RGK Small GTP-binding Proteins Interact with the Nucleotide Kinase Domain of Ca2+-channel β-Subunits via an Uncommon Effector Binding Domain
2007; Elsevier BV; Volume: 282; Issue: 15 Linguagem: Inglês
10.1074/jbc.m606423200
ISSN1083-351X
AutoresPascal Béguin, Alvin Yu Jin Ng, Carola Krause, Ramasubbu N. Mahalakshmi, Mei Yong Ng, Walter Hunziker,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoRGK proteins (Kir/Gem, Rad, Rem, and Rem2) form a small subfamily of the Ras superfamily. Despite a conserved GTP binding core domain, several differences suggest that structure, mechanism of action, and functional regulation differ from Ras. RGK proteins down-regulate voltage-gated calcium channel activity by binding in a GTP-dependent fashion to the Cavβ subunits. Mutational analysis combined with homology modeling reveal a novel effector binding mechanism distinct from that of other Ras GTPases. In this model the Switch 1 region acts as an allosteric activator that facilitates electrostatic interactions between Arg-196 in Kir/Gem and Asp-194, -270, and -272 in the nucleotide-kinase (NK) domain of Cavβ3 and wedging Val-223 and His-225 of Kir/Gem into a hydrophobic pocket in the NK domain. Kir/Gem interacts with a surface on the NK domain that is distinct from the groove where the voltage-gated calcium channel Cavα1 subunit binds. A complex composed of the RGK protein and the Cavβ3 and Cav1.2 subunits could be revealed in vivo using coimmunoprecipitation experiments. Intriguingly, docking of the RGK protein to the NK domain of the Cavβ subunit is reminiscent of the binding of GMP to guanylate kinase. RGK proteins (Kir/Gem, Rad, Rem, and Rem2) form a small subfamily of the Ras superfamily. Despite a conserved GTP binding core domain, several differences suggest that structure, mechanism of action, and functional regulation differ from Ras. RGK proteins down-regulate voltage-gated calcium channel activity by binding in a GTP-dependent fashion to the Cavβ subunits. Mutational analysis combined with homology modeling reveal a novel effector binding mechanism distinct from that of other Ras GTPases. In this model the Switch 1 region acts as an allosteric activator that facilitates electrostatic interactions between Arg-196 in Kir/Gem and Asp-194, -270, and -272 in the nucleotide-kinase (NK) domain of Cavβ3 and wedging Val-223 and His-225 of Kir/Gem into a hydrophobic pocket in the NK domain. Kir/Gem interacts with a surface on the NK domain that is distinct from the groove where the voltage-gated calcium channel Cavα1 subunit binds. A complex composed of the RGK protein and the Cavβ3 and Cav1.2 subunits could be revealed in vivo using coimmunoprecipitation experiments. Intriguingly, docking of the RGK protein to the NK domain of the Cavβ subunit is reminiscent of the binding of GMP to guanylate kinase. Small GTPases comprise four major branches, Rab, Ras, Arf, and Rho. The RGK 3The abbreviations used are: RGK proteins, Kir/Gem, Rad, Rem, Rem2; HEK, human embryonic kidney; NK, nucleotide kinase; SH3, Src homology 3; VDCC, voltage-gated calcium channel; WT, wild type; Y2H, yeast two hybrid; IP, immunoprecipitate; HA, hemagglutinin. 3The abbreviations used are: RGK proteins, Kir/Gem, Rad, Rem, Rem2; HEK, human embryonic kidney; NK, nucleotide kinase; SH3, Src homology 3; VDCC, voltage-gated calcium channel; WT, wild type; Y2H, yeast two hybrid; IP, immunoprecipitate; HA, hemagglutinin. family belongs to the Ras superfamily and includes Rad (1Reynet C. Kahn C.R. Science. 