Minireview 357

Deciphering the alphabet of G : the structure of the ␣, ␤, ␥ heterotrimer Alfred Wittinghofer

The recent independent structure elucidations of two be a common form of –protein interaction, as it heterotrimeric G proteins represent a milestone in our has also been found in a complex between an immuno- understanding of the regulation of this important class globulin and immunoglobulin-binding domain [7,8]. The of signal switch molecules. The results show how the structure of the Rap-Raf complex also demonstrates that introduction of GTP into the heterotrimer produces two the biological data that have been gathered so far on Ras signalling molecules: the G␣-GTP and G␤,␥ subunits. function can be reconciled with the three-dimensional structure. It showed that residues which had been called Address: Max-Planck-Institut für molekulare Physiologie, effector residues even before the effector was known are Rheinlanddamm 201, 44139 Dortmund, Germany. indeed involved in the interaction with the RBD of Raf. Structure 15 April 1996, 4:357–361 A further accomplishment has been the structure deter- mination of Mss4, which is reported to be the GEF for a © Current Biology Ltd ISSN 0969-2126 Ras-related protein [9].

Recent structures in the field of GTP-binding proteins The ␣ subunits of G proteins It has been a very exciting year for those of us interested in Despite the success described above, the most remarkable the structure/function relationships of GTP-binding pro- progress over the last three years has occurred in the field teins. We have witnessed the appearance of a number of of heterotrimeric G proteins, which consist of ␣, ␤ remarkable structures that have provided explanations for and ␥ subunits. Two independent groups have solved many biochemical observations gathered over the years. various complexes of the ␣ subunits [10–14]. Now, with Some of these have been awaited for a long time, such as the recent structure elucidations of the heterotrimeric the complex of EF-Tu with aminoacyl-tRNA, for which complex, by both groups, we have seen a further develop- the first microcrystals were reported 22 years ago [1]. This ment in the unraveling of the molecular alphabet [15,16]. structure [2] portrays a molecular mimicry in which a protein–RNA complex or a protein alone interact with the G proteins are molecular switches that are used in the ribosome in a similar manner. During the elongation cycle transduction of a signal from the outside world into the of protein synthesis this achieves either codon dependent cell interior (Fig. 1) [17]. The signal is initiated by the delivery of aminoacyl-tRNA via EF-Tu or codon-indepen- binding of a ligand to a heptahelical receptor, so named dent translocation via EF-G. The structure of the complex because it traverses the membrane seven times as trans- between EF-Tu and EF-Ts was also long overdue, as it membrane helices. The N terminus is located outside and was also crystallized a number of years ago [3]. This struc- the C-terminal end inside the cell. It is estimated that ture [4] shows, for the first time, the interaction between a around 1000 different receptors exist (most of which are GTP-binding protein and its guanine-nucleotide-exchange involved in olfaction) and almost as many specific ligands. factor (GEF). The structure is remarkable both for the The binding of the ligand somehow activates the potential crystallographic solution itself, which was not at all straight- of the receptor to act as a GEF towards the heterotrimeric forward, but also for the message that it carries. It shows . The results from a number of site-directed that the GEF does not kick out the nucleotide allosteri- mutagenesis experiments, using peptides derived from cally, but rather pokes itself into the guanine-nucleo- both the receptor and the ␣ subunit, have indicated the tide- and messes up not only the Mg2+-binding main points of interaction between these two molecules. site, but also that of the base. These are the second and third cytoplasmic loops and the C-terminal end on the receptor and the N- and C-terminal Another interesting structure published within the last ends of ␣. This interaction increases the dissociation rate year came right on the heel of the discovery that the of the G␣-bound nucleotide, which otherwise is extremely downstream target or effector molecule of the proto- slow. Once GDP has been released from the complex of oncogene product Ras is the c-Raf [5]. The activated receptor and , the structure of the complex between Rap1A, a Ras-homo- empty nucleotide-binding site on ␣ can rapidly bind any logous protein, and the Ras-binding domain (RBD) of Raf, guanine nucleotide that happens to be around. However, was the first structure between a GTP-binding protein only the binding of GTP, which is more prevalent in the and its effector to be determined [6]. It showed how the cell anyway, has drastic consequences for the tetrameric antiparallel ␤ strand of the G domain of Ras, located at the (or maybe even pentameric, as the ligand remains bound edge of the molecule, is used to create an apparent inter- to the activated receptor) complex: the heterotrimeric protein ␤ sheet between the two proteins. This appears to G protein is released from the receptor and splits into 358 Structure 1996, Vol 4 No 4

