- Email: [email protected]
Structural aspects of heterotrimeric G-protein signaling
480
Structural aspects of heterotrimeric G-protein signaling Andrew
Bohm*, Rachelle Gaudett
and Paul B Siglerr
Recently, structures of heterotrimeric G-protein subunits have been determined in isolation, in conjunction with each other, and in complex with their regulators. information, these structures
Along with biochemical
suggest how G-protein
are oriented relative to the membrane to seven-transmembrane mechanisms
surface and relative
helix receptors.
for receptor-catalyzed
subunits
modeling studies. In this review, we discuss results derived from these recent determinations, and we comment on the implications they have for our knowledge of receptor catalyzed nucleotide exchange.
They also suggest
nucleotide
exchange.
Addresses The Department of Molecular Biophysics and Biochemistry, and The Howard Hughes Medical institute, Yale University, 280 Whitney Ave, JWG 423, New Haven, CT 08511, USA *e-mail: [email protected] te-mail: rgaudetQbiomed.med.yale.edu Se-mail: [email protected] Current Opinion in Biotechnology 1997, 8:480-487 http://biomednet.com/elecref/0958168900800480 0 Current Biology Ltd ISSN 0958-l 669 Abbreviations RGS regulators of G-protein signalling rms root mean square 7TM seven-transmembrane helix
Introduction Heterotrimeric G-proteins consist of a, f3 and y subunits. Signal transduction pathways employing these proteins are ubiquitous transducers of extracellular signals in all eukaryotes, including primitive unicellular organisms. There are hundreds of systems of this type in the human body and, in addition to mediating the cellular response to many hormones and neurotransmitters, these systems are directly responsible for the senses of sight, smell and taste [l]. Activation of seven-transmembrane helix (7TM) receptors results in a conformational change that allows the receptors to act as nucleotide-exchange factors for Ga subunits. Once GDP has been replaced with GTP, the tightly associated Ga and G& subunits of the G protein separate from each other and from the 7TM receptor, leaving both Ga and GPy free to interact with and modulate downstream components of signaling cascades (Figure 1). Both Ga and GPy are regulated by other proteins. Ga subunits are regulated by a growing family of proteins [2*,3]; these ‘regulators of G-protein signaling’ (RGS) bind Ga and accelerate the rate of GTP hydrolysis to GDP, shortening the lifetime of Ga’s active, GTP-bound state. GPy subunits are regulated by phosducin, a protein that tightly binds G& and prevents interaction with Ga and/or downstream effecters [4’]. The past year has seen the structure determination of a variety of functionally important complexes involving these proteins. Our understanding of these systems has also been expanded through structure-based mutagenesis and
Structures of Ga Ga subunits are peripheral membrane proteins, typically myristoylated and often also palmitoylated at, and near, the amino terminus. These modifications anchor Ga to the membrane surface, where 7TM receptors and many downstream effecters reside. To date, only two of the 15 known mammalian Ga subunits have been structurally characterized. The structures of both Gta, the visual system G protein responsible for mediating the response from the 7TM photoreceptor rhodopsin to its downstream effector cGMP phosphodiesterase, and Gial, a Ga subunit which responds to a variety of receptors and causes downregulation of adenylyl cyclase, have been solved in their GTP-, GDP-, and GDP.AlFd--bound states [S-9]. The past year has added another structure to the series: a Giar GlyZOS+Ala mutant containing GDP and a bound phosphate ion [lo*]. This structure mimics the state of Ga just after GTP hydrolysis and, taken together with the structures of Ga bound to the transition state analog, GDP.AlFa; helps to clarify the mechanism of GTP hydrolysis, and highlights the importance of Mgz+ in this process. The Gly226+Ala mutant is catalytically impaired because its affinity for Mgz+ is reduced, Although Mg2+ was present in the crystallization setup, it was not seen in the crystal structure. The additional methyl group at position 226 causes the switch II region to adopt a previously unseen conformation that allows the hydrolyzed y-phosphate to remain bound, and suggests how switch II might look in the transient, ternary complex. The specific stereochemistry of GTP hydrolysis has been the subject of a number of excellent reviews [l l*,lZ], and will not be discussed here. Instead, emphasis will be placed on the overall architecture of G-protein subunits so that the general implications of their interactions can be better appreciated. The guanine nucleotide for which G proteins are named is held near the interface of two domains: a Ras-like GTPase domain, and a second, predominantly helical domain unique to Ga. Three regions within the GTPase domain, termed switch I, II and III, are conformationally sensitive to the state of the nucleotide (GDP versus GTP), and all three form part of the interface with GPy and/or downstream effecters [13,14]. In Ga’s GTP-bound state, the switch regions are held in place by contacts to the nucleotide’s y-phosphate. In the Gpy-free GDP-bound state, the switch regions are generally more flexible, as measured by both the root mean square (rms) deviation between analogous GDP-bound structures and
Structural aspects of heterotrimeric
G-protein signalling Bohm, Gaudet and Sigler
481
Figure 1
Hormones Neurotransmitters Odorants
GTP
!3y effecters i.e. GRKs
+ a effecters i.e. Adenvlvl cvclase -I
I
-I-
-.--
Phosphodlesterase
Raf kinase Adenylyl cyclase
7
\
PLCS Ion channels
c 1997 Current Op,n,an ,n Biotechnology
Schematic diagram of G-protein coupled signal transduction pathways. The G-protein a and Py subunits cycle from their active states (at the bottom of the figure) to their inactive state (heterotrimer, top) through nucleotide exchange at the level of the receptor (R). Active Ga and GPy subunits regulate effecters and may themselves be regulated by RGS proteins (Ga) and phosducin (Gfi$. The phosducin block (shown with a bar) is released by phosducin’s phosphotylation (P) by protein kinase A.
crystallographic
B-factors, the crystallographically refined parameters that indicate the degree of thermal motion of atoms. The nucleotide-dependent change in plasticity of these regions reflects their role in the regulation of Ga-Gpy interactions, and is probably also important in Ga-effector interactions.
has only been solved in its Ga-bound state [18]. The GPlyl structures are all very similar, but are different from the structure of G&yz, in which the coiled-coil is in a different orientation (Figure 3). This change could play a part in the observed specificity of different GPy isotypes for different effecters and receptors [19,20].
Structure of Gpy
G/3 belongs to the P-propeller family of proteins [21]. GP’s ‘propeller’ is composed of a sevenfold repeat of four-stranded P-sheet units. These sheets are arranged radially around a small central hole (Figure 2a). GP’s structural symmetry arises from an internally repeated ‘WD-40’ sequence motif, a -40 residue motif often ending with WD (amino acid single letter code) that occurs in a functionally diverse family of proteins [22]. Although GPy is the first WD40-containing protein to be structurally characterized, it is not the first structure with the P-propeller fold, and it is interesting to note that there may be an evolutionary connection between the WD-40 containing proteins and the larger family of P-propellers.
GPy subunits are very tightly associated and cannot generally be separated except under denaturing conditions. Six mammalian isoforms of GP and 12 mammalian isoforms of Gy have been described. Whereas the Gj3 subunits are highly conserved, with an average pairwise sequence identity of 72%, Gy subunits are significantly more variable with only -45% pairwise sequence identity. Of the numerous possible GPy combinations, two have been structurally characterized. GPlyl, the species found in the mammalian visual system, has been solved alone [15**] (Figure Za), in complex with Ga [16**] (Figure Zb), and in complex with phosducin [17**] (Figure 2~). G&y*
482
Protein engineering
Figure 2
(b)GaPyRN
J
(@WY
CarboxyterminalY prenylationand .. carboxymethvlation
(C 1PhosducinlGpyP PN w/
Phosducin
P Phosducin ?$, carboxy tenninal doI
(e1
Helical domai m
RGSUGia 0
i-
aR17a
-E
hr
:
SWItChI
aN
aT182
Structural aspects of heterotrimeric
G-protein signalling Bohm, Gaudet
and Sigler
403
Figure 2
Ribbon the ‘top! P-sheet
diagrams
of the recently
Gy (shown and strands
solved
G-protein
in grey) first forms a coiled A-D
of the third WD-40
repeat
and the structure corresponding to the WD-40 with the negatively charged membrane surface.
(a) The GPT subunits.
structures. coil with Gp’s (shown
amino terminus
GP (shown
and then snakes
in dark grey) are labeled
in white)
forms a P-propeller,
along the bottom
to emphasize
the difference
between
one at the the carboxyl
terminus
l-4
individual
here from of a P-sheets
sequence repeat. The positively charged region opposite the coiled coil is postulated to interact Gy’s post-translational modifications are inserted into the membrane. (b) The heterotrimeric
G-protein. GF5-yis shown in the same orientation as in (a). Go (shown in dark grey) binds to the top surface of GPy through (shown in white). The amino-terminal helix of Ga interacts with GPy on its membrane-binding side, placing the heterotrimer’s modifications,
viewed
of GP. Strands
of Gy and one at the amino terminus
of Ga, in proximity
its switch regions two fatty acid
to each other and to the membrane.
