Real-time NMR Study of Guanine Nucleotide Exchange and Activation of RhoA by PDZ-RhoGEF

Small guanosine triphosphatases (GTPases) become activated when GDP is replaced by GTP at the highly conserved nucleotide binding site. This process is intrinsically very slow in most GTPases but is significantly accelerated by guanine nucleotide exchange factors (GEFs). Nucleotide exchange in small GTPases has been widely studied using spectroscopy with fluorescently tagged nucleotides. However, this method suffers from effects of the bulky fluorescent moiety covalently attached to the nucleotide. Here, we have used a newly developed real-time NMR-based assay to monitor small GTPase RhoA nucleotide exchange by probing the RhoA conformation. We compared RhoA nucleotide exchange from GDP to GTP and GTP analogues in the absence and presence of the catalytic DH-PH domain of PDZ-RhoGEF (DH-PHPRG). Using the non-hydrolyzable analogue guanosine-5′-O-(3-thiotriphosphate), which we found to be a reliable mimic of GTP, we obtained an intrinsic nucleotide exchange rate of 5.5 × 10−4 min−1. This reaction is markedly accelerated to 1179 × 10−4 min−1 in the presence of DH-PHPRG at a ratio of 1:8,000 relative to RhoA. Mutagenesis studies confirmed the importance of Arg-868 near a conserved region (CR3) of the Dbl homology (DH) domain and revealed that Glu-741 in CR1 is critical for full activity of DH-PHPRG, together suggesting that the catalytic mechanism of PDZ-RhoGEF is similar to Tiam1. Mutation of the single RhoA (E97A) residue that contacts the pleckstrin homology (PH) domain rendered the mutant 10-fold less sensitive to the activity of DH-PHPRG. Interestingly, this mutation does not affect RhoA activation by leukemia-associated RhoGEF (LARG), indicating that the PH domains of these two homologous GEFs may play different roles. Small guanosine triphosphatases (GTPases) become activated when GDP is replaced by GTP at the highly conserved nucleotide binding site. This process is intrinsically very slow in most GTPases but is significantly accelerated by guanine nucleotide exchange factors (GEFs). Nucleotide exchange in small GTPases has been widely studied using spectroscopy with fluorescently tagged nucleotides. However, this method suffers from effects of the bulky fluorescent moiety covalently attached to the nucleotide. Here, we have used a newly developed real-time NMR-based assay to monitor small GTPase RhoA nucleotide exchange by probing the RhoA conformation. We compared RhoA nucleotide exchange from GDP to GTP and GTP analogues in the absence and presence of the catalytic DH-PH domain of PDZ-RhoGEF (DH-PHPRG). Using the non-hydrolyzable analogue guanosine-5′-O-(3-thiotriphosphate), which we found to be a reliable mimic of GTP, we obtained an intrinsic nucleotide exchange rate of 5.5 × 10−4 min−1. This reaction is markedly accelerated to 1179 × 10−4 min−1 in the presence of DH-PHPRG at a ratio of 1:8,000 relative to RhoA. Mutagenesis studies confirmed the importance of Arg-868 near a conserved region (CR3) of the Dbl homology (DH) domain and revealed that Glu-741 in CR1 is critical for full activity of DH-PHPRG, together suggesting that the catalytic mechanism of PDZ-RhoGEF is similar to Tiam1. Mutation of the single RhoA (E97A) residue that contacts the pleckstrin homology (PH) domain rendered the mutant 10-fold less sensitive to the activity of DH-PHPRG. Interestingly, this mutation does not affect RhoA activation by leukemia-associated RhoGEF (LARG), indicating that the PH domains of these two homologous GEFs may play different roles. IntroductionThe small GTPase 3The abbreviations used are: GTPaseguanosine triphosphataseGTPγSguanosine-5′-O-(3-thiotriphosphate)GMPPNPguanylyl imidodiphosphatemant-GTPN-methylanthraniloyl guanosine triphosphateGEFguanine nucleotide exchange factorDHDbl homologyPHpleckstrin homologyDH-PHPRGcatalytic DH-PH domain of PDZ-RhoGEFLARGleukemia-associated RhoGEF1H-15N HSQC1H-15N heteronuclear single quantum coherenceTCEPtris(2-carboxyethyl)phosphine. RhoA, a member of the Ras superfamily, plays a crucial role in cellular processes including proliferation, movement, cell shape, as well as cell-cell and cell-matrix interactions (1.Etienne-Manneville S. Hall A. Nature. 