1993; 262: 1441-1444Crossref PubMed Scopus (277) Google Scholar), Rem (2Finlin B.S. Andres D.A. J. Biol. Chem. 1997; 272: 21982-21988Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), Rem2 (3Finlin B.S. Shao H. Kadono-Okuda K. Guo N. Andres D.A. Biochem. J. 2000; 347: 223-231Crossref PubMed Scopus (88) Google Scholar, 4Béguin P. Mahalakshmi R.N. Nagashima K. Cher D.H. Kuwamura N. Yamada Y. Seino Y. Hunziker W. Biochem. J. 2005; 390: 67-75Crossref PubMed Scopus (62) Google Scholar), and Kir/Gem (5Maguire J. Santoro T. Jensen P. Siebenlist U. Yewdell J. Kelly K. Science. 1994; 265: 241-244Crossref PubMed Scopus (162) Google Scholar, 6Cohen L. Mohr R. Chen Y.Y. Huang M. Kato R. Dorin D. Tamanoi F. Goga A. Afar D. Rosenberg N. et al.Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12448-12452Crossref PubMed Scopus (82) Google Scholar). Although the core region involved in nucleotide binding is conserved with other Ras superfamily members, RGK proteins show several significant structural and functional differences to Ras (7Kelly K. Trends Cell Biol. 2005; 15: 640-643Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). These include an unconventional G3 motif indicative of an unusual mechanism for GTP hydrolysis (3Finlin B.S. Shao H. Kadono-Okuda K. Guo N. Andres D.A. Biochem. 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Nagashima K. Gonoi T. Shibasaki T. Takahashi K. Kashima Y. Ozaki N. Geering K. Iwanaga T. Seino S. Nature. 2001; 411: 701-706Crossref PubMed Scopus (239) Google Scholar), and the lack of lipid modification for membrane association. An important physiological function of RGK proteins is the regulation of VDCC activity (4Béguin P. Mahalakshmi R.N. Nagashima K. Cher D.H. Kuwamura N. Yamada Y. Seino Y. Hunziker W. Biochem. J. 2005; 390: 67-75Crossref PubMed Scopus (62) Google Scholar, 9Béguin P. Mahalakshmi R.N. Nagashima K. Cher D.H. Ikeda H. Yamada Y. Seino Y. Hunziker W. J. Mol. Biol. 2006; 355: 34-46Crossref PubMed Scopus (80) Google Scholar, 10Béguin P. Mahalakshmi R.N. Nagashima K. Cher D.H. Takahashi A. Yamada Y. Seino Y. Hunziker W. J. Cell Sci. 2005; 118: 1923-1934Crossref PubMed Scopus (69) Google Scholar, 11Béguin P. Nagashima K. Gonoi T. Shibasaki T. Takahashi K. Kashima Y. Ozaki N. Geering K. Iwanaga T. Seino S. 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Biol. 2000; 16: 521-555Crossref PubMed Scopus (1905) Google Scholar, 18Dolphin A.C. J. Bioenerg. Biomembr. 2003; 35: 599-620Crossref PubMed Scopus (305) Google Scholar). The Cavβ subunit is a cytosolic protein with an SH3 and NK (also referred to as guanylate kinase) domain (19Chen Y.H. Li M.H. Zhang Y. He L.L. Yamada Y. Fitzmaurice A. Shen Y. Zhang H. Tong L. Yang J. Nature. 2004; 429: 675-680Crossref PubMed Scopus (256) Google Scholar, 20Van Petegem F. Clark K.A. Chatelain F.C. Minor Jr., D.L. Nature. 2004; 429: 671-675Crossref PubMed Scopus (347) Google Scholar), and several isoforms and splice variants have been described (18Dolphin A.C. J. Bioenerg. Biomembr. 2003; 35: 599-620Crossref PubMed Scopus (305) Google Scholar). RGK proteins bind the Cavβ subunit, leading to the down-regulation of channel activity due either to the inhibition of channels present in the plasma membrane (13Finlin B.S. Mosley A.L. Crump S.M. Correll R.N. Ozcan S. Satin J. Andres D.A. J. Biol. Chem. 2005; 280: 41864-41871Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 15Chen H. Puhl III, H.L. Niu S.L. Mitchell D.C. Ikeda S.R. J. Neurosci. 