(c)
The phosducinlG& complex. GPy is again in the same orientation as in (a) and (b). Phosducin’s amino-terminal domain binds the ‘top’ of GPy, with the phosphorylation site (Ser73) pointing out, away from GPy. The negatively charged carboxy-terminal domain of phosducin interacts with the positively charged side of the P-propeller, preventing Gpy’s interaction with the membrane. (d) The heterotrimer as viewed from the left side of (b). This view best shows
the interaction
of
Ga’s
switch
II region
with the top of GPy. Regions
implicated
in receptor
binding
by mutagenic
and biochemical studies are highlighted (Go’s amino terminus, strand PS, a4j36 loop and carboxy terminus, Gps sixth and seventh WD-40 repeats and Gy’s carboxy terminus; shaded ellipsoids). The carboxy terminus of Ga (missing seven residues in this structure) was extended (white) to show the approximate position of the pertussis toxin ADP-ribosylation site (PTX). (e) The RGS4/Giat complex. Ga is in the same orientation as in (d). Ga’s switch II region is held tightly against the GTPase domain by the AIF4- ion which occupies the expected position of the y phosphate during GTP hydrolysis. The position of switch II is similar to that seen in the Go-GTP@ and Ga-GDP.AIF4structures, but different from that in the Gpy-bound Ga-GDP structure (see Id]) where switch II is pulled away from the GTPase domain to interact with GPy. The positions of the catalytic arginine and glutamine (labeled according to the Gist numbering) are shown, as well as RGS4’s asparagine 184, which interacts with and probably stabilizes the position of several residues in Ga’s active site. The amino-terminal helix of Ga has been truncated in this figure. It makes crystal contacts in this structure, and points in a different direction from that seen than in both structures of the GaPy heterotrimer. Helix aN adopted yet another conformation in the structure of the GDP-bound state of Gist (not shown). The magnified region has been rotated slightly to facilitate viewing of the active site.
Gy subunits are normally modified at their carboxyl-terminus with either a farnesyl or geranyl-geranyl moiety which, along with frequently occurring carboxymethylation, anchors GPy to the cell membrane [23]. Gy forms a coiled coil with the amino terminus of GP, and continues in an extended conformation along what will be referred to here as the ‘bottom’ of the P-propeller. The surface of GPy includes a large, positively charged patch on the side of the P-propeller opposite the coiled-coil. This patch, which would be expected to interact favorably with the negatively charged phospholipid membrane, is adjacent to Gy’s acyl modification, strongly implicating this region in Gpy’s membrane interaction.
free state until GTP binds the G protein, and causes Ga and GPy to separate from each other and from the receptor [31]. While the two crystal structures of heterotrimeric G proteins cannot unambiguously explain how receptors induce nucleotide release, the structures do explain how GTP binding results in separation of Ga and GPy. In the absence of the nucleotide’s y-phosphate, the switch regions of Ga, especially switch II, are free to interact with GPy, and are drawn away from the bulk of Ga. When GTP binds, switch II snaps away from GPy, back into the fold of Ga.GTP thereby depriving the interface of these critical components and disengaging Ga.GTP from GPy.
Structures
Structure of GPy with its regulator,
of the Gc@y heterotrimer
Two GaPy heterotrimeric complexes have been solved crystallographically [16”,18] (Figures Zb, Zd, and 3). While GPy is essentially unchanged upon Ga binding, the switch I, switch II, and amino-terminal regions of Ga.GDP undergo a significant Gpy-induced conformational rearrangement. There are two regions of interaction between GPy and Ga: Ga’s switch I and switch II bind to the ‘top’ face of Gpy’s P-propeller, and the myristoylated amino-terminal helix of Ga binds to one side of Gpy’s positively charged patch. The heterotrimer also contains a positively charged region for favorable membrane interaction. This region overlaps the residues that mutagenic and peptide data have implicated in Gapy’s interaction with 7TM receptors [24-26,27~~,28°*,29,30] (Figure Zd). Catalysis of nucleotide exchange by activated 7TM receptors is a two-step process. First, receptors bind GDPbound heterotrimers and cause nucleotide release. The receptor-G-protein complex then sits in this nucleotide-
phosducin
Under conditions of bright light, phosducin regulates sensitivity of the visual system by sequestering GPy in a tight bimolecular complex and releasing G& from the retinal rods’ internal disk membrane into the cytosol [32]. Since photoactivated rhodopsin cannot catalyze nucleotide exchange on Ga in the absence of GPy, the signal is essentially stopped at the G-protein level. Under low light conditions the phosducin-Gpy complex is destabilized by protein kinase A phosphorylation of phosducin, releasing GPy and restoring sensitivity [4’]. Although phosducin is best known for its role in vision, it is likely to regulate other systems as well since phosducin is present in many tissues, and indiscriminately binds a variety of GPy subunits [33,34]. Phosducin has two domains, each of which has a distinct role. Phosducin’s amino-terminal domain binds the ‘top’ face of GPy (the face that interacts with Ga’s switch regions). This domain competes with Ga, and prevents heterotrimer formation. The amino-terminal domain also
404
Protein
Figure
engineering
the switch regions of Ga, but does not appear to contribute catalytic residues to the hydrolytic machinery. Rather, RGS proteins seem to work by stabilizing conformational changes in Ga’s existing catalytic residues that are unique to the transition state. Most notable among these residues are the conserved, catalytically active, switch I arginine at position 178 and switch II glutamine at position 204 (Figure Ze). While the catalytic glutamine is stabilized both indirectly through RGS contacts to switch II, and directly by Asp128 from RGS4, there are no direct contacts between RGS4 and the catalytic arginine. Arg178 is stabilized primarily by a network of interactions whereby three of RGS4’s helices make contacts to switch I residue Thr182.
3
Structures of downstream heterotrimeric G proteins
Superposition of the two published crystal structures of heterotrimeric G-proteins. Gial &ys [16] is shown in grey. The complex of G&y, with the GtaIGia, chimera [16**] is shown in white. Structures were superimposed using only residues from the GP’s @-propeller. A flexible loop behind Ga’s amino-terminal helix (residues 127-l 36) was not included in the superposition. The largest structural difference is in the position of the coiled-coil. This superposition causes the bulk of the two Ga subunits to be rotated with respect to each other about an axis running roughly parallel to Ga’s amino-terminal helix. Ga’s switch II and helix aN regions are not rotated, suggesting that they are more tightly coupled to GPy than they are to the remainder of Ga. One can then imagine that if the receptor moves the bulk of Ga relative to GPy, these tightly bound regions might stay coupled to Gfly, opening a cleft betwwen the switch regions and the remainder of Gaand thereby facillitating nucleotide
release.
the exposed phosphorylation site, Ser73, that regulates phosducin’s affinity for GPy [3.5]. Phosducin’s carboxy-terminal domain is responsible for dissociating GPy from the cell membrane. This domain is electrostatically negative, and binds the positively charged patch on GPy, thereby preventing Gpy’s favorable membrane interaction (Figure 2~). Truncation mutants of phosducin that lack the carboxy-terminal domain have been shown to bind GPy without causing membrane dissociation [36]. contains
Structure of Ga with its regulator
RGS
The structure of Gial.GDP.AlF4has been solved in complex with its regulator, RGS4 [37**]. RGS proteins generally bind more tightly to GDPeAlFd--bound Ga (the transition state analog complex) than they do to Ga subunits in their GTP- or GDP-bound states [2’,3,38], hence they appear to accelerate Ga’s rate of GTP hydrolysis by stabilizing the transition state. RGS4 binds
effecters
of
Free GPy subunits, and activated, GTP-bound Ga subunits interact with a variety of downstream effecters. Most notable among the Ga targets are adenylyl cyclases and cGMP phosphodiesterase. The structure of a catalytically inactive fragment of an adenylyl cyclase has been solved [39’], and combined with binding site data derived from Ga and cyclase mutants [13,40], leads to an emerging structural model of the Ga-cyclase complex. Downstream molecules that interact with GPy include phospholipase Cp, adenylyl cyclases, Na+ and K+ ion channels, and a variety of tyrosine and serine/threonine kinases [41]. Many of the kinases that interact with GPy contain pleckstrin homology domains at or near the regions shown to bind GPy subunits [42], and it is likely that the mode of kinase-Gpy interaction is somewhat conserved among the various Gpy-regulated kinases. The Gpy-binding pleckstrin homology domain from the P-adrenergic receptor kinase has recently been solved by NMR (D Cowburn, personal communication).
