Crystal Structure of the Adenylyl Cyclase Activator Gsα

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Science  12 Dec 1997:
Vol. 278, Issue 5345, pp. 1943-1947
DOI: 10.1126/science.278.5345.1943


The crystal structure of Gs α, the heterotrimeric G protein α subunit that stimulates adenylyl cyclase, was determined at 2.5 Å in a complex with guanosine 5′-O-(3-thiotriphosphate) (GTPγS). Gs α is the prototypic member of a family of GTP-binding proteins that regulate the activities of effectors in a hormone-dependent manner. Comparison of the structure of Gs α·GTPγS with that of Gi α·GTPγS suggests that their effector specificity is primarily dictated by the shape of the binding surface formed by the switch II helix and the α3-β5 loop, despite the high sequence homology of these elements. In contrast, sequence divergence explains the inability of regulators of G protein signaling to stimulate the GTPase activity of Gs α. The βγ binding surface of Gs α is largely conserved in sequence and structure to that of Gi α, whereas differences in the surface formed by the carboxyl-terminal helix and the α4-β6 loop may mediate receptor specificity.

The Gs and Gi subfamilies of heterotrimeric G protein α subunits, although highly homologous, differ profoundly with respect to effector, regulator, and receptor specificity (1, 2). For example, Gs α binds to and activates all isoforms of adenylyl cyclase (3), whereas Gi α 1 and its close paralogs inhibit only certain isoforms of the effector. The GTPase activities of Gi subfamily members are stimulated by members of the RGS (regulators of G protein signaling) protein family; the GTPase activity of Gs α is not affected by any known RGS protein (4). Distinct subfamilies of G protein–coupled receptors activate either Gs or Gi. To better understand the origins of these functional differences, we have determined the three-dimensional structure of GTPγS-activated Gs α alone and in complex with its effector, adenylyl cyclase (5). Comparison of the structure of Gs α with those of previously determined Gi subfamily members (6,7) offers substantial insight into the molecular basis of specificity in heterotrimeric G proteins.

Deficiencies in Gs α function have serious biological repercussions. Adenosine diphosphate (ADP) ribosylation of the active site residue Arg201 by cholera toxin (8, 9) leads to irreversible inhibition of the GTPase activity of Gs α. The resulting constitutive activation of adenylyl cyclase in gastrointestinal epithelium is responsible for the diarrhea and dehydration that are the hallmarks of cholera. Similarly, mutation of Arg201 or the catalytic residue Gln227 contributes to the growth of tumors of the pituitary and thyroid glands and causes the McCune-Albright syndrome (10, 11). Heterozygous deficiency of Gs α is the basis for pseudohypoparathyroidism (type IA) (11-13).

The structure of the active Gs α·GTPγS complex was determined at 2.5 Å resolution by molecular replacement with the use of the atomic coordinates of Gi α·GTPγS (7) as the search model (Table 1). The structure has been refined to conventional and free crystallographic R factors of 23 and 29%, respectively. Each asymmetric unit of the crystal contains a nonphysiological Gs α dimer oriented parallel to the a axis of the crystal. The dimer interface buries 2500 Å2 of surface area and is additionally stabilized by 10 phosphate anions derived from the crystallization medium. These phosphates contribute an additional 900 Å2 of buried surface area (Fig.1A). Residues 1 through 33 and 392 through 394 (of 394 residues; the numbering is based on the long alternative splice variant) are disordered at the NH2- and COOH-termini of the crystal structure. Residues 65 through 88 of subunit A and residues 70 through 86 of subunit B in linker 1 of the protein are also disordered.