2005; 25: 9762-9772Crossref PubMed Scopus (78) Google Scholar) or interference with cell surface expression of the Cavα1 subunit (4Béguin P. Mahalakshmi R.N. Nagashima K. Cher D.H. Kuwamura N. Yamada Y. Seino Y. Hunziker W. Biochem. J. 2005; 390: 67-75Crossref PubMed Scopus (62) Google Scholar, 9Béguin P. Mahalakshmi R.N. Nagashima K. Cher D.H. Ikeda H. Yamada Y. Seino Y. Hunziker W. J. Mol. Biol. 2006; 355: 34-46Crossref PubMed Scopus (80) Google Scholar, 10Béguin P. Mahalakshmi R.N. Nagashima K. Cher D.H. Takahashi A. Yamada Y. Seino Y. Hunziker W. J. Cell Sci. 2005; 118: 1923-1934Crossref PubMed Scopus (69) Google Scholar, 11Béguin P. Nagashima K. Gonoi T. Shibasaki T. Takahashi K. Kashima Y. Ozaki N. Geering K. Iwanaga T. Seino S. Nature. 2001; 411: 701-706Crossref PubMed Scopus (239) Google Scholar, 21Sasaki T. Shibasaki T. Beguin P. Nagashima K. Miyazaki M. Seino S. J. Biol. Chem. 2005; 280: 9308-9312Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). It was thought that RGK proteins interact through the β-interacting domain of the Cavβ subunit, thus preventing association between the Cavα1 and Cavβ subunits (11Béguin P. Nagashima K. Gonoi T. Shibasaki T. Takahashi K. Kashima Y. Ozaki N. Geering K. Iwanaga T. Seino S. Nature. 2001; 411: 701-706Crossref PubMed Scopus (239) Google Scholar, 21Sasaki T. Shibasaki T. Beguin P. Nagashima K. Miyazaki M. Seino S. J. Biol. Chem. 2005; 280: 9308-9312Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). However, this assumption has been challenged since the β-interacting domain appears to be buried in the Cavβ subunit and, rather, may play a role in the maintenance of a correct conformation (19Chen Y.H. Li M.H. Zhang Y. He L.L. Yamada Y. Fitzmaurice A. Shen Y. Zhang H. Tong L. Yang J. Nature. 2004; 429: 675-680Crossref PubMed Scopus (256) Google Scholar, 20Van Petegem F. Clark K.A. Chatelain F.C. Minor Jr., D.L. Nature. 2004; 429: 671-675Crossref PubMed Scopus (347) Google Scholar). In addition, a complex containing Rem, the Cavβ subunit and a glutathione S-transferase-α-interacting domain fusion protein was recently demonstrated (22Finlin B.S. Correll R.N. Pang C. Crump S.M. Satin J. Andres D.A. J. Biol. Chem. 2006; 281: 23557-23566Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The Cavβ subunit interacts with RGK proteins bound to GTP but not GDP and, thus, represents a bona fide effector (4Béguin P. Mahalakshmi R.N. Nagashima K. Cher D.H. Kuwamura N. Yamada Y. Seino Y. Hunziker W. Biochem. J. 2005; 390: 67-75Crossref PubMed Scopus (62) Google Scholar, 9Béguin P. Mahalakshmi R.N. Nagashima K. Cher D.H. Ikeda H. Yamada Y. Seino Y. Hunziker W. J. Mol. Biol. 2006; 355: 34-46Crossref PubMed Scopus (80) Google Scholar, 10Béguin P. Mahalakshmi R.N. Nagashima K. Cher D.H. Takahashi A. Yamada Y. Seino Y. Hunziker W. J. Cell Sci. 2005; 118: 1923-1934Crossref PubMed Scopus (69) Google Scholar, 11Béguin P. Nagashima K. Gonoi T. Shibasaki T. Takahashi K. Kashima Y. Ozaki N. Geering K. Iwanaga T. Seino S. Nature. 2001; 411: 701-706Crossref PubMed Scopus (239) Google Scholar). However, RGK proteins may not be canonical small GTPases that switch between active GTP-bound and inactive GDP-bound forms. RGK proteins lack critical residues in the G2 (i.e. T35) and G3 (i.e. DXXG60) elements that are important for GTP hydrolysis in other Ras superfamily members (7Kelly K. Trends Cell Biol. 2005; 15: 640-643Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Indeed, although most other small GTPases show readily detectable intrinsic GTPase activity, GTP hydrolysis by RGK proteins is exceptionally low (3Finlin B.S. Shao H. Kadono-Okuda K. Guo N. Andres D.A. Biochem. J. 2000; 347: 223-231Crossref PubMed Scopus (88) Google Scholar, 6Cohen L. Mohr R. Chen Y.Y. Huang M. Kato R. Dorin D. Tamanoi F. Goga A. Afar D. Rosenberg N. et al.Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12448-12452Crossref PubMed Scopus (82) Google Scholar), and they are readily isolated from cells in the GTP-bound form (10Béguin P. Mahalakshmi R.N. Nagashima K. Cher D.H. Takahashi A. Yamada Y. Seino Y. Hunziker W. J. Cell Sci. 2005; 118: 1923-1934Crossref PubMed Scopus (69) Google Scholar). Thus, it is conceivable that in cells RGK proteins are constitutively bound to GTP. In this case inactivation of RGK proteins would have to depend on an atypical mechanism for GTP hydrolysis or actively regulated nucleotide exchange. Alternatively, inactivation could be accomplished by sequestering the RGK protein away from its site of action, an attractive possibility given that RGK proteins undergo highly regulated nuclear transport (4Béguin P. Mahalakshmi R.N. Nagashima K. Cher D.H. Kuwamura N. Yamada Y. Seino Y. Hunziker W. Biochem. J. 2005; 390: 67-75Crossref PubMed Scopus (62) Google Scholar, 9Béguin P. Mahalakshmi R.N. Nagashima K. Cher D.H. Ikeda H. Yamada Y. Seino Y. Hunziker W. J. Mol. Biol. 2006; 355: 34-46Crossref PubMed Scopus (80) Google Scholar, 10Béguin P. Mahalakshmi R.N. Nagashima K. Cher D.H. Takahashi A. Yamada Y. Seino Y. Hunziker W. J. Cell Sci. 2005; 118: 1923-1934Crossref PubMed Scopus (69) Google Scholar). Despite these unusual features relative to other small GTPases, little is known about the structure of RGK proteins and how they interact with effectors. Using extensive in vitro mutagenesis combined with in vitro and in vivo binding assays, we identified amino acids in Kir/Gem and the β3 subunit that are critical for their association and used this information to generate a structural model of a complex between the RGK protein and its effector. Our data indicate that RGK proteins and the VDCC α-subunit bind to distinct surfaces on the nucleotide kinase (NK) domain of the β-subunit and reveal an unusual effector binding mechanism for these small GTP-binding proteins. Moreover, a complex composed of an RGK protein, the Cavβ3 subunit, and the intact Cav1.2 subunit was revealed in vivo. These findings provide novel insights into the mechanism by which RGK proteins regulate VDCC activity. Yeast Two-hybrid Screen—The yeast two-hybrid (Y2H) assay was carried out with full-length Kir/Gem W269G and Cavβ3 (residues 49-379) as described (23Ozaki N. Shibasaki T. Kashima Y. Miki T. Takahashi K. Ueno H. Sunaga Y. Yano H. Matsuura Y. Iwanaga T. Takai Y. Seino S. Nat. Cell Biol. 2000; 2: 805-811Crossref PubMed Scopus (390) Google Scholar). Tissue Culture and Transfections—COS-1 and HEK-293T cells were grown and transiently transfected with different cDNAs as detailed (10Béguin P. Mahalakshmi R.N. Nagashima K. Cher D.H. Takahashi A. Yamada Y. Seino Y. Hunziker W. J. Cell Sci. 2005; 118: 1923-1934Crossref PubMed Scopus (69) Google Scholar) and used for experiments 24-38 h after transfection. Molecular Biology and Biochemistry—Point mutations in mouse Kir/Gem, rat β3, and rat Cav1.2 were created by PCR. A mouse Rho kinase β cDNA was assembled from different IMAGE clones (i.e. 534680, 5008498, 5149049, 30619707). All constructs were verified by DNA sequencing. Epitope-tagged proteins, the preparation of cell homogenates, and coprecipitation and pulldown experiments have been described (4Béguin P. Mahalakshmi