Figure 1

The switch cycle of heterotrimeric G proteins. The amount of signal produced from this α active system is proportional to the GDP G -GDP receptor inactive receptor =GEF dissociation rate kdiss and inversely Gβγ proportional to kcat, the rate of GTP cleavage. kdiss Association of GTP with the complex between GDP activated receptor and ‘empty’ G protein is hormone not rate limiting. + H+Pi βγ α kcat G G

HO2 GTP G βγ + Gα–GTP

Downstream effectors

the G␣-GTP subunit and the ␤,␥ subunit complexes. Tu and G, which have 2 or 4 additional domains, respec- Although the exact order of these events is not clear, the tively [22–26]. The other domain of G␣ is termed the ␤,␥ subunit always stays together and cannot be separated helical domain, because of its secondary structure ele- except under denaturing conditions. ments. It is inserted into the G domain in the area that in Ras is called the effector region. For a few GTP-binding A number of years ago it was thought that the ␣ subunits proteins, the difference between the GDP-bound and alone, in the GTP-bound form, interact with downstream GTP-bound conformation has also been elucidated. targets. These targets include adenylate cyclase and phos- Although the trigger for the conformational change in all phodiesterase ( that produce or destroy second of these is the release of two main-chain interactions messenger molecules) and K+- and Ca2+-specific ion chan- between the ␥-phosphate and the so-called switch I and nels. Now it is clear that the other part of the signal-trans- switch II regions ([27]; Fig. 2), the details of these changes ducing molecule, the ␤,␥ subunit, is also a mediator of a have never been seen as clearly as in the G␣ proteins hormonal signal as it can activate similar effector mol- [10–13]. Furthermore, these structures showed that, in ecules. The whole signaling reaction is terminated, as far addition to switch I and II, G␣ proteins have a third as the G protein is concerned, by the GTPase reaction of region, switch III, that also changes conformation on ␣. This restores the starting situation as, following GTP exchange of nucleotide. Another surprise came when both − hydrolysis, G␣ GDP rebinds the ␤,␥ subunit and remains groups solved the structure of AlF4 (which was anticipated inactive, waiting for another activated receptor molecule to mimic the ␥-phosphate group) bound to G␣-GDP ⋅ to come along and kick out the nucleotide. [12,14]. The structures showed that the bound GDP AlF4 mimics the transition state in the GTPase reaction and A lot has been found out in the last three years about the therefore gave many clues as to the mechanism of this structure of the ␣ subunit. The first structures to be much-disputed reaction [12,14, 28–30]. solved were those of the ␣ subunit of transducin (G␣t) in the active GTP-analogue and the inactive GDP-bound The ␤,␥ subunit states. This was carried out in a collaboration between the Now, the same two labs have published the structure of Heidi Hamm and Paul Sigler laboratories [10,11]. Some- the complete heterotrimeric G protein [15,16], and in time later, the structures of active and inactive conforma- addition the Sigler/Hamm group has determined the tions of Gi␣1 were solved by the Stephen Sprang/Alfred structure of ␤,␥ alone [31]. The results for the ␤,␥ subunit Gilman collaboration [12,13]. These structures [10–13] are surprising. It forms a propeller-like structure in which showed that the ␣ subunits contain two domains. One was the blades (seven, in this case) of the propeller are formed termed the Ras-like G domain and is responsible for by four-stranded ␤ sheets that are arranged around a taper- binding nucleotides. This domain is very similar to that ing water-filled central shaft. Such a propeller structure found in the small GTP-binding proteins of the Ras has been found in enzymes such as neuramidinase, galac- superfamily such as Ras [18,19], Arf [20], [21], and tose oxidase and methanol dehydrogenase. The repeating also to the G domain of the polypeptide elongation factors nature of the G␤ sequence had been recognized by Minireview Heterotrimeric G proteins Alfred Wittinghofer 359