The interaction of 7TM receptors with heterotrimeric G proteins Site directed mutagenesis, peptide binding, and proteolytic digestion studies of heterotrimeric G proteins and 7TM receptors have significantly improved our understanding of the interaction between these proteins. G-protein binding is mediated by the three carboxyterminal intracellular loops of the receptor [ZS], and the receptor makes contacts with all three subunits of the heterotrimeric G protein. Three regions of Ga contain residues that effect receptor interaction: Ga’s amino-terminal helix, exposed residues on helix a5 and strand p6, and Ga’s carboxy terminus [27”,29]. These receptor binding regions, along with Gy’s carboxy terminus [24,30], and the sixth or seventh WD repeats of GP [28], which have also been implicated in receptor binding, all map to the heterotrimer face proposed to interact with the membrane surface [16**,26]. This same surface also contains the site of pertussis toxin catalyzed ADP ribosylation of G proteins, a modification which decouples G proteins from 7TM receptors [43].
Structural aspects of heterotrlmrric
Mechanisms for receptor catalyzed nucleotide release There are two likely avenues for receptor catalyzed nucleotide release after GTP hydrolysis. Either GDP goes out guanine-side first, through a movement of the @--or5 loop, or it leaves phosphate-side first, through further conformational changes in the switch regions. Point mutations in the @-a5 region increase the rate of receptor-independent nucleotide exchange [44], suggesting that the nucleotide goes out guanine side first. Both a5 and p6 have been implicated in receptor binding, putting the receptor in relatively close proximity to guanine nucleotide [27**]. The alternative route, however, should also be considered. Whereas a5 and p6 are rigid structural elements with many contacts to the remainder of Ga, the switch regions are flexible, and easily subject to conformational change. Furthermore the crystal structures of the small G protein EF-Tu in complex with its exchange factor EF-Ts show the exchange factor acting near the phosphate, not the guanine ring [45*,46]. The key to 7TM-induced nucleotide exchange could be Ga’s hinge-like amino-terminal helix, which has been seen in a variety of conformations in the Gpy-free structures of Gial [9,37”]. The receptor’s interaction on both sides of Ga’s flexible amino-terminal region could induce a change in the heterotrimer whereby the bulk of Ga and GPy are displaced with respect to each other. If this were to happen, G& could draw the switch regions even farther from Ga, further opening the GTP binding cleft, and facilitating nucleotide release. This model is consistent with the requirement for GPy in receptor-mediated nucleotide exchange [47] and with an apparent flexibility in the heterotrimer suggested by a conformational difference seen between the two heterotrimeric crystal structures [16*,18]. When the P-propellers of the two heterotrimer structures are superimposed, their corresponding Ca atoms deviate by an rms difference of only -0.43A. This superposition causes Gta’s amino-tertiinal helix, switch II, and switch I regions to overlap quite well with their Gial counterparts (Figure 3). The remainder of Ga, however, is not in the same orientation in the two structures. It is rotated by -4.5’, suggesting a degree of flexibility. A rotation of this sort, if magnified by interaction with the receptor, could facilitate nucleotide release.
405
transmembrane helices, but higher resolution data are needed if we are to understand the mechanism by which 7TM receptors catalyze nucleotide exchange in a stimulus-dependent manner. The past year has seen significant advances in our ability to express and purify 7TM receptors [49,50]. Also, recent technical advances in cryoelectron microscopy have pushed the limits of this technique firmly into the realm of atomic resolution [Sl]. Based on these advances, as well as the advent of new methods for forming three-dimensional crystals of membrane proteins [SZ], it seems chat the first atomic resolution structure of a 7TM receptor might not be too far away.
Acknowledgements Thanks to John Sondek, Takemasa Kawasbimaand YongWang for helpful discussionsand to Steve Sprangfor providingcoordinaresof the GiailRGS4 complex. This work was iupp&ted- by a gr& from the National Insritute of Health to Paul B Sigler (GM22324); Rachelle Gaudet is supported by an National Science and Engineering Research Council of Canada 1967 fellowship.
References and reco-nded
reading
Papers of particular interest, published within the annual period of review, have been highlighted’as:
. of special inter& . . of outstanding intereet 1.
Gautam N, Stratbmann.ME Simon Ml: Dlvorsity of G-Protolns in slgnrl lranrductlon. Science’1 991, 262:602-806.
2. .
Berman DM, Kazasa T, Gilman AG: Thr GTPsw-rctlvstlng protein RGW stsbllkes the tnnsitlon state for nuclootlda hydrolysis I Biol Chem 1996, 271:27209-27212. This work shows that RGS4 can act catalyticallyas a GTPase-avtivating protein for Ga subunits.The authors also describe RGS4’e preference for GmGDPAlF4-, the transition-stateanalog. See also references 13,381. Describes the GTPase activatingactivityof RGS proteins, and the mechanism of their action. 3.
Watson N, Linder ME, Druey KM, Kehrl JH, Blumer Kl: RGS family membem: OfPass-actlvstlng protelnr for heterotrlmeric Gprotein a-subunits. Nature 1996, 383:172-l 76.
4. .
Willardson EM, Wilkins JF, Tateuro Y, Bitenaky MW: Regulation of phosducin phosphorylation in retinal rods by Ce*+/celmodullndependent edenylyl cyclerc Proc Nat/ Acad SC; USA 1996, 93:1476-1479. This paper proposes a light-dependent mechanism for phosducin’s regutation by PKA and phosphatase. In doing so, they explsin how phosducin regulates the light sensitivityof rod cells. 6.
Noel JP, Hamm HE, Sigler PB: The MA crystel structure of transducln-acomplexed with GTPyS. Narure 1993, 366:864-663.
6.
Lambright DG, Noel JP, Hamm HE, Sigler PB: Structural determinants for the rctlvrtlon of the a-subunit of I heterotrlmerlc G-protein. Nature 1994, 389:821 e826.
7.
Sondek J, LambrightDG, Noel JP, Hamm E, Sigler PB: GTPasa mechanism of G-proteins from the 1.7 f crystal structure of transducin a-GDPAlF,. Nature 1994, 372:276-279.
8.
Coleman DE, Berghuis AM, Lee E, tinder ME, Gilmsn AG, Sprang SR: Structures of ectlve conformations of Gia, end the mechanism of GTP hydrolysis. Science 1994, 265:1406-l 412.
9.
Mixon MB, Lee E, Coleman DE, Berghuis AM, Gilman AG, Sprang SR: Tertiary and quartemety structurel chrnges In Gia, induced by GTP hydrolysis. Science 1996, 270:054-080.
10. .
Berghuis AM, Lee E, Raw As, Oilman AC, Sprang SR: Structun of the GDP-PI complex of Gly203-1Alr Gia,: a mimlck of the
Conclusions The flurry of recent G-protein crystal structures have shown us, at atomic resolution, how GTP hydrolysis works, how heterotrimers associate and dissociate in response to nucleotide, and how Ga and GPy are regulated by other proteins. In contrast to the rapid progress on the G-protein front, however, there is still no high resolution picture of a 7TM receptor. Electron cryomicroscopy of two-dimensional crystals of frog rhodopsin has produced -6A resolution maps [48], the best view so far. These maps are sufficient to determine the orientation of the
G-protein signalllng Bohm, Gaudet and Sigler
486
Protein
engineering
tarnary product complex of Ga-catalyzed GTP hydrolysis. Structure 1996, 4:1277-l 290. This is the latest structure in the Ga series. It mimics the state of Ga just after GTP hydrolysis. Taken together with references 15-91 (the GTPyS, GDP-, end GDP.AIFd--bound states of Gta and Gia) this structure clarifies the GTP hydrolysis mechanism. 11.
Kjeldgsard M, Nyborg J, Clark BFC: The GTP binding motif: variations on a theme. FASEB J 1996, 10:1347-l 368. k extensive review of nucleotide binding by G proteins, incorporating work on heterotrimeric G proteins as well as small G proteins like Rss and EF-Tu.
tations, the authors present a model for receptor catalyzed nucleotide exchange. 28. .
Taylor JM, Jocob-Mosier GG, Lawton RG, VanDort M, Neubig RR: Receptor and membrane interaction sites on Op. J Biol Chem 1996, 271~3336-3339. The authors show crosslinking between a peptide derived from the B_adrenergic receptor and the sixth or seventh WD-40 repeats of GP. 29.
Hamm HE, Deretic D, Arendt A, Hargrave PA, Koenig B, Hofmann KP: Site of G protein binding to rhodopsin mapped with synthetic peptides from the a subunit Science 1988, 241~832-835.
30.
Gierschik P, Scheer A: S-prenylated cystain analogues inhibit receptor-mediated G protein activation in native human granulocyte and reconstituted retinal rod outer segment membranes. Biochemistry 1995, 34:4952-4961.
31.
Fung BK-K: Characterization of transducin from bovine retinal rod outer segments. J Biol Chem 1963, 268:10495-l 0502.