R.N. Nagashima K. Cher D.H. Kuwamura N. Yamada Y. Seino Y. Hunziker W. Biochem. J. 2005; 390: 67-75Crossref PubMed Scopus (62) Google Scholar, 10Béguin P. Mahalakshmi R.N. Nagashima K. Cher D.H. Takahashi A. Yamada Y. Seino Y. Hunziker W. J. Cell Sci. 2005; 118: 1923-1934Crossref PubMed Scopus (69) Google Scholar). For coimmunoprecipitations, cell lysate (400 μg) was prepared in a buffer containing either Tween 0.2% for Cavβ3/RGK interaction studies or in Triton X-100 0.5% for Cavβ3/RGK/Cav1.2 association analysis. The lysate was incubated with 4 μl of anti-FLAG (M2), anti-Myc (Sigma), or anti-HA (Roche Applied Science)-agarose beads and 30 μl of protein A-Sepharose beads (Amersham Biosciences) for 4 h at 4 °C. After washing, the complexes were eluted and subjected to SDS-PAGE (7-8%), and Western blot analysis using either mouse monoclonal anti-FLAG (M2; Sigma), anti-Myc (Roche Applied Science), or rat anti-HA (Roche Applied Science) antibody was performed. Wild-type (WT) or mutated glutathione S-transferase-Kir/Gem fusion proteins (residues 20-295) (11Béguin P. Nagashima K. Gonoi T. Shibasaki T. Takahashi K. Kashima Y. Ozaki N. Geering K. Iwanaga T. Seino S. Nature. 2001; 411: 701-706Crossref PubMed Scopus (239) Google Scholar) were dialyzed in phosphate-buffered saline and used for GTP/GDP binding assays using the nitrocellulose binding assay (24Moyers J.S. Bilan P.J. Zhu J. Kahn C.R. J. Biol. Chem. 1997; 272: 11832-11839Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). 1 μg of fusion protein was incubated with saturating nucleotide concentrations (100 μm GDP and 3 μm GTP) or concentrations approximating the Kd (10 μm GDP (6Cohen L. Mohr R. Chen Y.Y. Huang M. Kato R. Dorin D. Tamanoi F. Goga A. Afar D. Rosenberg N. et al.Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12448-12452Crossref PubMed Scopus (82) Google Scholar) and 0.5 μm GTP), yielding similar results (data not shown). Immunocytochemistry—Analysis of the subcellular localization and cell surface expression of the Cav1.2 was done as described (4Béguin P. Mahalakshmi R.N. Nagashima K. Cher D.H. Kuwamura N. Yamada Y. Seino Y. Hunziker W. Biochem. J. 2005; 390: 67-75Crossref PubMed Scopus (62) Google Scholar, 9Béguin P. Mahalakshmi R.N. Nagashima K. Cher D.H. Ikeda H. Yamada Y. Seino Y. Hunziker W. J. Mol. Biol. 2006; 355: 34-46Crossref PubMed Scopus (80) Google Scholar, 10Béguin P. Mahalakshmi R.N. Nagashima K. Cher D.H. Takahashi A. Yamada Y. Seino Y. Hunziker W. J. Cell Sci. 2005; 118: 1923-1934Crossref PubMed Scopus (69) Google Scholar). Rabbit anti-Myc (Upstate Biotechnology) and mouse anti-FLAG (Sigma) were used for double labeling, and a rat anti-HA monoclonal (Roche Applied Science) was included for triple-labeling experiments. Specimens were visualized with an Axiocam microscope (Carl Zeiss) at 100× magnification. Molecular Modeling—Molecular modeling, energy calculations, and three-dimensional rendering of protein structures were performed using the Sybyl software package (Tripos, Inc.). Most molecular dynamics simulations were performed at 300 K at an interval of 1000 fs and 1-fs steps using unless otherwise stated, the parameters from Kollman (25Weiner S.J. Kollman P.A. Nguyen D.J. Case D.A. J. Comput. Chem. 1986; 7: 230-252Crossref PubMed Scopus (3582) Google Scholar) for the force field and charges. Energy minimization was performed using the method by Powell and Fletcher (26Fletcher R. Powell M.D.J. Comput. J. 1963; 6: 163-168Crossref Google Scholar). The structure of Kir/Gem was taken from the crystallized structure of Gem bound to GDP (PDB 2G2Y). The Switch 1 region (95GVHDSMDDSD-CEVLG109) was rebuilt based on the structure of the GTP-bound form of Rap2A as a template (PDB 2RAP) (27Menetrey J. Cherfils J. Proteins. 