Crystal Structures of the G Protein GiR1 Complexed with GDP and Mg2+: A Crystallographic Titration Experiment‡

The effect of Mg2+ binding on the conformation of the inactive GDP-bound complex of the heterotrimeric G protein R subunit GiR1 has been investigated by X-ray crystallography. Crystal structures of the GiR1‚GDP complex were determined after titration with 5, 10, 100, and 200 mM Mg2+. Comparison of these structures with that of the Mg2+-free complex revealed Mg2+ bound at the same site as observed in the structure of the active, GiR1‚GTPγS‚Mg-bound complex of GiR1, with a similar coordination scheme except for the substitution of a water molecule for an oxygen ligand of the γ-phosphate of GiR1‚GTPγS‚ Mg2+. In contrast to the GDP‚Mg2+ complex of GtR and of other G proteins, switch I residues of GiR1 participate in Mg2+ binding and undergo conformational changes as a consequence of Mg2+ binding. Partial order is induced in switch II, which is disordered in the Mg2+-free complex, but no order is observed in the switch III region. This contrasts with the GDP‚Mg2+ complex of GtR in which both switch II and III switch are ordered. Mg2+ binding also induces binding of an SO4 molecule to the active site in a manner which may mimic a GiR1‚GDP‚PO4‚Mg product complex. Implications of these findings are discussed. The R subunits of heterotrimeric G proteins (GR) are GTPases which, in concert with G protein heterodimers composed of â and γ subunits (Gâγ), transduce intracellular signals from membrane-bound receptors to downstream effector molecules (1, 2). Biological activities which employ GRâγ proteins include vision, hormone-mediated responses, and synaptic nerve signal transmission. GRâγ proteins couple activated membrane-bound receptors to downstream effector molecules by cycling between inactive (nonsignaling) and active (signaling) states. In the inactive state, GRâγ proteins exist as heterotrimeric GRâγ‚GDP complexes with GDP bound tightly to the GR subunit. Upstream signaling events begin when ligand-activated receptors catalyze the exchange of GDP bound to the GR subunit for GTP‚Mg2+. Binding of GTP‚Mg2+ to the GR subunit results in dissociation of the heterotrimer, forming free Gâγ complex and the active, GR‚GTP‚Mg species. Gâγ and the active GR‚GTP‚Mg complex may then bind to and regulate downstream effectors. Upon hydrolysis of GR‚GTP‚Mg to GR‚GDP by the GTPase activity of GR [in some cases assisted by stimulatory proteins (3, 4)], the inactive GR‚GDP complex dissociates from effector and combines with Gâγ to re-form the nonsignaling GRâγ‚GDP complex. The GRâγ‚GDP complex may then rebind to receptor and undergo further rounds of the signal transduction cycle. GR subunits belong to the G protein superfamily of GTPases, members of which contain a conserved guanine nucleotide binding domain and cycle between inactive GDPbound and active GTP-bound states (5, 6). GiR1, the subject of this report, is activated by R2-adrenergic (7) and m2 muscarinic receptors (8) and inhibits isoforms I, V, and VI of adenylyl cyclase (9, 10). The conformational changes that occur within G proteins have been observed by X-ray crystallographic analysis of G proteins activated with either GDP or nonhydrolyzable GTP analogues and Mg2+. Comparison of the active and inactive structures of Ras (11-13) and other G proteins (14, 15) has revealed two polypeptide segments that differ significantly in conformation in the GDPand GTP-bound states. These two regions, dubbed switch I and switch II, contribute residues that are involved in binding the γ-phosphate of GTP and Mg2+ (e.g., Figure 1). Structures of G proteins complexed with effectors (16, 17) or other proteins (18, 19) reveal that switch I and switch II are also involved in nucleotidedependent protein-protein interactions. In particular, the structure of GsR complexed with the catalytic domain of its effector, adenylyl cyclase, indicates that switch II directly interacts with the effector (17). Switch I and II thus constitute the heart of the conformational switching mechanism which both senses the presence or absence of a γ-phosphate group within the active site and controls the interaction of G proteins with other molecules. Crystallographic determinations of the active and inactive structures of the heterotrimeric G protein R subunits transducin (GtR) and GiR1 have revealed the conformational changes that switch I and switch II undergo, and have identified an additional conformationally active region, switch III (20-23). Curiously, although the switch regions ‡ X-ray coordinates for the GiR1‚GDP‚Mg complex have been deposited in the Brookhaven Protein Data Bank under the file name 1bof. * Address correspondence to this author at the Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75235-9050. E-mail: [email protected]. Phone: 214 648-5008. Fax: 214 648-6336. § Department of Biochemistry. | Howard Hughes Medical Institute. 14376 Biochemistry 1998, 37, 14376-14385 S0006-2960(98)01030-7 CCC: $15.00 © 1998 American Chemical Society Published on Web 09/15/1998 and other features of GtR and GiR1 are essentially identical in the active GTPS‚Mg2+-bound1 complexes, the inactive, GDP-bound complexes are distinctly different. In particular, switch II and switch III are completely disordered in GiR1‚ GDP whereas they are well-ordered in GtR‚GDP. The dissimilarity between these inactive, GDP-bound structures implies that their mechanism of dissociation from their respective effectors may also differ. Alternatively, a possible reason for these differences is that GtR‚GDP (crystallized in 200 mM Mg2+) has Mg2+ bound to the active site, whereas GiR1‚GDP (crystallized in the absence of Mg2+) does not. G proteins are sensitive to the presence or absence of Mg2+. All members of the G protein family require Mg2+ in order to catalyze hydrolysis of GTP (1, 24). Structures of G proteins complexed with nonhydrolyzable GTP analogues and Mg2+ (including GtR and GiR1) show