15. ..
Sondek J, Bohm A, Lambright DG, Hamm *HE, Sigler PB: Crystal structure of a G-protein By dimer at 2.1 A resolution. Nature 1996, 379:369-374. The structure of GP,yt in the absence of Ga. This paper contains a detailed map of contacts between GP and GY, and details of the structure formed by the WD-40 sequence motif.
32.
Yoshida T, Willardson BM, Wilkins JF, Jensen GJ, Thornton BD, Bitensky MW: The Phosphorylation stata of phosducin determines its ability to block transducin subunit interaction and inhibit transducin binding to activated rhodopsin. J Biol Chem 1994.269:24050-24057.
16. ..
Lambright DG, Songlek J, Bohm A, Skiba NP, Hamm HE, Sigler PB: The 2.OA crystal structure of a hetarotrimeric Gprotein. Nature 1996, 379:31 l-31 9. Structure of G/31yl complexed to a Gta/Gial chimera This paper details the interactions between Ga and Go. It also presents the model for Gapy’s interaction with the membrane.
33.
Denner S, Lohse MJ: Phosducin is a ubiquitious regulator of GpY subunits. Proc Nat/ Acad Sci USA 1996, 93:10145-l 0150.
34.
Muller S, Straub A, Schoder S, Bauer PH, Lohse MJ: Interactions of phosducin with defined G-protein by-subunits. J Biol Chem 1996, 271 :l1781-l 1786.
Gaudet R, Bohm A, Sigler PB: Crystal structure at 2.4 A resolution of the comolex of transducin Or and its reaulator. _ Phosductn. Cell 1996,’ 87:577-588. ’’ This structure explains how phosducin competes with Ga for Gby. Electrostatic calculations predict how G!3y binds the membrane in the &ence of Ga. and how phosducin disrupts this interaction.
35.
Lee RH, Brown BM, Lolley RN: Protein kinase A phosphorylates retinal phosducin on serine 73 in situ. J Biol Chem 1990, 26515860-l 5866.
36.
Tanaka H, Kuo C-H, Matsuda T, Fukada Y, Hayashi F, Ding Y, lrie Y, Miki N: MEKA/Phosducin attenuates hydrophobicitv of transducin bg subunits without binding to farnesyl moiety. Biochem Siophys Res Comm 1996, 223:567-591.
12.
Hilgenfeld R: How do the GTPases 1995, 2:3-6.
really work? Struct Biol
13.
Berlot CH, Bourne HR: Identification of effector-activating residues of Gsa. Cell 1992, 68:91 l-922.
14.
Skiba NP, Bae H, Hamm HE: Mapping of effector binding sites of transducin alpha-subunit using G-alpha t/G alpha il chimeras. J Biol Chem 1996. 271:413-424.
1 7. ..
10.
Well MA, Coleman DE, Lee E, Iniguez-Lluhi JA, Posner BA, Gilman AG, Sprang SR: The Structure of the G Protein heterotrimer GialPly2. Ce// 1995.83:1047-l 056.
19.
Kleuss C, Scherubl H, Hescheler J, Schultz G, Wittig B: Selectivity of signal tranrducion determined by ysubunits of hetarotrimeric G proteins. Science 1993, 259:832-834.
20.
Muller S, Hekmen M, Lohse MJ: Specific enhancement of padrenergic receptor kinase activity by defined G-protain p and y subunits. Proc Nat/ Acad Sci USA 1993,9&l 0439-l 0443.
21.
Murzin AG: Structural principles for the propeller assembly of P-sheets: the preference for seven-fold fymmetry. Proteins 1992, 14:191-201.
22.
Neer El, Schmidt CJ, Nsmbudripsd R, Smith TF: The ancient regulatory-protein family of WD-repeat proteins. Nature 1994, 3711297-300.
23.
Wedegaertner PB, Wilson PT, H Bourne HR: Lipid modifications of trimeric G-proteins. J Biol Chem 1995, 270:503-506.
24.
Kisselev OG, Ermolaeva MV, Gautsm N: A farnesylatad domain in the G protein y subunit is a specific determinant of receptor coupling. J Biol Chem 1994, 269:21399-21402.
25.
Hargrave PA, Hsmm HE, Hofmann KP: Interaction of rhodopsin with the G-protein, transducin. Bioessays 1993, 15:43-50.
26.
Lichtarge 0, Bourne HR, Cohen FE: Evolutionarily conserved GaPy binding surfaces support a model of the G proteinreceotor wmolex Proc Nat/ Acad Sci USA 1996, 93:750775ii.
37. ..
Tesmer JJ, Berman DM, Gilman AG, Sprang SR: Structure of RGS4 bound to AIF4-activated Gia,: stabilization of the transition state for GTP hydrolysis. Cell 1997, 69:251-261. Although only the RGS domain of RGS4 is seen, the structure explains how RGS proteins bind and accelerate GTPase activity of Ga subunits. 36.
39.
Zhang G, Lui Y, Ruoho AE, Hurley JH: Structure of the adenylyl cyclase catalytic core. Nature 1997, 386:247-253. ;he two cytoplasmic domains of adenylyl cyclase are homologous to each other. This structure of a catalytically inactive homodimer of one of the two cytoplssmic domains (llC2) presents a picture of the overall fold, but not the catalytic machinery of the ATP+cAMP reaction. 40.
Yan S-Z, Huang Z-H, Hurley JH, Tang W-J: Gsa binding site and activation of mammalian adenylyl cyclase. J Biol Chem 1997, in press.
41.
Claphsm D, Neer El: New roles for G-protein By-dimers in transmembrane signalling. Nature 1993, 365403-406.
42.
lnglese J, Koch W, Touhara K, Lefkowitz RJ: GpY interactions with PH domains and Ras-MAPK signalling pathways. Fends Biochem Sci 1995, 20:151-l 56.
43.
Van Dop C, Yamanaka G, Steinberg F, Sekura RD, Man&k CR, Stryer L, Bourne HR: ADP-ribosylation of transducin by pertussis toxin blocks the light-stimulated hydrolysis of GTP and cGMP in retinal photoreceptors. J Biol Chem 1984, 269:2326.
44.
Taroh I, Herzmerk P, Nakomoto JM, Van Dop C, Boume HR: Rapid GDP release from Gsa in patienfs with gain and loss of endocrine function. Nature 1994, 371 :164-l 68.
27. ..
Onrust R. Herzmark P, Chi P, Garcia PD, Lichtarge 0, Kingsley C, Boume HR: Receptor and py binding sites in the alpha subunit of the retinal G-protein transducin. Science 1997, . _^275:381-384. . This paper describes a vanety ot slte-dlrected mutants ot tia that disrupt interactions with Gpy and with 7TM receptors. On the basis of these mu-
Chen C-K, Wieland T, Simon Ml: RGS-r, a retinal specific RGS protein, binds an intermediate conformation of transducin and enhances recycling. Proc Nat/ Acad Sci USA 1996, 93:1288512889.
Structural
aspects
45.
Kawashima T. Berthet-Colominas C. Wulff M. &sack S. Leberman R: ‘The structure of the &reri&a co/i EFiTuEF-Ts complex at 2.5 A resolution. Nature 1996, 379:51 l-51 6. The structure of the small G protein EF-Tu in complex with its soluble exchange factor EF-Ts suggests a mechanism whereby nucleotide exchange is accelerated by disruption of of the Mga+ binding site.
.
46.
Wang Y, Jiang Y, Meyering-Vos M, Sprinzl M, Sigler PB: Crystal structure of the EF-TuEF-Ts complex from Thermus thermophilus. Nat Struct Bioll997, in press.
47
Birnbaumer L: G Proteins in signal transduction. Pharmacol Toxicol 1990, 30:675-705.
46.
Schertler GFX, Hargrave PA: Projection structure of frog rhodopsin in two crystal forms. Proc Nat/ Acad Sci USA 1995, 92:11576-l 1562.
Annu Rev
of heterotrimeric
G-protein
signalling
Bohm, Gaudet
and Sigler
407
49.
Reeves PJ, Thunond RL, Khorana HG: Structure and function in rhodopsin: high level expression of a synthetic bovine opsin gene and its mutants in stable mammalian call lines. Proc Nat/ Acad SC; USA 1996,93:11487-l 1492.
50.
Kiefer H, Krieger J, Olszewski JD, Von Heijne G, Prestwich GD, Breer H: Exoression of an olfactorv receotor in Escheric/ria co/i: purification; reconstitution, and ligand binding. Biochemistry 1996, 35:16077-l 6004.
51.
Dorset DL: Electron crystallography. 769.
52.
Landau EM, Rosenbusch JP: Lipidic cubic phases: a novel concept for the crystallization of membrane proteins. Proc Nat/ Acad Sci USA 1996, 93:14532-l 4535.
