: 635-646Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 42.Chook Y.M. Blobel G. Nature. 1999; 399: 230-237Crossref PubMed Scopus (287) Google Scholar). The Rab binding domain of Rabphilin and the Rac/Arf effector Arfaptin are also helical, but the contacts that each make with their G proteins are quite different (43.Ostermeier C. Brunger A.T. Cell. 1999; 96: 363-374Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). The results of Flynn et al. (28.Flynn P. Mellor H. Palmer R. Panayotou G. Parker P.J. J. Biol. Chem. 1998; 273: 2698-2705Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) suggesting the possibility of multiple G protein-interacting sites on PRK1 and further data identifying PRKs as targets for both Rac1 and RhoA (19.Lu Y. Settleman J. Genes Dev. 1999; 13: 1168-1180Crossref PubMed Scopus (112) Google Scholar, 20.Vincent S. Settleman J. Mol. Cell. Biol. 1997; 17: 2247-2256Crossref PubMed Scopus (172) Google Scholar) stimulated this work, in which we set out to determine the role of the multiple HR1 domains in PRK1/G protein interactions. We demonstrate here that the PRK1 HR1 repeats have different affinities for both Rho and Rac. We present the structure of PRK1 HR1b and a map of the contacts it makes in complex with Rac1. We also present data to show the necessity of the C terminus of Rac for high affinity binding to PRK1 and mutagenesis data that indicate the site of HR1b interaction on Rac. Expression Constructs—All proteins were expressed as GST fusion proteins from the pGEX vectors (Amersham Biosciences). The construct expressing residues 75-132 of PAK has been described previously (44.Owen D. Mott H.R. Laue E.D. Lowe P.N. Biochemistry. 2000; 39: 1243-1250Crossref PubMed Scopus (61) Google Scholar) and that expressing residues 198-439 of RhoGAP was a kind gift from Prof. Alan Hall (45.Lancaster C.A. Taylorharris P.M. Self A.J. Brill S. Vanerp H.E. Hall A. J. Biol. Chem. 1994; 269: 1137-1142Abstract Full Text PDF PubMed Google Scholar). The HR1a expression construct consisted of PRK1 residues 1-106, HR1b residues 122-199, and HR1ab residues 1-199. Rac1 Q61L and all other mutants were expressed in pGEX2T from constructs encoding residues 1-191 cloned into the BamHI and EcoRI sites. The pGEX2T-Rac1 Q61L construct was used as a template to generate the C-terminal deletion constructs. Oligonucleotide primers were designed to amplify the required coding sequence, flanked directly by a 5′ BamHI site and a 3′ stop codon followed by an EcoRI site to facilitate subcloning into pGEX2T. The resulting PCR products were ligated into pCR2.1 (Invitrogen), and their sequences were verified using an automated DNA sequencer (Applied Biosystems Inc.) The coding fragments were then excised using BamHI and EcoRI and ligated into pGEX2T linearized with the same enzymes. Rac1 Mutagenesis—Site-directed mutagenesis of the pGEX2T-Q61L Rac1 expression construct encoding residues 1-191 was performed using the QuikChange Site-directed Mutagenesis kit (Stratagene). The sequence of the Rac1 coding region of all mutants was verified using an automated DNA sequencer (Applied Biosystems Inc.). Recombinant Protein Production—GST fusion proteins were expressed in Escherichia coli BL21. Stationary cultures were diluted 1 in 10, grown at 37 °C to an A600 of 0.8, and induced with 0.1 mm isopropyl-β-d-thiogalactopyranoside for 5 h. Proteins were affinity-purified using glutathione-agarose beads (Sigma). Fusions of HR1a, -b, -ab, PAK GBD, and RhoGAP were eluted from the glutathione-agarose beads and used directly in SPAs. GST-Rac1 variants were cleaved from their GST tag while attached to the glutathione-agarose beads with thrombin (Novagen). Protein concentrations for all proteins were evaluated using their A280, amino acid compositions, and the extinction coefficients of tyrosine, phenylalanine, tryptophan, and the guanine nucleotide (46.Mach H. Middaugh C.R. Lewis R.V. Anal. Biochem. 1992; 200: 74-80Crossref PubMed Scopus (419) Google Scholar). The integrity of each protein was determined by mass spectrometry. Labeled proteins for NMR spectroscopy were produced by growing the E. coli in a medium based on MOPS buffer, containing 50% labeled Celtone with 15NH4Cl and/or [13C]glucose (Spectra Stable Isotopes). Labeled HR1b was cleaved from its GST tag with factor Xa (Roche Applied Science), and the resulting protein was further purified on a Superdex-30 gel filtration column (Amersham Biosciences) in NMR buffer (20 mm Tris-HCl, pH 7.4, 20 mm NaCl, 0.05% NaN3). Nucleotide exchange of G proteins was performed as described previously (44.Owen D. Mott H.R. Laue E.D. Lowe P.N. Biochemistry. 