2002; 420: 629-635Crossref PubMed Scopus (3797) Google Scholar). RhoA acts as a molecular switch, cycling between the inactive GDP- and activated GTP-bound states (Fig. 1A). Akin to other small GTPases, RhoA contains a phosphate binding loop (P-loop) and two switch regions that undergo conformational changes upon nucleotide cycling and mediate interactions with effector proteins. Together these three regions interact with the nucleotide phosphate groups and a magnesium ion that is required for high affinity nucleotide binding (low nm to sub nmKd). GTPase-activating proteins catalyze nucleotide hydrolysis, thus inactivating GTPases, whereas guanine nucleotide exchange factors (GEFs) activate GTPases by stimulating nucleotide exchange (2.Bos J.L. Rehmann H. Wittinghofer A. Cell. 2007; 129: 865-877Abstract Full Text Full Text PDF PubMed Scopus (1271) Google Scholar). PDZ-RhoGEF, like its homologues LARG and p115-RhoGEF, is specific to RhoA and is activated by the Gα12/13 subunit of G-protein coupled receptors via its regulator of G-protein signaling domain. Interestingly, PDZ-RhoGEF single nucleotide polymorphisms have been associated with type II diabetes (3.Fu M. Sabra M.M. Damcott C. Pollin T.I. Ma L. Ott S. Shelton J.C. Shi X. Reinhart L. O'Connell J. Mitchell B.D. Baier L.J. Shuldiner A.R. Diabetes. 2007; 56: 1363-1368Crossref PubMed Scopus (25) Google Scholar) and lung cancer (4.Gu J. Wu X. Dong Q. Romeo M.J. Lin X. Gutkind J.S. Berman D.M. Cancer. 2006; 106: 2716-2724PubMed Google Scholar).RhoGEFs are multidomain proteins (5.Schmidt A. Hall A. Genes Dev. 2002; 16: 1587-1609Crossref PubMed Scopus (975) Google Scholar), most of which contain a conserved catalytic Dbl homology (DH) domain with an associated pleckstrin homology (PH) domain (6.Hoffman G.R. Cerione R.A. FEBS Lett. 2002; 513: 85-91Crossref PubMed Scopus (117) Google Scholar, 7.Derewenda U. Oleksy A. Stevenson A.S. Korczynska J. Dauter Z. Somlyo A.P. Otlewski J. Somlyo A.V. Derewenda Z.S. Structure. 2004; 12: 1955-1965Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Interactions between the DH domains and Rho GTPases induce structural changes of the nucleotide binding pocket and stabilize the nucleotide-free form of Rho (6.Hoffman G.R. Cerione R.A. FEBS Lett. 2002; 513: 85-91Crossref PubMed Scopus (117) Google Scholar). Although the DH domain is required for GEF activity, the PH domain plays multiple roles including stabilizing the DH domain, directing its subcellular localization, and regulating GEF activity (6.Hoffman G.R. Cerione R.A. FEBS Lett. 2002; 513: 85-91Crossref PubMed Scopus (117) Google Scholar, 7.Derewenda U. Oleksy A. Stevenson A.S. Korczynska J. Dauter Z. Somlyo A.P. Otlewski J. Somlyo A.V. Derewenda Z.S. Structure. 2004; 12: 1955-1965Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). DH domains are comprised of a helix bundle in which three highly conserved regions (CR1, CR2, and CR3) constitute the core domain. The DH domain interacts extensively, through CR1 and CR3, with the switch regions of the cognate GTPase as illustrated in the crystal structure of PDZ-RhoGEF (DH-PHPRG) in complex with RhoA (1XCG) (7.Derewenda U. Oleksy A. Stevenson A.S. Korczynska J. Dauter Z. Somlyo A.P. Otlewski J. Somlyo A.V. Derewenda Z.S. Structure. 