Acta Cryst B 1996, 52:753-
Structural aspects of heterotrimeric G-protein signaling Andrew
Bohm*, Rachelle Gaudett
and Paul B Siglerr
Recently, structures of heterotrimeric G-protein subunits have been determined in isolation, in conjunction with each other, and in complex with their regulators. information, these structures
Along with biochemical
suggest how G-protein
are oriented relative to the membrane to seven-transmembrane mechanisms
surface and relative
helix receptors.
for receptor-catalyzed
subunits
modeling studies. In this review, we discuss results derived from these recent determinations, and we comment on the implications they have for our knowledge of receptor catalyzed nucleotide exchange.
They also suggest
nucleotide
exchange.
Addresses The Department of Molecular Biophysics and Biochemistry, and The Howard Hughes Medical institute, Yale University, 280 Whitney Ave, JWG 423, New Haven, CT 08511, USA *e-mail: [email protected] te-mail: rgaudetQbiomed.med.yale.edu Se-mail: [email protected] Current Opinion in Biotechnology 1997, 8:480-487 http://biomednet.com/elecref/0958168900800480 0 Current Biology Ltd ISSN 0958-l 669 Abbreviations RGS regulators of G-protein signalling rms root mean square 7TM seven-transmembrane helix
Introduction Heterotrimeric G-proteins consist of a, f3 and y subunits. Signal transduction pathways employing these proteins are ubiquitous transducers of extracellular signals in all eukaryotes, including primitive unicellular organisms. There are hundreds of systems of this type in the human body and, in addition to mediating the cellular response to many hormones and neurotransmitters, these systems are directly responsible for the senses of sight, smell and taste [l]. Activation of seven-transmembrane helix (7TM) receptors results in a conformational change that allows the receptors to act as nucleotide-exchange factors for Ga subunits. Once GDP has been replaced with GTP, the tightly associated Ga and G& subunits of the G protein separate from each other and from the 7TM receptor, leaving both Ga and GPy free to interact with and modulate downstream components of signaling cascades (Figure 1). Both Ga and GPy are regulated by other proteins. Ga subunits are regulated by a growing family of proteins [2*,3]; these ‘regulators of G-protein signaling’ (RGS) bind Ga and accelerate the rate of GTP hydrolysis to GDP, shortening the lifetime of Ga’s active, GTP-bound state. GPy subunits are regulated by phosducin, a protein that tightly binds G& and prevents interaction with Ga and/or downstream effecters [4’]. The past year has seen the structure determination of a variety of functionally important complexes involving these proteins. Our understanding of these systems has also been expanded through structure-based mutagenesis and
Structures of Ga Ga subunits are peripheral membrane proteins, typically myristoylated and often also palmitoylated at, and near, the amino terminus. These modifications anchor Ga to the membrane surface, where 7TM receptors and many downstream effecters reside. To date, only two of the 15 known mammalian Ga subunits have been structurally characterized. The structures of both Gta, the visual system G protein responsible for mediating the response from the 7TM photoreceptor rhodopsin to its downstream effector cGMP phosphodiesterase, and Gial, a Ga subunit which responds to a variety of receptors and causes downregulation of adenylyl cyclase, have been solved in their GTP-, GDP-, and GDP.AlFd--bound states [S-9]. The past year has added another structure to the series: a Giar GlyZOS+Ala mutant containing GDP and a bound phosphate ion [lo*]. This structure mimics the state of Ga just after GTP hydrolysis and, taken together with the structures of Ga bound to the transition state analog, GDP.AlFa; helps to clarify the mechanism of GTP hydrolysis, and highlights the importance of Mgz+ in this process. The Gly226+Ala mutant is catalytically impaired because its affinity for Mgz+ is reduced, Although Mg2+ was present in the crystallization setup, it was not seen in the crystal structure. The additional methyl group at position 226 causes the switch II region to adopt a previously unseen conformation that allows the hydrolyzed y-phosphate to remain bound, and suggests how switch II might look in the transient, ternary complex. The specific stereochemistry of GTP hydrolysis has been the subject of a number of excellent reviews [l l*,lZ], and will not be discussed here. Instead, emphasis will be placed on the overall architecture of G-protein subunits so that the general implications of their interactions can be better appreciated. The guanine nucleotide for which G proteins are named is held near the interface of two domains: a Ras-like GTPase domain, and a second, predominantly helical domain unique to Ga. Three regions within the GTPase domain, termed switch I, II and III, are conformationally sensitive to the state of the nucleotide (GDP versus GTP), and all three form part of the interface with GPy and/or downstream effecters [13,14]. In Ga’s GTP-bound state, the switch regions are held in place by contacts to the nucleotide’s y-phosphate. In the Gpy-free GDP-bound state, the switch regions are generally more flexible, as measured by both the root mean square (rms) deviation between analogous GDP-bound structures and
Structural aspects of heterotrimeric
G-protein signalling Bohm, Gaudet and Sigler
481
Figure 1
Hormones Neurotransmitters Odorants
GTP
!3y effecters i.e. GRKs
+ a effecters i.e. Adenvlvl cvclase -I
I
-I-
-.--
Phosphodlesterase
Raf kinase Adenylyl cyclase
7
\
PLCS Ion channels
c 1997 Current Op,n,an ,n Biotechnology
Schematic diagram of G-protein coupled signal transduction pathways. The G-protein a and Py subunits cycle from their active states (at the bottom of the figure) to their inactive state (heterotrimer, top) through nucleotide exchange at the level of the receptor (R). Active Ga and GPy subunits regulate effecters and may themselves be regulated by RGS proteins (Ga) and phosducin (Gfi$. The phosducin block (shown with a bar) is released by phosducin’s phosphotylation (P) by protein kinase A.
crystallographic
B-factors, the crystallographically refined parameters that indicate the degree of thermal motion of atoms. The nucleotide-dependent change in plasticity of these regions reflects their role in the regulation of Ga-Gpy interactions, and is probably also important in Ga-effector interactions.
has only been solved in its Ga-bound state [18]. The GPlyl structures are all very similar, but are different from the structure of G&yz, in which the coiled-coil is in a different orientation (Figure 3). This change could play a part in the observed specificity of different GPy isotypes for different effecters and receptors [19,20].
Structure of Gpy
G/3 belongs to the P-propeller family of proteins [21]. GP’s ‘propeller’ is composed of a sevenfold repeat of four-stranded P-sheet units. These sheets are arranged radially around a small central hole (Figure 2a). GP’s structural symmetry arises from an internally repeated ‘WD-40’ sequence motif, a -40 residue motif often ending with WD (amino acid single letter code) that occurs in a functionally diverse family of proteins [22]. Although GPy is the first WD40-containing protein to be structurally characterized, it is not the first structure with the P-propeller fold, and it is interesting to note that there may be an evolutionary connection between the WD-40 containing proteins and the larger family of P-propellers.
GPy subunits are very tightly associated and cannot generally be separated except under denaturing conditions. Six mammalian isoforms of GP and 12 mammalian isoforms of Gy have been described. Whereas the Gj3 subunits are highly conserved, with an average pairwise sequence identity of 72%, Gy subunits are significantly more variable with only -45% pairwise sequence identity. Of the numerous possible GPy combinations, two have been structurally characterized. GPlyl, the species found in the mammalian visual system, has been solved alone [15**] (Figure Za), in complex with Ga [16**] (Figure Zb), and in complex with phosducin [17**] (Figure 2~). G&y*
482
Protein engineering
Figure 2
(b)GaPyRN
J
(@WY
CarboxyterminalY prenylationand .. carboxymethvlation
(C 1PhosducinlGpyP PN w/
Phosducin
P Phosducin ?$, carboxy tenninal doI
(e1
Helical domai m
RGSUGia 0
i-
aR17a
-E
hr
:
SWItChI
aN
aT182
Structural aspects of heterotrimeric
G-protein signalling Bohm, Gaudet
and Sigler
403
Figure 2
Ribbon the ‘top! P-sheet
diagrams
of the recently
Gy (shown and strands
solved
G-protein
in grey) first forms a coiled A-D
of the third WD-40
repeat
and the structure corresponding to the WD-40 with the negatively charged membrane surface.
(a) The GPT subunits.
structures. coil with Gp’s (shown
amino terminus
GP (shown
and then snakes
in dark grey) are labeled
in white)
forms a P-propeller,
along the bottom
to emphasize
the difference
between
one at the the carboxyl
terminus
l-4
individual
here from of a P-sheets
sequence repeat. The positively charged region opposite the coiled coil is postulated to interact Gy’s post-translational modifications are inserted into the membrane. (b) The heterotrimeric
G-protein. GF5-yis shown in the same orientation as in (a). Go (shown in dark grey) binds to the top surface of GPy through (shown in white). The amino-terminal helix of Ga interacts with GPy on its membrane-binding side, placing the heterotrimer’s modifications,
viewed
of GP. Strands
of Gy and one at the amino terminus
of Ga, in proximity
its switch regions two fatty acid
to each other and to the membrane.