Figure 1

The structure of Gs α·GTPγS. (A) A dimer of Gs α·GTPγS was observed in the asymmetric unit of the crystals and is depicted here as a ribbon and coil diagram looking down the noncrystallographic twofold axis. The 16 phosphate anions are drawn as red tetrahedrons. Most of the anions bind within a groove at the dimer interface between the α5 helices. The two phosphate anions that bind near the NH2-termini of each molecule of Gs α form crystal contacts. GTPγS (yellow) and Mg2+ (black) are represented by ball-and-stick models and are located in the nucleotide binding pocket. Helices are green, β strands are purple, and coils are gray. This and the other ribbon diagrams were generated with MOLSCRIPT (40) and rendered with RASTER3D (41). (B) Superposition of Gi α(transparent rose) on the structure of Gs α·GTPγS (solid gray). Only the nucleotide bound to Gs α is shown. The approximate locations of two of the three major insertions in the Gs α sequence relative to Gi α (i2 and i3) are indicated in white (see text). The two proteins superimpose with a rmsd of 1.0 Å for 260 Cα atom pairs. Their structures are essentially identical at the GTP binding site and are most divergent in various loops at the periphery of the molecule, most notably at the α3-β5 and α4-β6 loops. (C) Sequence alignment of representative proteins from three Gα subfamilies: bovine Gs α (Protein Information Resource accession number A23813), murine Gq α (A38414), and bovine Gi α 1 (A23631) (42). Secondary structure has been assigned on the basis of the structures of Gs α and Gi α 1·GTPγS (7). The three conformationally flexible switch elements are indicated by red blocks. The arrow marks the site in Gs α at which the long and short splice variants differ in length by 14 amino acids. Green amino acid letters indicate residues in Gs α that contact adenylyl cyclase, whereas red amino acid letters indicate potential adenylyl cyclase binding residues in Gi αidentified by alanine-scanning mutagenesis (21). The general locations of the i1, i2, and i3 insertions are also indicated.

Table 1

Summary of data collection and refinement statistics. The short-splice form of bovine G was expressed with a COOH-terminal hexahistidine tag in Escherichia coli and purified to homogeneity on a nickel-nitrilotriacetic acid column, followed by hydroxyapatite and Mono Q fast protein liquid chromatography, essentially as described by Lee et al. (32). Purified G was concentrated to 12 mg ml−1 (270 μM) and incubated with 800 μM GTPγS in a buffer containing 20 mM Na Hepes (pH 8.0), 5 mM MgCl2, 1 mM EDTA, and 5 mM dithiothreitol (DTT). Crystals of G were obtained at 20°C by hanging drop vapor diffusion. Hanging drops containing 6 μl total of a 1:1 mixture of activated G and well solution were suspended over a 500-μl well containing 90 to 100% saturated KH2PO4 or 2.5 M NaH2PO4. The crystals, which form as bundles of 20-μm thick plates, belong to space group P212121 and contain two molecules of G per asymmetric unit. For data collection, individual crystals were harvested in a solution containing 2.5 M NaH2PO4, 25 mM sodium citrate (pH 4.5), 5 mM MgSO4, 2 mM DTT, 1 mM EDTA, 150 μM GTPγS, and 15% glycerol as the cryoprotectant. The crystals were subsequently frozen in liquid propane and were maintained at −180°C during data collection. Diffraction data were collected from two crystals with the use of 0.908 Å radiation from the A1 beam line at the Cornell High-Energy Synchrotron Source (CHESS). The data were integrated and scaled with the HKL package (33), and the structure was solved by molecular replacement with the use of AMORE (34) as implemented by the CCP4 program suite (35). A cross-rotation function using Giα1·GTP·γS (7) as the search model revealed only one significant peak. Subsequently, two translationally related molecules of G·GTPγS were located by the translation function. These two subunits constitute a noncrystallographic dimer oriented along the a axis of the unit cell. The initial atomic model was built by substituting G side chains with their equivalents in G using the program O (36), and subsequent manual model building was alternated with conventional and simulated annealing refinement in X-PLOR 3.851 (37, 38). The two molecules of G were restrained by their noncrystallographic symmetry only for the first several rounds of refinement. The two subunits superimpose with a rmsd of 0.3 Å. The backbone conformations of 92% of the amino acids are within the most favored regions of the Ramachandran plot; there are no residues in disallowed regions (39). The model includes one molecule of Mg2+ and GTPγS per G subunit and 16 phosphate anions. The average B factor is 26.5 Å2.