2004; 12: 1955-1965Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). A highly conserved glutamate in CR1 and a conserved basic residue near CR3 (Fig. 2) have been implicated in the formation of the GEF-GTPase complex and in the nucleotide exchange catalysis in many RhoGEFs (6.Hoffman G.R. Cerione R.A. FEBS Lett. 2002; 513: 85-91Crossref PubMed Scopus (117) Google Scholar, 7.Derewenda U. Oleksy A. Stevenson A.S. Korczynska J. Dauter Z. Somlyo A.P. Otlewski J. Somlyo A.V. Derewenda Z.S. Structure. 2004; 12: 1955-1965Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 8.Oleksy A. Opaliński Ł. Derewenda U. Derewenda Z.S. Otlewski J. J. Biol. Chem. 2006; 281: 32891-32897Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Although mutations of the CR1 and CR3 region were found to affect the GEF activity of Tiam1 and Trio (6.Hoffman G.R. Cerione R.A. FEBS Lett. 2002; 513: 85-91Crossref PubMed Scopus (117) Google Scholar, 8.Oleksy A. Opaliński Ł. Derewenda U. Derewenda Z.S. Otlewski J. J. Biol. Chem. 2006; 281: 32891-32897Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 9.Liu X. Wang H. Eberstadt M. Schnuchel A. Olejniczak E.T. Meadows R.P. Schkeryantz J.M. Janowick D.A. Harlan J.E. Harris E.A. Staunton D.E. Fesik S.W. Cell. 1998; 95: 269-277Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar), the importance of the CR1 for PDZ-RhoGEF activity has not been investigated.FIGURE 2Sequence alignment of DH-PH domains of human RhoGEFs. PDZ-RhoGEF (O15085, residues 734–923), LARG (Q9NZN5, residues 787–977), p115-RhoGEF (Q92888, residues 416–605), Dbl-GEF (Q92974, residues 235–432), p63-RhoGEF (Q86VW2, residues 160–336), Dbs-GEF (O15068, residues 631–811), and Tiam1 (Q13009, residues 1,040–1,234) were aligned using Clustal_W2. Hydrophobic, acidic, basic, and polar residues are indicated in red, blue, magenta, and green, respectively. The stars mark residues conserved throughout Rho guanine nucleotide exchange factors. Colons correspond to conserved substitutions and, periods correspond to semiconserved substitutions. Conserved residues examined in this study (Glu-741, Arg-868, and Ser-1065) are boxed. CR1, CR2, and CR3 are three highly conserved regions of the DH domain.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The PH domain is comprised of seven antiparallel β-strands topped by a helix containing a partially conserved residue (Ser-1065 in PDZ-RhoGEF) that interacts with Glu-97 of RhoA (7.Derewenda U. Oleksy A. Stevenson A.S. Korczynska J. Dauter Z. Somlyo A.P. Otlewski J. Somlyo A.V. Derewenda Z.S. Structure. 2004; 12: 1955-1965Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The DH domain alone is ∼41-fold less active than the DH-PH domain of PDZ-RhoGEF (7.Derewenda U. Oleksy A. Stevenson A.S. Korczynska J. Dauter Z. Somlyo A.P. Otlewski J. Somlyo A.V. Derewenda Z.S. Structure. 2004; 12: 1955-1965Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), but it is also less stable; thus the importance of the RhoA PH domain interaction for GEF activity is not clear.We recently developed an NMR-based, real-time assay to measure the kinetics of GTP hydrolysis by the small GTPase Rheb by monitoring nucleotide-dependent changes in the NMR spectra of the GTPase protein (10.Marshall C.B. Ho J. Buerger C. Plevin M.J. Li G.Y. Li Z. Ikura M. Stambolic V. Sci. Signal. 