Figure 2

Schematic view of the structure of a N heterotrimeric G protein and of its interaction with the membrane and membrane-bound 123 heptahelical receptor, as inferred from the three-dimensional structures [15,16]. Regions extracellular loops in G␣ and the receptor thought to be involved in G protein activation are hatched. heptahelical receptor MEMBRANE

myristoyl farnesyl

cytoplasmic loops N 1232 3 C C C

bβ

G domain

shaft switch I,II

γ GDP

Helical domain

N

N

Neer et al. [32] as the so-called WD40 repeat (so named would be unlikely to lead to a properly folded stable because the repeating core unit contains a conserved WD protein. A similar situation might hold for other repeating sequence motif, in addition to a conserved GH motif, and motifs such the armadillo which is a motif found with a is approximately 40 residues in length), which is found in variable number of repeats in a number of proteins [34]. a number of regulatory proteins in higher eucaryotes [32,33]. The number of repeats varies between these reg- The ␥-subunit is an extended molecule, the only intrachain ulatory proteins, and also between the propeller proteins hydrogen bonds resulting in the formation of two helices. whose structures have so far been solved. It had been sug- The N-terminal helix of ␥ forms a coiled coil with the gested that the WD40 motif forms a small three-stranded N-terminal end of the ␤ subunit which, again, had been ␤ sheet which may not be stable by itself, but is stabilized predicted [35,36]. The way the ␥ subunit is folded (or not by interaction with other WD40 repeats, possibly in a folded) and how it forms extensive hydrophobic inter- closed circular structure [32]. Although it is remarkable actions with ␤ explains the finding that ␥ cannot be isolated how close the prediction came, the structure shows that in the absence of ␤ and that ␤ is only moderately stable the order of ␤ sheets within one blade of the propeller in the absence of ␥ [37]. Knowing the structure of the does not coincide with the order predicted from the con- ␤ subunit, it might be interesting to express the propeller sensus WD40 repeat. That is, the last (outermost) ␤ strand structure of the ␤ subunit alone, leaving out the N-terminal of each antiparallel sheet (blade) is the first element of the and C-terminal ends, to see whether it forms a stable sequence repeat, whereas the rest of the sequence within domain and whether it can interact with the ␣ subunit. one repeat actually specifies the three inner strands of the next blade of the propeller. It seems that this rearrange- The heterotrimer ment ensures the stability of the circular structure. This The structures of the heterotrimeric complex as deter- structure also explains why the expression of a single mined by both groups are rather similar. Due to their WD40 repeat as defined by the sequence comparison structure determination of the ␤,␥ subunit alone, the 360 Structure 1996, Vol 4 No 4