2000; 39: 1243-1250Crossref PubMed Scopus (61) Google Scholar). Scintillation Proximity Assays (SPA)—Affinities of Rac1 and RhoA proteins for GST-HR1 domains, GST-PAK-(75-132), and GST-RhoGAP were measured using SPAs. GST fusion protein was attached to a fluoromicrosphere via an anti-GST antibody in the presence of Q61L Rac/Q63L Rho·[3H]GTP. Binding of the G protein to the GST fusion protein brings the labeled nucleotide close enough to the scintillant to obtain a signal. Apparent Kd values for Q61L Rac·[3H]GTP, Q63L Rho·[3H]GTP, and proteins incorporating further mutations were measured as described previously (47.Thompson G. Owen D. Chalk P.A. Lowe P.N. Biochemistry. 1998; 37: 7885-7891Crossref PubMed Scopus (120) Google Scholar, 48.Graham D.L. Eccleston J.F. Lowe P.N. Biochemistry. 1999; 38: 985-991Crossref PubMed Scopus (69) Google Scholar) by varying the concentration of Rac/Rho·[3H]GTP at a constant concentration of GST fusion protein. These assays were performed with 30 nm GST fusion protein. By using this method the upper and lower limits of the Kd values that can be measured are 1000 and 1 nm, respectively. For each affinity determination, data points were obtained for at least 10 different Rac1 concentrations. Binding curves were fitted using the appropriate binding isotherms (47.Thompson G. Owen D. Chalk P.A. Lowe P.N. Biochemistry. 1998; 37: 7885-7891Crossref PubMed Scopus (120) Google Scholar, 48.Graham D.L. Eccleston J.F. Lowe P.N. Biochemistry. 1999; 38: 985-991Crossref PubMed Scopus (69) Google Scholar) to obtain Kd values. NMR Spectroscopy—NMR samples were produced to a final concentration of ∼1 mm in 500 μl with 10% D2O. Experiments were recorded on Bruker DRX spectrometers operating at 500 or 800 MHz, and all experiments were recorded at 298 K. 15N-HSQC, three-dimensional 15N-separated NOESY (mixing time 100 ms), 15N-separated TOCSY (DIPSI-2 mixing time 60 ms), and HNHA experiments were recorded on uniformly 15N-labeled HR1b. 13C-HSQC, HQQC, three-dimensional HNCA, HN(CO)CA, CBCA(CO)NH, HNCACB, H(CC)(CO)NH, HCCHTOCSY (DIPSI-2 mixing time 18.4 ms), and 13C-separated NOESY (mixing time 100 ms) experiments were recorded on 13C,15N-labeled HR1b (see Ref. 49.Ferentz A.E. Wagner G. Q. Rev. Biophys. 2000; 33 (and references therein): 29-65Crossref PubMed Scopus (207) Google Scholar). NMR data were processed using AZARA and analyzed using ANSIG (50.Kraulis P.J. Domaille P.J. Campbellburk S.L. Vanaken T. Laue E.D. Biochemistry. 1994; 33: 3515-3531Crossref PubMed Scopus (288) Google Scholar). 3JHN-HA couplings were measured from an HNHA experiment. Backbone torsion angles were also estimated from CA, CO, CB, N, and HA chemical shifts using the program TALOS (51.Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2727) Google Scholar). These were used as restraints for ϕ and Ψ with errors of ±30°, or twice the standard deviation from TALOS, whichever was larger. In those cases where the 3JHN-HA could be measured, the error on the ϕ angle was set to ±10°. The titration of unlabeled Rac1 into 15N-labeled HR1b was performed in the same buffer conditions as the HR1b spectra. Titration points were measured at 1:0.2, 1:0.4, 1:0.6, 1:0.8, and 1:1, HR1b:Rac1. Chemical shift changes were calculated as a final deviation δHN = √(δN)2 + (10 × δH)2, where δN = change in 15N chemical shift and δH = change in 1H chemical shift. Structure Calculations—Structures were calculated using CNS 1.0 and ARIA 1.0 (52.Linge J.P. O'Donoghue S.I. Nilges M. Methods Enzymol. 2001; 339: 71-90Crossref PubMed Scopus (329) Google Scholar), where the ambiguity of NOEs is reduced for each iteration by comparing the NOE tables with structures from the previous iteration. 20 structures were calculated for each iteration, and 7 were used for ARIA analysis. The total contribution of the accepted NOEs to the cross-peak volume was decreased during the 9 iterations from 1.01 (keeping all possibilities) to 0.8 (rejecting the possibilities that contribute less than 20% of the cross-peak volume). The parameters used for the calculation were essentially as described in Linge et al. (52.Linge J.P. O'Donoghue S.I. Nilges M. Methods Enzymol. 