2009; 2: ra3Crossref PubMed Scopus (48) Google Scholar). In this report, the NMR GTPase assay was extended to monitor intrinsic and PDZ-RhoGEF-mediated RhoA nucleotide exchange using native GDP and GTP as substrates. Our data show that PDZ-RhoGEF favors RhoA activation by catalyzing the exchange from GDP to GTP, severalfold more efficiently than the reverse reaction. Our mutagenesis studies demonstrate that Arg-868 near the CR3 and Glu-741 in the CR1 are critical for the full activity of PDZ-RhoGEF. We also found that RhoA Glu-97 is required for full activation by DH-PHPRG, suggesting that the PH domain plays an important role in catalysis. While investigating PDZ-RhoGEF-mediated RhoA nucleotide exchange reactions using different nucleotides, we discovered that GTPγS better mimics GTP than the analogues mant-GTP or GMPPNP. IntroductionThe small GTPase 3The abbreviations used are: GTPaseguanosine triphosphataseGTPγSguanosine-5′-O-(3-thiotriphosphate)GMPPNPguanylyl imidodiphosphatemant-GTPN-methylanthraniloyl guanosine triphosphateGEFguanine nucleotide exchange factorDHDbl homologyPHpleckstrin homologyDH-PHPRGcatalytic DH-PH domain of PDZ-RhoGEFLARGleukemia-associated RhoGEF1H-15N HSQC1H-15N heteronuclear single quantum coherenceTCEPtris(2-carboxyethyl)phosphine. RhoA, a member of the Ras superfamily, plays a crucial role in cellular processes including proliferation, movement, cell shape, as well as cell-cell and cell-matrix interactions (1.Etienne-Manneville S. Hall A. Nature. 2002; 420: 629-635Crossref PubMed Scopus (3797) Google Scholar). RhoA acts as a molecular switch, cycling between the inactive GDP- and activated GTP-bound states (Fig. 1A). Akin to other small GTPases, RhoA contains a phosphate binding loop (P-loop) and two switch regions that undergo conformational changes upon nucleotide cycling and mediate interactions with effector proteins. Together these three regions interact with the nucleotide phosphate groups and a magnesium ion that is required for high affinity nucleotide binding (low nm to sub nmKd). GTPase-activating proteins catalyze nucleotide hydrolysis, thus inactivating GTPases, whereas guanine nucleotide exchange factors (GEFs) activate GTPases by stimulating nucleotide exchange (2.Bos J.L. Rehmann H. Wittinghofer A. Cell. 2007; 129: 865-877Abstract Full Text Full Text PDF PubMed Scopus (1271) Google Scholar). PDZ-RhoGEF, like its homologues LARG and p115-RhoGEF, is specific to RhoA and is activated by the Gα12/13 subunit of G-protein coupled receptors via its regulator of G-protein signaling domain. Interestingly, PDZ-RhoGEF single nucleotide polymorphisms have been associated with type II diabetes (3.Fu M. Sabra M.M. Damcott C. Pollin T.I. Ma L. Ott S. Shelton J.C. Shi X. Reinhart L. O'Connell J. Mitchell B.D. Baier L.J. Shuldiner A.R. Diabetes. 2007; 56: 1363-1368Crossref PubMed Scopus (25) Google Scholar) and lung cancer (4.Gu J. Wu X. Dong Q. Romeo M.J. Lin X. Gutkind J.S. Berman D.M. Cancer. 2006; 106: 2716-2724PubMed Google Scholar).RhoGEFs are multidomain proteins (5.Schmidt A. Hall A. Genes Dev. 2002; 16: 1587-1609Crossref PubMed Scopus (975) Google Scholar), most of which contain a conserved catalytic Dbl homology (DH) domain with an associated pleckstrin homology (PH) domain (6.Hoffman G.R. Cerione R.A. FEBS Lett. 2002; 513: 85-91Crossref PubMed Scopus (117) Google Scholar, 7.Derewenda U. Oleksy A. Stevenson A.S. Korczynska J. Dauter Z. Somlyo A.P. Otlewski J. Somlyo A.V. Derewenda Z.S. Structure. 2004; 12: 1955-1965Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Interactions between the DH domains and Rho GTPases induce structural changes of the nucleotide binding pocket and stabilize the nucleotide-free form of Rho (6.Hoffman G.R. Cerione R.A. FEBS Lett. 2002; 513: 85-91Crossref PubMed Scopus (117) Google Scholar). Although the DH domain is required for GEF activity, the PH domain plays multiple roles including stabilizing the DH domain, directing its subcellular localization, and regulating GEF activity (6.Hoffman G.R. Cerione R.A. FEBS Lett. 2002; 513: 85-91Crossref PubMed Scopus (117) Google Scholar, 7.Derewenda U. Oleksy A. Stevenson A.S. Korczynska J. Dauter Z. Somlyo A.P. Otlewski J. Somlyo A.V. Derewenda Z.S. Structure. 