1999; 37: 465-473Crossref PubMed Scopus (14) Google Scholar). Because Arg-196 is not resolved in PDP 2G2Y, its structure was also rendered. The GTP and Mg2+ were from PDB 2RAP were superimposed onto the structure of Kir/Gem using charges assigned by Gasteiger Huckel parameters for GTP and a formal charge of 2+ for the magnesium ion. Molecular dynamics simulations followed by energy refinement using the Tripos Force Field was performed to render the Switch 1 region in the new GTP-bound form of Kir/Gem. The model for the Cavβ3 was derived from the crystal structure of the Cavβ subunit bound to the interacting fragment of the α-subunit (PDB file 1VYT). and the same secondary structure assignments and nomenclature were used (19Chen Y.H. Li M.H. Zhang Y. He L.L. Yamada Y. Fitzmaurice A. Shen Y. Zhang H. Tong L. Yang J. Nature. 2004; 429: 675-680Crossref PubMed Scopus (256) Google Scholar). Kir/Gem was docked onto Cavβ3 taking into account the wet lab experimental data. The largest surface containing the negatively charged residues on the NK domain important for Kir/Gem binding was used as a starting point. This surface showed good shape complementarity with that of the putative G2 domain of Kir/Gem. Modification of the rotamer conformation of the Kir/Gem Arg-196 side chain was required to position the amine group closely enough for it to interact with the carboxyl groups of Asp-194, -270, Asp-272 in Cavβ3. Molecular dynamics simulations and energy minimization determined the optimal position of the Arg-196 side chain with respect to the three Asp residues of Cavβ3. The side chains of Val-223 and His-225 in Kir/Gem fit nicely into a large hydrophobic pocket on the NK domain of the Cavβ subunit. Molecular dynamics simulations (100-fs interval) were performed on the sequence 221AAVQHN226 to investigate the reasons behind the results of the wet lab mutational analysis of Val-223 and His-225. Mutations were introduced in silico into Kir/Gem, and molecular dynamics simulations were carried out to explore the optimal conformations. For the R196K mutation, residues in a sphere radius of 6 Å were subjected to molecular dynamics simulations to explore differences in the conformations of Asp-194, 270, and -272. Identification of Residues in Kir/Gem Critical for the Interaction with Cavβ3—To identify amino acids important for the interaction with Cavβ3, we first generated a truncated form of Kir/Gem containing the core Ras homology domain (amino acids 74-251, Fig. 1A) with the G1-G5 motifs involved in GTP binding (Fig. 1A, gray). As assessed by pulldown experiments, this core domain was sufficient for the nucleotide-dependent interaction with the Cavβ3 subunit (data not shown). We then mutated clusters of three amino acids in the core domain to Ala and tested the interaction of the mutants with Cavβ3 using a Y2H assay. Individual Ala substitutions in the susceptible regions (Fig. 1B, red) led to the identification of nine residues whose mutation interfered with Cavβ3 binding (Fig. 1C). The lack of an interaction between the different Kir/Gem mutants and Cavβ3 was confirmed by coimmunoprecipitation experiments (Fig. 1D). Because calmodulin when bound to Kir/Gem may interfere with Cavβ3 binding (10Béguin P. Mahalakshmi R.N. Nagashima K. Cher D.H. Takahashi A. Yamada Y. Seino Y. Hunziker W. J. Cell Sci. 2005; 118: 1923-1934Crossref PubMed Scopus (69) Google Scholar, 11Béguin P. Nagashima K. Gonoi T. Shibasaki T. Takahashi K. Kashima Y. Ozaki N. Geering K. Iwanaga T. Seino S. Nature. 