(c)
The phosducinlG& complex. GPy is again in the same orientation as in (a) and (b). Phosducin’s amino-terminal domain binds the ‘top’ of GPy, with the phosphorylation site (Ser73) pointing out, away from GPy. The negatively charged carboxy-terminal domain of phosducin interacts with the positively charged side of the P-propeller, preventing Gpy’s interaction with the membrane. (d) The heterotrimer as viewed from the left side of (b). This view best shows
the interaction
of
Ga’s
switch
II region
with the top of GPy. Regions
implicated
in receptor
binding
by mutagenic
and biochemical studies are highlighted (Go’s amino terminus, strand PS, a4j36 loop and carboxy terminus, Gps sixth and seventh WD-40 repeats and Gy’s carboxy terminus; shaded ellipsoids). The carboxy terminus of Ga (missing seven residues in this structure) was extended (white) to show the approximate position of the pertussis toxin ADP-ribosylation site (PTX). (e) The RGS4/Giat complex. Ga is in the same orientation as in (d). Ga’s switch II region is held tightly against the GTPase domain by the AIF4- ion which occupies the expected position of the y phosphate during GTP hydrolysis. The position of switch II is similar to that seen in the Go-GTP@ and Ga-GDP.AIF4structures, but different from that in the Gpy-bound Ga-GDP structure (see Id]) where switch II is pulled away from the GTPase domain to interact with GPy. The positions of the catalytic arginine and glutamine (labeled according to the Gist numbering) are shown, as well as RGS4’s asparagine 184, which interacts with and probably stabilizes the position of several residues in Ga’s active site. The amino-terminal helix of Ga has been truncated in this figure. It makes crystal contacts in this structure, and points in a different direction from that seen than in both structures of the GaPy heterotrimer. Helix aN adopted yet another conformation in the structure of the GDP-bound state of Gist (not shown). The magnified region has been rotated slightly to facilitate viewing of the active site.
Gy subunits are normally modified at their carboxyl-terminus with either a farnesyl or geranyl-geranyl moiety which, along with frequently occurring carboxymethylation, anchors GPy to the cell membrane [23]. Gy forms a coiled coil with the amino terminus of GP, and continues in an extended conformation along what will be referred to here as the ‘bottom’ of the P-propeller. The surface of GPy includes a large, positively charged patch on the side of the P-propeller opposite the coiled-coil. This patch, which would be expected to interact favorably with the negatively charged phospholipid membrane, is adjacent to Gy’s acyl modification, strongly implicating this region in Gpy’s membrane interaction.
free state until GTP binds the G protein, and causes Ga and GPy to separate from each other and from the receptor [31]. While the two crystal structures of heterotrimeric G proteins cannot unambiguously explain how receptors induce nucleotide release, the structures do explain how GTP binding results in separation of Ga and GPy. In the absence of the nucleotide’s y-phosphate, the switch regions of Ga, especially switch II, are free to interact with GPy, and are drawn away from the bulk of Ga. When GTP binds, switch II snaps away from GPy, back into the fold of Ga.GTP thereby depriving the interface of these critical components and disengaging Ga.GTP from GPy.
Structures
Structure of GPy with its regulator,
of the Gc@y heterotrimer
Two GaPy heterotrimeric complexes have been solved crystallographically [16”,18] (Figures Zb, Zd, and 3). While GPy is essentially unchanged upon Ga binding, the switch I, switch II, and amino-terminal regions of Ga.GDP undergo a significant Gpy-induced conformational rearrangement. There are two regions of interaction between GPy and Ga: Ga’s switch I and switch II bind to the ‘top’ face of Gpy’s P-propeller, and the myristoylated amino-terminal helix of Ga binds to one side of Gpy’s positively charged patch. The heterotrimer also contains a positively charged region for favorable membrane interaction. This region overlaps the residues that mutagenic and peptide data have implicated in Gapy’s interaction with 7TM receptors [24-26,27~~,28°*,29,30] (Figure Zd). Catalysis of nucleotide exchange by activated 7TM receptors is a two-step process. First, receptors bind GDPbound heterotrimers and cause nucleotide release. The receptor-G-protein complex then sits in this nucleotide-
phosducin
Under conditions of bright light, phosducin regulates sensitivity of the visual system by sequestering GPy in a tight bimolecular complex and releasing G& from the retinal rods’ internal disk membrane into the cytosol [32]. Since photoactivated rhodopsin cannot catalyze nucleotide exchange on Ga in the absence of GPy, the signal is essentially stopped at the G-protein level. Under low light conditions the phosducin-Gpy complex is destabilized by protein kinase A phosphorylation of phosducin, releasing GPy and restoring sensitivity [4’]. Although phosducin is best known for its role in vision, it is likely to regulate other systems as well since phosducin is present in many tissues, and indiscriminately binds a variety of GPy subunits [33,34]. Phosducin has two domains, each of which has a distinct role. Phosducin’s amino-terminal domain binds the ‘top’ face of GPy (the face that interacts with Ga’s switch regions). This domain competes with Ga, and prevents heterotrimer formation. The amino-terminal domain also
404
Protein
Figure
engineering
the switch regions of Ga, but does not appear to contribute catalytic residues to the hydrolytic machinery. Rather, RGS proteins seem to work by stabilizing conformational changes in Ga’s existing catalytic residues that are unique to the transition state. Most notable among these residues are the conserved, catalytically active, switch I arginine at position 178 and switch II glutamine at position 204 (Figure Ze). While the catalytic glutamine is stabilized both indirectly through RGS contacts to switch II, and directly by Asp128 from RGS4, there are no direct contacts between RGS4 and the catalytic arginine. Arg178 is stabilized primarily by a network of interactions whereby three of RGS4’s helices make contacts to switch I residue Thr182.
3
Structures of downstream heterotrimeric G proteins
Superposition of the two published crystal structures of heterotrimeric G-proteins. Gial &ys [16] is shown in grey. The complex of G&y, with the GtaIGia, chimera [16**] is shown in white. Structures were superimposed using only residues from the GP’s @-propeller. A flexible loop behind Ga’s amino-terminal helix (residues 127-l 36) was not included in the superposition. The largest structural difference is in the position of the coiled-coil. This superposition causes the bulk of the two Ga subunits to be rotated with respect to each other about an axis running roughly parallel to Ga’s amino-terminal helix. Ga’s switch II and helix aN regions are not rotated, suggesting that they are more tightly coupled to GPy than they are to the remainder of Ga. One can then imagine that if the receptor moves the bulk of Ga relative to GPy, these tightly bound regions might stay coupled to Gfly, opening a cleft betwwen the switch regions and the remainder of Gaand thereby facillitating nucleotide
release.
the exposed phosphorylation site, Ser73, that regulates phosducin’s affinity for GPy [3.5]. Phosducin’s carboxy-terminal domain is responsible for dissociating GPy from the cell membrane. This domain is electrostatically negative, and binds the positively charged patch on GPy, thereby preventing Gpy’s favorable membrane interaction (Figure 2~). Truncation mutants of phosducin that lack the carboxy-terminal domain have been shown to bind GPy without causing membrane dissociation [36]. contains
Structure of Ga with its regulator
RGS
The structure of Gial.GDP.AlF4has been solved in complex with its regulator, RGS4 [37**]. RGS proteins generally bind more tightly to GDPeAlFd--bound Ga (the transition state analog complex) than they do to Ga subunits in their GTP- or GDP-bound states [2’,3,38], hence they appear to accelerate Ga’s rate of GTP hydrolysis by stabilizing the transition state. RGS4 binds
effecters
of
Free GPy subunits, and activated, GTP-bound Ga subunits interact with a variety of downstream effecters. Most notable among the Ga targets are adenylyl cyclases and cGMP phosphodiesterase. The structure of a catalytically inactive fragment of an adenylyl cyclase has been solved [39’], and combined with binding site data derived from Ga and cyclase mutants [13,40], leads to an emerging structural model of the Ga-cyclase complex. Downstream molecules that interact with GPy include phospholipase Cp, adenylyl cyclases, Na+ and K+ ion channels, and a variety of tyrosine and serine/threonine kinases [41]. Many of the kinases that interact with GPy contain pleckstrin homology domains at or near the regions shown to bind GPy subunits [42], and it is likely that the mode of kinase-Gpy interaction is somewhat conserved among the various Gpy-regulated kinases. The Gpy-binding pleckstrin homology domain from the P-adrenergic receptor kinase has recently been solved by NMR (D Cowburn, personal communication).