2004; 12: 1955-1965Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). DH domains are comprised of a helix bundle in which three highly conserved regions (CR1, CR2, and CR3) constitute the core domain. The DH domain interacts extensively, through CR1 and CR3, with the switch regions of the cognate GTPase as illustrated in the crystal structure of PDZ-RhoGEF (DH-PHPRG) in complex with RhoA (1XCG) (7.Derewenda U. Oleksy A. Stevenson A.S. Korczynska J. Dauter Z. Somlyo A.P. Otlewski J. Somlyo A.V. Derewenda Z.S. Structure. 2004; 12: 1955-1965Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). A highly conserved glutamate in CR1 and a conserved basic residue near CR3 (Fig. 2) have been implicated in the formation of the GEF-GTPase complex and in the nucleotide exchange catalysis in many RhoGEFs (6.Hoffman G.R. Cerione R.A. FEBS Lett. 2002; 513: 85-91Crossref PubMed Scopus (117) Google Scholar, 7.Derewenda U. Oleksy A. Stevenson A.S. Korczynska J. Dauter Z. Somlyo A.P. Otlewski J. Somlyo A.V. Derewenda Z.S. Structure. 2004; 12: 1955-1965Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 8.Oleksy A. Opaliński Ł. Derewenda U. Derewenda Z.S. Otlewski J. J. Biol. Chem. 2006; 281: 32891-32897Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Although mutations of the CR1 and CR3 region were found to affect the GEF activity of Tiam1 and Trio (6.Hoffman G.R. Cerione R.A. FEBS Lett. 2002; 513: 85-91Crossref PubMed Scopus (117) Google Scholar, 8.Oleksy A. Opaliński Ł. Derewenda U. Derewenda Z.S. Otlewski J. J. Biol. Chem. 2006; 281: 32891-32897Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 9.Liu X. Wang H. Eberstadt M. Schnuchel A. Olejniczak E.T. Meadows R.P. Schkeryantz J.M. Janowick D.A. Harlan J.E. Harris E.A. Staunton D.E. Fesik S.W. Cell. 1998; 95: 269-277Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar), the importance of the CR1 for PDZ-RhoGEF activity has not been investigated.The PH domain is comprised of seven antiparallel β-strands topped by a helix containing a partially conserved residue (Ser-1065 in PDZ-RhoGEF) that interacts with Glu-97 of RhoA (7.Derewenda U. Oleksy A. Stevenson A.S. Korczynska J. Dauter Z. Somlyo A.P. Otlewski J. Somlyo A.V. Derewenda Z.S. Structure. 2004; 12: 1955-1965Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The DH domain alone is ∼41-fold less active than the DH-PH domain of PDZ-RhoGEF (7.Derewenda U. Oleksy A. Stevenson A.S. Korczynska J. Dauter Z. Somlyo A.P. Otlewski J. Somlyo A.V. Derewenda Z.S. Structure. 2004; 12: 1955-1965Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), but it is also less stable; thus the importance of the RhoA PH domain interaction for GEF activity is not clear.We recently developed an NMR-based, real-time assay to measure the kinetics of GTP hydrolysis by the small GTPase Rheb by monitoring nucleotide-dependent changes in the NMR spectra of the GTPase protein (10.Marshall C.B. Ho J. Buerger C. Plevin M.J. Li G.Y. Li Z. Ikura M. Stambolic V. Sci. Signal. 2009; 2: ra3Crossref PubMed Scopus (48) Google Scholar). In this report, the NMR GTPase assay was extended to monitor intrinsic and PDZ-RhoGEF-mediated RhoA nucleotide exchange using native GDP and GTP as substrates. Our data show that PDZ-RhoGEF favors RhoA activation by catalyzing the exchange from GDP to GTP, severalfold more efficiently than the reverse reaction. Our mutagenesis studies demonstrate that Arg-868 near the CR3 and Glu-741 in the CR1 are critical for the full activity of PDZ-RhoGEF. We also found that RhoA Glu-97 is required for full activation by DH-PHPRG, suggesting that the PH domain plays an important role in catalysis. While investigating PDZ-RhoGEF-mediated RhoA nucleotide exchange reactions using different nucleotides, we discovered that GTPγS better mimics GTP than the analogues mant-GTP or GMPPNP.

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