2001; 411: 701-706Crossref PubMed Scopus (239) Google Scholar), corresponding mutations were also introduced into Kir/Gem W269G, which does not bind calmodulin, and tested for association with Cavβ3. Except for the W269G/V195A mutant (Fig. 1D, lane 15), no coprecipitation was observed. Because V195A (in either the WT or the W269G context) displayed residual binding to Cavβ3 in pulldown experiments (supplemental Data I), we concluded that this residue is less critical for the interaction. Kir/Gem migrates on SDS-PAGE as a doublet, and alkaline phosphatase treatment indicates that the slower migrating band is a phosphorylated form (data not shown). Interestingly, only the faster migrating, non-phosphorylated form of Kir/Gem binds Cavβ3. Furthermore, only the lower band was detected in Kir/Gem mutants that bind neither GTP nor GDP (see Fig. 3), indicating that phosphorylation is sensitive to conformation.FIGURE 3Identification of residues in Kir/Gem important for Cavβ3 binding as opposed to conformation. A, nucleotide binding. Fusion proteins between glutathione S-transferase and WT or mutated Kir/Gem were incubated with labeled GTP (a) or GDP (b), and the bound radioactivity was determined. c, Coomassie-stained SDS-PAGE gel of the fusion proteins to verify the amount of protein used in the assay. B, effect of substitutions of Arg-196, Val-223, and His-225 in Kir/Gem on the interaction with Cavβ3. a, lysate of COS-1 cells expressing WT or mutated Myc-Kir/Gem and FLAG-Cavβ3 were immunoprecipitated with FLAG antibodies to isolate Cavβ3, and associated Kir/Gem was revealed by Western blot using Myc antibody. b and c, cell lysate blotted with Myc or FLAG antibody to monitor Kir/Gem and Cavβ3 expression levels, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The spatial location of the residues critical for Cavβ3 binding was determined based on a structural model of Kir/Gem generated using the recently solved crystal structure of a GDP-bound form of Kir/Gem (PDB 2G3Y) (Fig. 2A). The solved Kir/Gem-GDP structure is overall very similar to that of the GDP- or GTP-bound Rap2A (28Pizon V. Chardin P. Lerosey I. Olofsson B. Tavitian A. Oncogene. 1988; 3: 201-204PubMed Google Scholar), which has 21% sequence identity with Kir/Gem and is the closest homologue to have been crystallized in its GTP-bound form (27Menetrey J. Cherfils J. Proteins. 1999; 37: 465-473Crossref PubMed Scopus (14) Google Scholar). The Switch 1 region in the GDP Kir/Gem structure was not completely resolved, indicating that this region may be disordered, an observation that is confirmed by the recently solved structure of GDP-bound Rad (29Yanuar A. Sakurai S. Kitano K. Hakoshima T. Genes Cells. 2006; 11: 961-968Crossref PubMed Scopus (15) Google Scholar). Thus, we used molecular dynamics simulations to render the Switch 1 region of Kir/Gem using the GTP-bound form of Rap2A, including GTP and Mg2+, as a template (Fig. 2A). These calculations indicate that in the GTP-bound form of Kir/Gem, GTP, and Mg2+ order the Switch 1 region via the coordination with negatively charged residues in Switch 1. The orientation of the long side chain Arg-196 was also not resolved in the crystal structure of GDP-bound Kir/Gem (Fig. 2A). The backbone structure of this region is assumed to be identical in the GDP- and