The interaction of 7TM receptors with heterotrimeric G proteins Site directed mutagenesis, peptide binding, and proteolytic digestion studies of heterotrimeric G proteins and 7TM receptors have significantly improved our understanding of the interaction between these proteins. G-protein binding is mediated by the three carboxyterminal intracellular loops of the receptor [ZS], and the receptor makes contacts with all three subunits of the heterotrimeric G protein. Three regions of Ga contain residues that effect receptor interaction: Ga’s amino-terminal helix, exposed residues on helix a5 and strand p6, and Ga’s carboxy terminus [27”,29]. These receptor binding regions, along with Gy’s carboxy terminus [24,30], and the sixth or seventh WD repeats of GP [28], which have also been implicated in receptor binding, all map to the heterotrimer face proposed to interact with the membrane surface [16**,26]. This same surface also contains the site of pertussis toxin catalyzed ADP ribosylation of G proteins, a modification which decouples G proteins from 7TM receptors [43].
Structural aspects of heterotrlmrric
Mechanisms for receptor catalyzed nucleotide release There are two likely avenues for receptor catalyzed nucleotide release after GTP hydrolysis. Either GDP goes out guanine-side first, through a movement of the @--or5 loop, or it leaves phosphate-side first, through further conformational changes in the switch regions. Point mutations in the @-a5 region increase the rate of receptor-independent nucleotide exchange [44], suggesting that the nucleotide goes out guanine side first. Both a5 and p6 have been implicated in receptor binding, putting the receptor in relatively close proximity to guanine nucleotide [27**]. The alternative route, however, should also be considered. Whereas a5 and p6 are rigid structural elements with many contacts to the remainder of Ga, the switch regions are flexible, and easily subject to conformational change. Furthermore the crystal structures of the small G protein EF-Tu in complex with its exchange factor EF-Ts show the exchange factor acting near the phosphate, not the guanine ring [45*,46]. The key to 7TM-induced nucleotide exchange could be Ga’s hinge-like amino-terminal helix, which has been seen in a variety of conformations in the Gpy-free structures of Gial [9,37”]. The receptor’s interaction on both sides of Ga’s flexible amino-terminal region could induce a change in the heterotrimer whereby the bulk of Ga and GPy are displaced with respect to each other. If this were to happen, G& could draw the switch regions even farther from Ga, further opening the GTP binding cleft, and facilitating nucleotide release. This model is consistent with the requirement for GPy in receptor-mediated nucleotide exchange [47] and with an apparent flexibility in the heterotrimer suggested by a conformational difference seen between the two heterotrimeric crystal structures [16*,18]. When the P-propellers of the two heterotrimer structures are superimposed, their corresponding Ca atoms deviate by an rms difference of only -0.43A. This superposition causes Gta’s amino-tertiinal helix, switch II, and switch I regions to overlap quite well with their Gial counterparts (Figure 3). The remainder of Ga, however, is not in the same orientation in the two structures. It is rotated by -4.5’, suggesting a degree of flexibility. A rotation of this sort, if magnified by interaction with the receptor, could facilitate nucleotide release.
405
transmembrane helices, but higher resolution data are needed if we are to understand the mechanism by which 7TM receptors catalyze nucleotide exchange in a stimulus-dependent manner. The past year has seen significant advances in our ability to express and purify 7TM receptors [49,50]. Also, recent technical advances in cryoelectron microscopy have pushed the limits of this technique firmly into the realm of atomic resolution [Sl]. Based on these advances, as well as the advent of new methods for forming three-dimensional crystals of membrane proteins [SZ], it seems chat the first atomic resolution structure of a 7TM receptor might not be too far away.
Acknowledgements Thanks to John Sondek, Takemasa Kawasbimaand YongWang for helpful discussionsand to Steve Sprangfor providingcoordinaresof the GiailRGS4 complex. This work was iupp&ted- by a gr& from the National Insritute of Health to Paul B Sigler (GM22324); Rachelle Gaudet is supported by an National Science and Engineering Research Council of Canada 1967 fellowship.
References and reco-nded
reading
Papers of particular interest, published within the annual period of review, have been highlighted’as:
. of special inter& . . of outstanding intereet 1.
Gautam N, Stratbmann.ME Simon Ml: Dlvorsity of G-Protolns in slgnrl lranrductlon. Science’1 991, 262:602-806.
2. .
Berman DM, Kazasa T, Gilman AG: Thr GTPsw-rctlvstlng protein RGW stsbllkes the tnnsitlon state for nuclootlda hydrolysis I Biol Chem 1996, 271:27209-27212. This work shows that RGS4 can act catalyticallyas a GTPase-avtivating protein for Ga subunits.The authors also describe RGS4’e preference for GmGDPAlF4-, the transition-stateanalog. See also references 13,381. Describes the GTPase activatingactivityof RGS proteins, and the mechanism of their action. 3.
Watson N, Linder ME, Druey KM, Kehrl JH, Blumer Kl: RGS family membem: OfPass-actlvstlng protelnr for heterotrlmeric Gprotein a-subunits. Nature 1996, 383:172-l 76.
4. .
Willardson EM, Wilkins JF, Tateuro Y, Bitenaky MW: Regulation of phosducin phosphorylation in retinal rods by Ce*+/celmodullndependent edenylyl cyclerc Proc Nat/ Acad SC; USA 1996, 93:1476-1479. This paper proposes a light-dependent mechanism for phosducin’s regutation by PKA and phosphatase. In doing so, they explsin how phosducin regulates the light sensitivityof rod cells. 6.
Noel JP, Hamm HE, Sigler PB: The MA crystel structure of transducln-acomplexed with GTPyS. Narure 1993, 366:864-663.
6.
Lambright DG, Noel JP, Hamm HE, Sigler PB: Structural determinants for the rctlvrtlon of the a-subunit of I heterotrlmerlc G-protein. Nature 1994, 389:821 e826.
7.
Sondek J, LambrightDG, Noel JP, Hamm E, Sigler PB: GTPasa mechanism of G-proteins from the 1.7 f crystal structure of transducin a-GDPAlF,. Nature 1994, 372:276-279.
8.
Coleman DE, Berghuis AM, Lee E, tinder ME, Gilmsn AG, Sprang SR: Structures of ectlve conformations of Gia, end the mechanism of GTP hydrolysis. Science 1994, 265:1406-l 412.
9.
Mixon MB, Lee E, Coleman DE, Berghuis AM, Gilman AG, Sprang SR: Tertiary and quartemety structurel chrnges In Gia, induced by GTP hydrolysis. Science 1996, 270:054-080.
10. .
Berghuis AM, Lee E, Raw As, Oilman AC, Sprang SR: Structun of the GDP-PI complex of Gly203-1Alr Gia,: a mimlck of the
Conclusions The flurry of recent G-protein crystal structures have shown us, at atomic resolution, how GTP hydrolysis works, how heterotrimers associate and dissociate in response to nucleotide, and how Ga and GPy are regulated by other proteins. In contrast to the rapid progress on the G-protein front, however, there is still no high resolution picture of a 7TM receptor. Electron cryomicroscopy of two-dimensional crystals of frog rhodopsin has produced -6A resolution maps [48], the best view so far. These maps are sufficient to determine the orientation of the
G-protein signalllng Bohm, Gaudet and Sigler
486
Protein
engineering
tarnary product complex of Ga-catalyzed GTP hydrolysis. Structure 1996, 4:1277-l 290. This is the latest structure in the Ga series. It mimics the state of Ga just after GTP hydrolysis. Taken together with references 15-91 (the GTPyS, GDP-, end GDP.AIFd--bound states of Gta and Gia) this structure clarifies the GTP hydrolysis mechanism. 11.
Kjeldgsard M, Nyborg J, Clark BFC: The GTP binding motif: variations on a theme. FASEB J 1996, 10:1347-l 368. k extensive review of nucleotide binding by G proteins, incorporating work on heterotrimeric G proteins as well as small G proteins like Rss and EF-Tu.
tations, the authors present a model for receptor catalyzed nucleotide exchange. 28. .
Taylor JM, Jocob-Mosier GG, Lawton RG, VanDort M, Neubig RR: Receptor and membrane interaction sites on Op. J Biol Chem 1996, 271~3336-3339. The authors show crosslinking between a peptide derived from the B_adrenergic receptor and the sixth or seventh WD-40 repeats of GP. 29.
Hamm HE, Deretic D, Arendt A, Hargrave PA, Koenig B, Hofmann KP: Site of G protein binding to rhodopsin mapped with synthetic peptides from the a subunit Science 1988, 241~832-835.
30.
Gierschik P, Scheer A: S-prenylated cystain analogues inhibit receptor-mediated G protein activation in native human granulocyte and reconstituted retinal rod outer segment membranes. Biochemistry 1995, 34:4952-4961.
31.
Fung BK-K: Characterization of transducin from bovine retinal rod outer segments. J Biol Chem 1963, 268:10495-l 0502.
15. ..