2001; 339: 71-90Crossref PubMed Scopus (329) Google Scholar) except that the length of the high temperature dynamics was increased to 6 ps and the cooling to a total of 39 ps (24 ps of cooling to 1000 K and 15 ps of cooling to 50 K). The HR1b/Rac1 modeling was performed with the protocol HADDOCK, interfaced to CNS 1.0 (53.Dominguez C. Boelens R. Bonvin A. J. Am. Chem. Soc. 2003; 125: 1731-1737Crossref PubMed Scopus (2087) Google Scholar). For all sets of residues, the solvent accessibility was estimated using the program NACCESS (54.Hubbard S.J. Thornton J.M. NACCESS. University College, London1993Google Scholar), and those residues that were less than 50% solvent-exposed were not used. The HR1b residues whose chemical shifts had changed by more than the mean shift change were the active residues. The passive residues were defined as those that were within 5 Å of the active residues. The Rac1 active residues were defined as those that caused a greater than 2.5-fold increase in the Kd. The passive residues were those that were within 5 Å of the active residues. NMR Structure of PRK1 HR1b—The backbone resonances of HR1b were assigned using standard triple resonance NMR experiments. Complete assignments were obtained except for the backbone amides of residues 155-159. Side chain assignments were obtained using HCCH-TOCSY, 15N-separated TOCSY, and H(CC)(CO)NH experiments. 2077 distance restraints were extracted from 15N-separated and 13C-separated NOESY spectra, of which 1443 were unambiguous at the start of the calculation and 634 were ambiguous. These were translated by ARIA into 1575 restraints (1040 unambiguous and 535 ambiguous) for the first iteration. The remaining 502 restraints were removed, either because they were non-unique or because they were ambiguous restraints with more than 50 assignment possibilities. At the end of the final iteration there were 1207 unambiguous and 400 ambiguous restraints. 52 ϕ restraints were included from the 3JHN-HA measurements and an additional 69 loose ϕ and Ψ restraints from TALOS calculations. Table I shows the structural statistics for the 25 lowest energy structures of 100 calculated. The final structures had no NOE violations of more than 0.5 Å and no dihedral violations of more than 5°. The structures are of good quality as judged from a Ramachandran plot (87% of the residues are in the most favored region). The backbone r.m.s.d. over all non-terminal residues (124-191) is 0.82 Å. The HR1b structure is shown in Fig. 2. It consists of two α-helices, which pack together into an anti-parallel coiled-coil. The packing between the helices is mediated by a regular array of Leu, Ile, and Ala side chains. The structures are well defined over both the α-helices. The turn between the helices, residues 152-160, is less well defined by the NMR data. The NH resonances within this loop were absent from the spectra, suggesting that they are undergoing exchange on a ms time scale.Table IExperimental restraints and structural statisticsNo. experimental restraints Unambiguous1207 Ambiguous400 Dihedral restraints121〈SA〉a〈SA〉 represents the average root mean square deviations for the ensemble〈SA〉cb〈SA〉c represents values for the structure that is closest to the meanCoordinate precision r.m.s.d. of backbone atoms 124—191 (Å)0.82 ± 0.160.66 r.m.s.d. of all heavy atoms 124—191 (Å)1.39 ± 0.141.49 r.m.s.d. of backbone atoms 124—153, 160—191 (Å)0.67 ± 0.160.35 r.m.s.d. of all heavy atoms 124—153, 160—191 (Å)1.36 ± 0.121.20 r.m.s.d. From the experimental restraints NOE distances (Å)0.013 ± 0.00360.015 Dihedral angles (°)0.058 ± 0.0400.083 From idealized geometry Bonds (Å)0.0014 ± 1.02 × 10-40.0012 Angles (°)0.321 ± 0.00640.308 Impropers (°)0.182 ± 0.0130.169Final energy EL-JcThe Lennard-Jones potential was not used at any stage in the refinement (kJ/mol)—335.7 ± 12.19—347.89Ramachandran analysis Residues in most favored regions87.0%89.3% Residues in additionally allowed regions7.1%6.7% Residues in generously allowed regions4.0%4.0% Residues in disallowed regions1.9%0.0%a 〈SA〉 represents the average root mean square deviations for the ensembleb 〈SA〉c represents values for the structure that is closest to the meanc The Lennard-Jones potential was not used at any stage in the refinement Open table in a new tab Selective Binding of HR1 Repeats to Rho Family G Proteins—PRK1 was originally isolated as a Rho effector protein (18.Amano M. Mukai H. Ono Y. Chihara K. Matsui T. Hamajima Y. Okawa K. Iwamatsu A. Kaibuchi K. Science. 1996; 271: 648-650Crossref PubMed Scopus (394) Google Scholar, 21.Watanabe G. Saito Y. Madaule P. Ishizaki T. Fujisawa K. Morii N. Mukai H. Ono Y. Kakizuka A. Narumiya S. Science. 1996; 271: 645-648Crossref PubMed Scopus (347) Google Scholar). However, as Drosophila PRK1 and -2 have also been described as Rac-interacting proteins (19.Lu Y. Settleman J. Genes Dev. 1999; 13: 1168-1180Crossref PubMed Scopus (112) Google Scholar, 20.Vincent S. Settleman J. Mol. Cell. Biol. 1997; 17: 2247-2256Crossref PubMed Scopus (172) Google Scholar), we decided to examine the binding affinities of the independent HR1 domains of PRK1 to both Rho and Rac. We used SPAs, which measure protein-protein interactions under equilibrium conditions. These have been used previously to quantify the affinity of
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