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Gs α, like its homologs Gi α (7) and Gt α (6), consists of a Ras-like domain joined through two linker polypeptides to an α-helical domain that is unique to heterotrimeric G proteins (2). The structure of the α-helical domain of Gs α, expressed as a recombinant protein, has been determined by nuclear magnetic resonance (NMR) (14) and is virtually identical to that of the corresponding domain in the intact subunit [root mean square deviation (rmsd) of 1.2 Å for 113 pairs of Cα atoms]. Both NMR and crystal structures demonstrate that the αB-αC loop is poorly ordered and that the proline at position 115 adopts the cis conformation. Relative to Gi α and Gt α, Gs α contains three major polypeptide insertions (Fig. 1, B and C). The first of these (i1) is near the NH2-terminus and is not visible in the Gs α structure. The second (i2) is incorporated into the linker 1 peptide (connecting α1 to αA). However, this insert is subject to alternative splicing; in the short splice variant of Gs α described here, linker 1 is only one residue longer than that in Gi α. Unlike Gi α, linker 1 in Gs α is partially disordered. The third (i3) is a 15-residue insertion between αG and α4 of the Ras-like domain. The i3 insertion folds into a flap that protrudes from the surface of the domain (Fig. 1B) and forms most of the noncrystallographic dimer interface. Conformational differences between the helical domains of Gs α and those of Gi α and Gt α have been noted previously (7, 14). None of these differences involve regions of the subunit that participate in interactions with βγ (for Gi α and Gt α) (15, 16), RGS proteins (for Gi α) (17), or adenylyl cyclase (for Gs α) (5). In contrast, structural variations among these subunits in the α3-β5 and α4-β6 loops of the Ras-like domains (Fig.2) are determinants of effector specificity (see below).

Figure 2

Superposition of the putative effector binding loops (α2-β4, α3-β5, and α4-β6) and the α5 helix from Gs α onto Gi α (42). The side chains from residues of Gs α are drawn as stick models with the use of conventional coloring. The backbone and side chains of Gi α are illustrated in transparent rose. The model of Gi α is derived from the structure of the Gi α 1·RGS4 complex (17), which has a completely ordered α5 helix. The superposition is essentially the same as that shown in Fig. 1B. The α2-β4 loops of each α subunit are essentially identical. The α3-β5 loop of Gs α, although structurally similar to that of Gi α, is rotated downward in the figure. This rotation creates a hydrophobic pocket on the back side of the β sheet, which is filled by the side chain of Met386 from the α5 helix, and moves the residue at position 282 in Gs α toward the conserved Phe238. In the Gs subfamily, residue 282 is a leucine, which helps to accommodate the shift of the α3-β5 loop. The α4-β6 loop of Gs α is longer than and shares no sequence identity with its counterpart in Gi α. The α3-β5 and α4-β6 loops are supported by a stacking interaction between Trp277 and His357, both of which are invariant in the Gssubfamily. The α5 helix of Gs α is bent, whereas that of Gi α extends straight into solvent. The large differences observed in the α4-β6 and α5 structures may help account for receptor specificity among closely related α subunits.

GTP is the organizing center for three switch elements (switches I through III) (Fig. 1, B and C) in Gα that undergo substantial conformational rearrangement upon GTP hydrolysis. These switch elements are also intimately involved in binding βγ, RGS family members, and effectors (5,15-17). The structure and orientation of all three switch elements are essentially identical in Gs α·GTPγS, Gi α·GTPγS (Fig. 1B), and Gt α·GTPγS. The nucleotide is bound within the narrow cleft formed by switch I and switch II (Fig. 1, A and B), exactly as observed in the structures of the other Gαhomologs. Indeed, the side chains that contact GTPγS and coordinate Mg2+ are identical to the corresponding residues in Gi α. Electron densities corresponding to Mg2+, one of its coordinating water molecules, and the presumptive hydrolytic water are observed. The second water ligand to Mg2+ is, however, evident in the Gs α·adenylyl cyclase complex (5). Arg201, which facilitates GTP hydrolysis by stabilizing the proposed pentavalent phosphate intermediate (7, 18) and is the site of ADP ribosylation by cholera toxin (8, 9), is exposed to solvent and partially ordered. Gln227, which is conserved in most members of the Ras superfamily and is required for catalytic activity in heterotrimeric G proteins, forms no direct contacts with either the γ-thiophosphate of GTPγS or the presumptive hydrolytic water. This is also the case in the structures of other Ras superfamily members bound to GTP analogs (2).