Sigler/Hamm group can draw the conclusion that its struc- whereas for the switch interface it is 1800 Å2 [16]. Thus, ture does not change on binding to G␣-GDP, the rms the latter is probably more important, although correla- deviation between free and bound ␤,␥ being 0.72 Å. It tions between size of interface and strength of interaction thus seems to behave as a rigid unit that is released when are difficult to determine. The ␥ subunit does not ␣ binds GTP and rebinds back onto ␣ when the GTPase contribute to the interface between ␣ and ␤,␥. timer has run out. The interaction between ␣ and ␤,␥ occurs via two distinct interfaces on ␣. One of these is the Receptor interaction and activation switch II region, which changes conformation between The ␣ subunits of many G␣ proteins are N-terminally GDP- and GTP-bound state, and the other is the N-termi- myristoylated and most are reversibly palmitoylated on nal region, the conformation of which is probably indepen- cysteine 3. The ␥ subunits contain a CaaX box at the dent of the nature of the nucleotide. The switch I region C terminus which is the signal for either farnesylation is the connecting linker between the ␣-helical domain and (a C15 thioether bond to cysteine) or geranylgeranylation the second ␤ strand of the G domain and is only margin- (C20), depending on the nature of residue X. These post- ally involved in the interaction between the subunits. In translational modifications are responsible for membrane Ras this is the so-called effector region, which makes attachment of the heterotrimer. Although the lipophilic GTP-dependent contact to the effector protein Raf. moieties are missing from the recombinant G␣ proteins, the results (especially of the Lambright et al. paper [16]) The conformational change in the Switch II region of ␣ suggest that N- and C-terminal ends are on the same face has been mapped very precisely and occurs in the region of the heterotrimer, as only a few residues are missing that makes most of the contacts with ␤. The Hamm/Sigler from the respective ends. This allows conclusions to be structure gives much more detailed information on the drawn about membrane attachment and receptor inter- nature of the interface and the residues involved (Fig. 2), action. This side is remarkably flat and its electrostatic probably due to the higher resolution and quality of the surface is predominantly neutral with some dispersed pos- crystals used. The two reports differ in the extent to itive charges; these properties seem to predispose it as the which the ␣ subunit in the heterotrimer resembles the membrane-interacting surface [16]. This is in line with a free ␣ in the GDP-bound conformation, partly because number of studies, using ADP-ribosylation by pertussis the N-terminal helix of Gt␣ [11] and the switch II region toxin and peptide inhibition, which implicate the N and of Gi␣1 [13] are not well defined. However, both conclude C termini and residues 311–329 of ␣ as important for the that the tightly packed interface between ␣ and ␤ is interaction with the receptor [17,38]. mostly formed by a number of hydrophobic side chains (from helix ␣2 of the ␣ subunit and the top of the ␤ pro- It has been argued that nucleotide release is caused by peller) and that the conformation of ␣ in the heterotrimer activated receptor inducing the formation of a cleft could not feasibly occur in the GTP-bound state. As the between the G and helical domain of the ␣ subunit, conformational change on GTP binding is known in great thereby releasing the nucleotide [10]. This speculation detail, we now have an explanation at the atomic level of rested on the assumption that G␣ proteins, in contrast to how GTP binding disrupts the heterotrimer and releases Ras-like proteins, always need activated receptors for G␣-GTP and ␤,␥ to do their respective signalling jobs. exchange to happen, which is not true for all G␣ proteins. However, spontaneous release of nucleotide from some The second interface between ␣ and ␤,␥ is formed G␣ proteins is just as fast as that of Ras-like proteins, between the N terminus of ␣ and the first blade of the which have no helical domain. It has also been speculated propeller, assigning a function to the N terminus of ␣ that that the receptor induces GDP release by promoting is in line with previous observations. In the structure subunit dissociation [15]. This might be difficult for the determinations of Gt␣ by the Hamm/Sigler group, N-ter- receptor to achieve given the large interface between the minally truncated protein was used because full-length ␣ and ␤ subunits. Furthermore, it appears from the struc- protein would not form crystals. In the structure of ture that subunit dissociation is a result of GTP binding to heterotrimeric transducin, a Gt␣/Gi␣1 chimera was used in the G␣ proteins and not due to direct association with the which residues 216–294 of Gt␣ were replaced by residues receptor. Provided the site of interaction between receptor 220–298 from Gi␣1, for the simple reason that the chimeric and the heterotrimer is as anticipated from the location of transducin can be expressed in Escherichia coli whereas the post-translational modifications and the electrostatic transducin proper cannot. In Gi␣1–GDP, the N terminus surface calculation, then it appears that the receptor acts at had been found to make a mini-domain with the C termi- a site distant from the nucleotide-binding site on ␣. This nus. This could potentially be due to crystal packing as it would mean that the mechanism for increasing nucleotide- ⋅ was not seen in heterotrimeric structure of either Gi␣1 or dissociation rate is different from the EF-Tu EF-Ts situa- transducin. Alternatively, it could indicate a large confor- tion in which EF-Ts is found to interfere directly with mational change on GDP binding. The area of the inter- nucleotide binding. Therefore, in order to understand the face between ␤,␥ and the N terminus of ␣ is 900 Å2, mechanism of activation of heterotrimeric G proteins it Minireview Heterotrimeric G proteins Alfred Wittinghofer 361

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