GTP-bound forms of Kir/Gem based on the absence of major structural variations between the GTP- and GDP-bound forms of Rap2A (Fig. 2C). Several of the residues that affect Cavβ3 binding when mutated are buried in the modeled GTP-bound Kir/Gem structure (Fig. 2C), raising the possibility that their mutation alters the conformation of Kir/Gem rather than binding per se. We, therefore, carried out GTP and GDP binding assays to monitor the conformation of the different Kir/Gem mutants. Except for R196A and V223A, the mutants (i.e. L90A, Y156A, L194A, V200A, F231A) displayed a dramatic reduction in both GTP (Fig. 3A, panel a) and GDP (Fig. 3A, panel b) binding, suggesting that they affect the conformation of Kir/Gem. Binding of nucleotides to H225A and V195A was by comparison only marginally affected. Interestingly, Arg-196, Val-223, and His-225 are located on the same surface in the modeled Kir/Gem (Fig. 2C), consistent with them being part of the effector binding domain. This region does not show major conformational differences between the GTP- and GDP-bound structures in Rap2A (Fig. 2B), and Arg-196, Val-223, and His-225 are in close proximity of the G4 and G5 motif. The location and orientation of Arg-196, Val-223, and His-225 is, thus, subject to the conformational constraints for nucleotide binding imposed by the G4 and G5 motifs. Selected amino acids whose substitution to Ala affected either nucleotide and Cavβ3 (Leu-90, Tyr-156) or only Cavβ3 (Arg-196) binding were analyzed in more detail in the Y2H (Fig. 2D) or coimmunoprecipitation (Fig. 3B) assay. Analysis of all known small G proteins showed the presence of Leu, Ile, or Met and Tyr or Phe at the positions corresponding to Kir/Gem Leu-90 and Tyr-196. In contrast to L90A and Y156A, L90I, L90M, and Y156F bound Cavβ3, indicating the importance of these residues for a conserved conformation among the small GTPase superfamily members. Arg-196, which when mutated to Ala selectively affects Cavβ3 binding of Kir/Gem, was substituted to all possible amino acids found in the other Ras superfamily members (30Colicelli J. Sci. STKE 2004. 2004; : RE13Google Scholar). Only the conservative R196K substitution retained binding of Cavβ3 to Kir/Gem (Figs. 2D and 3B, lanes 1-4), indicating that Arg-196 plays a crucial role for the selective interaction of RGK proteins, but not other small GTPases, with the β-subunit. Analysis of Val-223 revealed that conservative substitutions (i.e. V223W, V223F, V223I, V223M, V223Y, V223L) did not affect Cavβ3 binding in coimmunoprecipitation experiments (Fig. 3B), highlighting the requirement of a bulky hydrophobic residue at this position. The only exception was a substitution to Met (see below). Although conservative substitutions of His-225 by Trp and Phe retained binding, the H225R, H225K, and H225I mutants did not associate with Cavβ3. Thus, the large planar side chain rather than the positive charge of His-225 accounts for the association of Kir/Gem with Cavβ3. The critical role of Arg-196 and Val-223 of Kir/Gem for Cavβ3 association was corroborated in vivo. The colocalization of Kir/Gem and Cavβ3 to dendrite-like elongations and the Kir/Gem W269G-mediated nuclear accumulation of β3 were no longer observed in cells expressing the R196A or V223A mutants (supplemental Data II). Residues in Kir/Gem Critical for Cavβ3 Binding Are Conserved in Rad, Rem, and Rem2—Because all RGK proteins bind Cavβ subunits (see (7Kelly K. Trends Cell Biol. 2005; 15: 64
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