Of all Gα homologs, only Gs αand Golf activate adenylyl cyclase. It is evident from the structure of the complex between Gs α and the catalytic domains of adenylyl cyclase (5) that exclusion of Gi α from this site arises primarily from differences in the conformation, but not the amino acid composition, of the cyclase binding site. Residues that bind adenylyl cyclase are located in the α2 helix of switch II and the α3-β5 loop (5) (Figs. 1C and 3). Both segments were identified as potential adenylyl cyclase binding sites by alanine- and homolog-scanning mutagenesis (19,20). Of the nine Gs α side chains that directly interact with adenylyl cyclase, seven are invariant or highly conserved among the Gs α and Gi α proteins (Fig. 1C). The exceptions, Gln236 and Asn239, are replaced by histidine and glutamic acid, respectively. The side chain of Asn239stacks against the guanidinium group of Arg913 of type II adenylyl cyclase, and Gln236 serves as a hydrogen bond acceptor from Asn905. The corresponding amino acid side chains in Gi α could potentially participate in analogous interactions. Indeed, mutation of these residues in Gs α, along with Asp240, to their counterparts in Gi α, results in only a threefold reduction in the capacity to stimulate adenylyl cyclase activity (20). It is more likely that Gi α fails to activate adenylyl cyclase because its α3-β5 loop is displaced from the switch II helix, so that both elements cannot be simultaneously accommodated by the cyclase binding site. The relative shift is due to the substitution, in Gi α, of a bulky phenylalanine residue for Leu282 in the helical α3-β5 loop (Fig. 2). To avoid collision with the conserved Phe215(Gi α) in switch II, α3-β5 is translated by approximately 1.5 Å. The α3-β5 loop of Gs α is also stabilized by a stacking interaction between Trp277 and His357 in the α4-β6 loop. No such stabilizing contacts are possible in Gi α because Trp277 and His357 are replaced by Cys264 and Lys317, respectively, and because the α4-β6 loop of Gi α differs in sequence, length, and structure from the corresponding loop in Gs α. Indeed, substitution of the Gi α α4-β6 loop into Gs α abolishes activation of adenylyl cyclase (20) by removing stabilizing interactions and perhaps also by disrupting adjacent effector binding regions. However, substitution of the Gs α α4-β6 sequence into Gi α fails to confer cyclase-stimulating activity because the α4-β6 loop supports, but does not form part of, the cyclase binding site. Switch II of Gi α and, less convincingly, its α4-β6 loop have been implicated in formation of the inhibitory complex with type V and VI adenylyl cyclases (21) at a site distinct from that recognized by Gs α (22) (Fig.1C). Switch III, which is stabilized by ionic contacts with switch II, does not contribute to the adenylyl cyclase binding site. Located at the NH2-terminus of α3 distal to the α3-β5 loop, switch III adopts the same conformation in Gs α and Gi α. Accordingly, disruption of an ionic contact between switch III and switch II through mutation of Arg231 only modestly reduces the stimulatory activity of Gs α(23).

Figure 3

Interaction footprints of Gα regulators and effectors. Surfaces representing contact regions of βγ (blue), RGS4 (red), and adenylyl cyclase (green) are mapped onto the solvent-accessible surface of Gs α (43). A contact was defined as an interatomic distance of less than 4.0 Å. Residues that contact both adenylyl cyclase and βγ are colored cyan, those that contact both RGS4 and βγ are magenta, and those that interact with all three are dark gray. RGS4 and adenylyl cyclase have few if any significant steric overlaps; the gray areas thus represent cases where each protein contacts a different part of the same Gαresidue. The figure was generated with GRASP (43).

Gs α does not undergo a conformational change upon binding to the soluble domains of adenylyl cyclase. The rmsd between the 334 Cα atom pairs in the free and bound forms of Gs α is 0.5 Å. The effector-binding regions of Gs α, and perhaps those of other Gα subunits, are already maintained in a competent conformation in the GTPγS-bound form of the protein. In contrast, the switch II elements of both Gt α and Gi α, and probably Gs α, undergo substantial conformational changes when GDP is exchanged for GTPγS. Although the apparent affinity of adenylyl cyclase for Gs α· GDP is only 10 times less than for Gs α· GTPγS (24), Gs α·GDP binds with much greater affinity to βγ subunits than does Gs α·GTPγS (25). Consequently, full deactivation of adenylyl cyclase requires the rapid sequestration of Gs α·GDP in a high-affinity complex with βγ.