Sondek J, Bohm A, Lambright DG, Hamm *HE, Sigler PB: Crystal structure of a G-protein By dimer at 2.1 A resolution. Nature 1996, 379:369-374. The structure of GP,yt in the absence of Ga. This paper contains a detailed map of contacts between GP and GY, and details of the structure formed by the WD-40 sequence motif.
32.
Yoshida T, Willardson BM, Wilkins JF, Jensen GJ, Thornton BD, Bitensky MW: The Phosphorylation stata of phosducin determines its ability to block transducin subunit interaction and inhibit transducin binding to activated rhodopsin. J Biol Chem 1994.269:24050-24057.
16. ..
Lambright DG, Songlek J, Bohm A, Skiba NP, Hamm HE, Sigler PB: The 2.OA crystal structure of a hetarotrimeric Gprotein. Nature 1996, 379:31 l-31 9. Structure of G/31yl complexed to a Gta/Gial chimera This paper details the interactions between Ga and Go. It also presents the model for Gapy’s interaction with the membrane.
33.
Denner S, Lohse MJ: Phosducin is a ubiquitious regulator of GpY subunits. Proc Nat/ Acad Sci USA 1996, 93:10145-l 0150.
34.
Muller S, Straub A, Schoder S, Bauer PH, Lohse MJ: Interactions of phosducin with defined G-protein by-subunits. J Biol Chem 1996, 271 :l1781-l 1786.
Gaudet R, Bohm A, Sigler PB: Crystal structure at 2.4 A resolution of the comolex of transducin Or and its reaulator. _ Phosductn. Cell 1996,’ 87:577-588. ’’ This structure explains how phosducin competes with Ga for Gby. Electrostatic calculations predict how G!3y binds the membrane in the &ence of Ga. and how phosducin disrupts this interaction.
35.
Lee RH, Brown BM, Lolley RN: Protein kinase A phosphorylates retinal phosducin on serine 73 in situ. J Biol Chem 1990, 26515860-l 5866.
36.
Tanaka H, Kuo C-H, Matsuda T, Fukada Y, Hayashi F, Ding Y, lrie Y, Miki N: MEKA/Phosducin attenuates hydrophobicitv of transducin bg subunits without binding to farnesyl moiety. Biochem Siophys Res Comm 1996, 223:567-591.
12.
Hilgenfeld R: How do the GTPases 1995, 2:3-6.
really work? Struct Biol
13.
Berlot CH, Bourne HR: Identification of effector-activating residues of Gsa. Cell 1992, 68:91 l-922.
14.
Skiba NP, Bae H, Hamm HE: Mapping of effector binding sites of transducin alpha-subunit using G-alpha t/G alpha il chimeras. J Biol Chem 1996. 271:413-424.
1 7. ..
10.
Well MA, Coleman DE, Lee E, Iniguez-Lluhi JA, Posner BA, Gilman AG, Sprang SR: The Structure of the G Protein heterotrimer GialPly2. Ce// 1995.83:1047-l 056.
19.
Kleuss C, Scherubl H, Hescheler J, Schultz G, Wittig B: Selectivity of signal tranrducion determined by ysubunits of hetarotrimeric G proteins. Science 1993, 259:832-834.
20.
Muller S, Hekmen M, Lohse MJ: Specific enhancement of padrenergic receptor kinase activity by defined G-protain p and y subunits. Proc Nat/ Acad Sci USA 1993,9&l 0439-l 0443.
21.
Murzin AG: Structural principles for the propeller assembly of P-sheets: the preference for seven-fold fymmetry. Proteins 1992, 14:191-201.
22.
Neer El, Schmidt CJ, Nsmbudripsd R, Smith TF: The ancient regulatory-protein family of WD-repeat proteins. Nature 1994, 3711297-300.
23.
Wedegaertner PB, Wilson PT, H Bourne HR: Lipid modifications of trimeric G-proteins. J Biol Chem 1995, 270:503-506.
24.
Kisselev OG, Ermolaeva MV, Gautsm N: A farnesylatad domain in the G protein y subunit is a specific determinant of receptor coupling. J Biol Chem 1994, 269:21399-21402.
25.
Hargrave PA, Hsmm HE, Hofmann KP: Interaction of rhodopsin with the G-protein, transducin. Bioessays 1993, 15:43-50.
26.
Lichtarge 0, Bourne HR, Cohen FE: Evolutionarily conserved GaPy binding surfaces support a model of the G proteinreceotor wmolex Proc Nat/ Acad Sci USA 1996, 93:750775ii.
37. ..
Tesmer JJ, Berman DM, Gilman AG, Sprang SR: Structure of RGS4 bound to AIF4-activated Gia,: stabilization of the transition state for GTP hydrolysis. Cell 1997, 69:251-261. Although only the RGS domain of RGS4 is seen, the structure explains how RGS proteins bind and accelerate GTPase activity of Ga subunits. 36.
39.
Zhang G, Lui Y, Ruoho AE, Hurley JH: Structure of the adenylyl cyclase catalytic core. Nature 1997, 386:247-253. ;he two cytoplasmic domains of adenylyl cyclase are homologous to each other. This structure of a catalytically inactive homodimer of one of the two cytoplssmic domains (llC2) presents a picture of the overall fold, but not the catalytic machinery of the ATP+cAMP reaction. 40.
Yan S-Z, Huang Z-H, Hurley JH, Tang W-J: Gsa binding site and activation of mammalian adenylyl cyclase. J Biol Chem 1997, in press.
41.
Claphsm D, Neer El: New roles for G-protein By-dimers in transmembrane signalling. Nature 1993, 365403-406.
42.
lnglese J, Koch W, Touhara K, Lefkowitz RJ: GpY interactions with PH domains and Ras-MAPK signalling pathways. Fends Biochem Sci 1995, 20:151-l 56.
43.
Van Dop C, Yamanaka G, Steinberg F, Sekura RD, Man&k CR, Stryer L, Bourne HR: ADP-ribosylation of transducin by pertussis toxin blocks the light-stimulated hydrolysis of GTP and cGMP in retinal photoreceptors. J Biol Chem 1984, 269:2326.
44.
Taroh I, Herzmerk P, Nakomoto JM, Van Dop C, Boume HR: Rapid GDP release from Gsa in patienfs with gain and loss of endocrine function. Nature 1994, 371 :164-l 68.
27. ..
Onrust R. Herzmark P, Chi P, Garcia PD, Lichtarge 0, Kingsley C, Boume HR: Receptor and py binding sites in the alpha subunit of the retinal G-protein transducin. Science 1997, . _^275:381-384. . This paper describes a vanety ot slte-dlrected mutants ot tia that disrupt interactions with Gpy and with 7TM receptors. On the basis of these mu-
Chen C-K, Wieland T, Simon Ml: RGS-r, a retinal specific RGS protein, binds an intermediate conformation of transducin and enhances recycling. Proc Nat/ Acad Sci USA 1996, 93:1288512889.
Structural
aspects
45.
Kawashima T. Berthet-Colominas C. Wulff M. &sack S. Leberman R: ‘The structure of the &reri&a co/i EFiTuEF-Ts complex at 2.5 A resolution. Nature 1996, 379:51 l-51 6. The structure of the small G protein EF-Tu in complex with its soluble exchange factor EF-Ts suggests a mechanism whereby nucleotide exchange is accelerated by disruption of of the Mga+ binding site.
.
46.
Wang Y, Jiang Y, Meyering-Vos M, Sprinzl M, Sigler PB: Crystal structure of the EF-TuEF-Ts complex from Thermus thermophilus. Nat Struct Bioll997, in press.
47
Birnbaumer L: G Proteins in signal transduction. Pharmacol Toxicol 1990, 30:675-705.
46.
Schertler GFX, Hargrave PA: Projection structure of frog rhodopsin in two crystal forms. Proc Nat/ Acad Sci USA 1995, 92:11576-l 1562.
Annu Rev
of heterotrimeric
G-protein
signalling
Bohm, Gaudet
and Sigler
407
49.
Reeves PJ, Thunond RL, Khorana HG: Structure and function in rhodopsin: high level expression of a synthetic bovine opsin gene and its mutants in stable mammalian call lines. Proc Nat/ Acad SC; USA 1996,93:11487-l 1492.
50.
Kiefer H, Krieger J, Olszewski JD, Von Heijne G, Prestwich GD, Breer H: Exoression of an olfactorv receotor in Escheric/ria co/i: purification; reconstitution, and ligand binding. Biochemistry 1996, 35:16077-l 6004.
51.
Dorset DL: Electron crystallography. 769.
52.
Landau EM, Rosenbusch JP: Lipidic cubic phases: a novel concept for the crystallization of membrane proteins. Proc Nat/ Acad Sci USA 1996, 93:14532-l 4535.
Acta Cryst B 1996, 52:753-