that Mg2+ binds to the active site and interacts with switch I, and the â/γ-phosphate groups (14, 15). Similarly, structures of G proteins complexed with GDP and Mg2+ reveal Mg2+ bound to the same position; however, the conformation of switch I is altered such that it is no longer involved in Mg2+ binding. Many nonheterotrimeric G proteins, such as EF-Tu and Ras, bind Mg2+ in the GDP-bound state, with Mg2+ increasing their affinity for GDP (25-27 and references cited therein). It has been proposed that some nucleotide exchange mechanisms release GDP, in part, by disrupting Mg2+ binding (28-30), and that physiologically Mg2+ may maintain the inactive state by acting as a nucleotide dissociation inhibitor (27). This activity would prevent nucleotide exchangesand hence unwarranted activation by GTPsin the absence of a nucleotide exchange factor (27). For these proteins, the transition between the active and the inactive state is mediated by the presence or absence of the γ-phosphate group of GTP alone. In contrast, Mg2+ binds to heterotrimeric G protein GR‚ GDP and GRâγ‚GDP complexes with weak affinity, does not increase GDP binding affinity, and is not required in order to maintain the inactive, GDP-bound state (31). However, as noted, the structure of the GDP-bound complex of the heterotrimeric G protein R subunit transducin (GtR) (crystallized in 200 mM Mg2+) (21) reveals Mg2+ bound to the active site in a manner that is similar to that observed in other G protein GDP‚Mg2+ complexes. This finding indicates that, in at least one case, Mg2+ can bind to a GR‚GDP complex, although it is not known whether Mg2+ binds to this complex at physiological concentrations of Mg2+. In contrast, the structure of the GDP-bound complex of the homologous heterotrimeric G protein R subunit GiR1 (crystallized in the absence of Mg2+) exhibits an empty Mg2+ binding site, and is thus a rare example of a GDP-bound G protein structure which contains no Mg2+ and is not complexed with other proteins (23). [The structure of the EF-G‚GDP complex also contains no Mg2+; this molecule does not bind GDP well, and appears to undergo nucleotide exchange without the need for an exchange factor (32).] The lack of order of switch II and III in GiR1‚GDP, in contrast to GtR‚GDP‚Mg, could be a consequence of the absence of Mg2+ in the GiR1‚GDP complex. It is possible that, like GtR, Mg2+ may bind to GiR1‚GDP and might then stabilize these regions, providing an alternate model of the inactive state. Alternatively, failure of Mg2+ to induce order in these regions would strengthen, in the case of GiR1, the hypothesis that these regions become completely disordered after loss of the γ-phosphate group of GTP and that this loss of order promotes dissociation from effector. The possibility that Mg2+ binding may induce conformational changes in GiR1‚GDP may also reveal some of the effects of Mg2+ during activation of GiR1. Full activation of G proteins following exchange of GDP for GTP appears to require Mg2+ bound to the active site. Conformational changes upon addition of Mg2+ to isolated GR‚GTP subunits can be observed by changes in fluorescence intensity (33, 34) and decreased sensitivity to trypsin digestion (35, 36). GR subunits bearing certain mutations (GsR: G226A, GiR1: G203A or G42V) behave normally as isolated R subunits (that is, they bind GTP and have normal GTPase activity and unaltered ability to modulate effectors), but have lost the ability to dissociate from the GRâγ‚GTP complex (3739). These mutant GR subunits have greatly reduced affinity for Mg2+ and appear to have lost the ability to adopt the fully active conformation. It is inferred from these observations that Mg2+ must bind to the GRâγ‚GTP complex in order to effect release of GR‚GTP from the heterotrimer. Magnesium ion may thus play a structural role in the mechanism of conformational switching to the active GR‚GTP‚Mg state in addition to its role in catalysis. To determine whether Mg2+ can bind to crystalline GR‚ GDP complexes, to elucidate its mode of binding, and to investigate its potential role in conformational changes, we have solved a series of structures of GiR1‚GDP complexed with Mg2+. The experiment was carried out as an X-ray crystallographic titration experiment in which crystals of the GiR1‚GDP complex containing 5, 10, 100, and 200 mM Mg2+ were chosen for structure determination in order to map out the lower and upper limits of potential Mg2+ effects. These structures have been compared to the structures of the Mg2+-free GiR1‚GDP complex, the GiR1‚GTPγS‚Mg and GtR‚GTPγS‚Mg complexes, and other G protein structures. The titrated structures reveal that Mg2+ does bind to GiR1‚ GDP but that the mode of binding differs from that observed in other G protein GDP‚Mg2+-bound complexes. Conformational changes in switch I and limited ordering of switch II are observed; however, switch II and switch III remain essentially disordered. In addition, Mg2+ promotes binding of SO4 to the active site, thus forming a GiR1‚GDP‚Mg‚ SO4 complex which may mimic a GiR1‚GDP‚Mg‚PO4 ternary product complex formed following GTP hydrolysis. Implications of these effects with respect to Mg2+-induced changes during activation, events following hydrolysis of GTP, and loss of effector binding affinity are discussed. EXPERIMENTAL PROCEDURES Crystallization and Data Collection. Nonmyristoylated, nonpalmitoylated recombinant rat GiR1 (354 residues, 40.5 kDa) was synthesized in E. coli and purified as described previously (40). Crystals of GiR1‚GDP‚Mg were grown in ammonium sulfite under conditions identical to those used to obtain crystals of the Mg2+-free GiR1‚GDP complex (41), except that either 5 or 10 mM MgSO4 was included in the crystallization solution. Crystals do not grow in solutions 1 Abbreviations: DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; GTPγS, guanosine 5′-O-3-thiotriphosphate. GiR1‚GDP‚Mg Crystal Structures Biochemistry, Vol. 37, No. 41, 1998 14377