Although the effector binding sites of Gs αand Gi α (or Gt α) differ, the residues in the extensive βγ binding surfaces observed in Gi α 1 (Ile184, Phe199, Lys210, Trp211, Cys214, and Phe215) (15) are largely conserved in Gs α. Superposition of the α subunits from both the αiβ1γ2heterotrimer and the Gs α·adenylyl cyclase complex demonstrates that βγ and adenylyl cyclase bind to extensively overlapping sites on the α subunit (Fig. 3) and are similarly oriented with respect to the plasma membrane. Thus, βγ is a potent competitor with adenylyl cyclase for the GDP-bound form of Gs α. Although the α3-β5 and α4-β6 loops have been implicated in effector binding and specificity, they do not participate in interactions with βγ (Fig. 3).

In contrast to the mechanism of effector recognition, the selectivity of RGS proteins for Gi α subunits (compared to Gs α) can be attributed to differences in the amino acid composition rather than the conformation of the RGS binding site. The crystal structure of RGS4 complexed with Gi α 1·GDP· AlF4 demonstrates that RGS proteins activate GTP hydrolysis by binding to and stabilizing all three switch elements of Gi α in their transition state conformation (17). All RGS molecules characterized thus far are capable of accelerating the GTPase activities of Gi αor Gq α subfamily members (or both) but not of Gs α (4). Because the backbone conformations of the switch I and II elements are essentially identical in Gi α and Gs α, the specificity of RGS for heterotrimeric G protein subfamilies is largely dictated by the identity of side chains within the switch elements. Six residues of Gi α 1 that come in contact with RGS4 are not conserved in Gs α: Lys180 (Leu203 in Gs α), Thr182(Ser205), and Val185 (Phe208) in switch I; and Ser206 (Asp229), Lys209 (Arg232), and His213(Gln236) in switch II. These substitutions deter binding of RGS proteins to Gs α by steric overlap, charge repulsion, or the creation of small cavities in the interface (for example, the substitution of Thr182 by Ser205). Although each substitution alone does not seem sufficient to disrupt a potential RGS·Gs α complex, their cumulative effects apparently are.

Surprisingly, the “footprint” of RGS4, as mapped onto the surface of Gs α, does not substantially overlap with that of adenylyl cyclase (Fig. 3). Superposition of Gi α 1· RGS4 on Gs α·adenylyl cyclase reveals only minor potential steric conflicts between adenylyl cyclase and RGS4, although no substantial interface exists between them. Assuming an analogous interaction between switch II of Gi α and as-yet-unidentified domains of type V and VI adenylyl cyclase, RGS4 could potentially accelerate GTP hydrolysis while Gi α is still bound to adenylyl cyclase. This in turn suggests the possibility that RGS proteins and adenylyl cyclase, perhaps along with heterotrimeric G proteins and their receptors, exist as discrete complexes on the membrane of the cell after activation of G protein–coupled receptors.

The fidelity of signal transduction depends on the capacity of G protein–coupled receptors to distinguish among the unique structural features of various Gα subunits. Although the surface contacted by the receptor probably includes segments of the β subunit and the NH2-terminus of Gα, the COOH-terminus of Gα contributes importantly to receptor selectivity [reviewed in (26)]. Evidence gained from alanine-scanning mutagenesis (27) and patterns of evolutionary conservation (28) also argue for inclusion of the α4-β6 loop and the α5 helix in the receptor binding surface. The α5 helix of Gs α is kinked at its midsection and bends around the underlying β sheet (Figs. 1A and 2). The α4-β6 loop is in close proximity to the COOH-terminus of α5. The contact is stabilized by insertion of Met386 into a hydrophobic pocket formed by the NH2-terminal residues of β5 and β6. Together, the α4-β6 loop and the α5 helix form a plane on the back side of Gs α that may interact with receptors (29-31). In contrast, the corresponding α5 helix in Gi α (as visualized in the structures of the Gi α 1·GDP·β1γ2and Gi α 1·GDP·AlF4 ·RGS4 complexes) is relatively straight and extends away from the central β sheet of the Ras-like domain (Fig. 2). The relative position of the α4-β6 loop of Gs α also differs from that of the cognate loop of Gi α. The divergence of these two structural elements from those of Gi α and Gt α may therefore contribute to receptor selectivity.

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