- Email: [email protected]
GEFs, GAPs, GDIs and effectors: taking a closer (3D) look at the regulation of Ras-related GTP-binding proteins
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GEFs, GAPs, GDIs and effectors: taking a closer (3D) look at the regulation of Ras-related GTP-binding proteins Matthias Geyer* and Alfred Wittinghofert Cell biology depends on the interactions of macromolecules, such as protein-DNA, protein-protein or protein-nucleotide interactions. GTP-binding proteins are no exception to the rule. They regulate cellular processes as diverse as protein biosynthesis and intracellular membrane trafficking. Recently, a large number of genes encoding GTP-binding proteins and the proteins that interact with these molecular switches have been cloned and expressed. The 3D structures of some of these have also been elucidated.
Addresses *Max-Planck-lnstitut fLir medizinische Forschung, Abteilung Biophysik, JahnstraBe 29, 69120 Heidelberg, Germany; e-mail: [email protected] tMax-Planck-lnstitut f~ir rnolekulare Physiotogie, Abteilung Strukturelle Biologic, Rheinlanddarnm 201, Postfach 10 26 64 44139, Dortmund, Germany; e-maih [email protected] Current Opinion in Structural Biology 1997, 7:786-792
http://biomednet.corn/elecref/O959440XO0700786 © Current Biology Ltd ISSN 0959-440X Abbreviations FTase GAP GDF GDI GEF NTF2 PTase RBD rmsd
farnesyl transferase GTPase-activating protein GDI dissociation factor GDP-dissociation inhibitor guanine nucleotide exchange factor nuclear transport factor 2 prenyl transferase Ras-binding domain root mean square deviation
Introduction A mammalian cell contains an estimated 50-100 different GTP-binding proteins, which function as molecular switches involved in regulating many different biological processes ranging from protein biosynthesis and membrane trafficking to communicating signals from the outside of the cell to its interior. The Ras superfamily of GTP-binding proteins are small, 20-25 kDa proteins, which bind guanine nucleotides very tightly and cycle between a presumed inactive GDP-bound and an active GTP-bound state [1,2]. GTP-binding proteins contain a set of five conserved sequence elements by which they can be easily identified as being a member of this class of proteins. These elements are necessary for guanine nucleotide and Mg 2+ binding, for GTPase activity and for the conformational switch. In addition, almost all Ras-related proteins contain a C-terminal cysteine-containing motif such as the CXC or the CAAX ('CAAX box' using single-letter amino acid code, where X represents any amino acid) motifs which
serve as attachment points for one or two prenyl groups such as farnesyl or geranylgeranyl. This post-translational modification is necessary for membrane insertion and biological activity. The enzymes required for post-translational modifications, such as farnesyl transfcrase, are therefore important regulators of functions. On the basis of sequence similarities, the superfamily can be subdivided further into the Ras, Rho/Rac, Rab, Ran, Rad and Arf subfamilies, and these subfamilies can be correlated very well with the different biological functions of their members [3]. Atf is myristoylated at the N terminus and Ran is not at all post-translationally modified. Three-dimensional structures of GTP-binding proteins and some of their regulators and effectors have been elucidated recently. The implications for the molecular mechanisms of their action are the focus of this review. The GTPase
regulation
cycle
The Ras-rclated proteins bind guanine nucleotidcs very tightly--with binding constants of up to 1011 M - l - a n d the dissociation rate constants for bound nucleotides are very low. Guanine nucleotide exchange factors (GEFs) increase the rate of nucleotide dissociation by several orders of magnitude and thus facilitate loading with GTP. During the process, protein-bound GDP is released and GTP, which is more abundant in the cell than GDP, binds instead. In the GTP-bound state, Ras-related proteins interact with so-called downstream targets or effectors, which in turn communicate with other partners located further downstream in the signalling cascade. Effectors are defined as proteins that interact much more tightly with the GTP-bound form than with the GDP-bound form of the Ras-related proteins. This interaction is terminated by hydrolysis of protein-bound GTP to GDP, which restores the GDP-bound form and terminates interaction with the effector. The intrinsic GTPase reaction is usually very slow (the half life is in the order of minutes to hours), but it can be stimulated by several orders of magnitude (half life milliseconds to seconds) by GTPase-activating proteins (GAPs) [4]. The schematic GTPase cycle is shown in Figure 1. Before GTP-binding proteins can enter the GTPasc cycle, their localisation at the cell membrane is facilitated by post-translational prenylation. This is mediated by prenyl transferases (PTases) as shown in Figure 1. Certain Ras-related proteins such as Rab and Rho/Rac are additionally regulated by guanine nucleotide dissociation inhibitors (GDIs). GDIs have been found to inhibit GDP dissociation; their true function is to extract the membrane-inserted, post-translationally modified protein from the membrane by binding to the prenylated C-ter-
GEFs, GAPs, GDIs and effectors Geyer and Wittinghofer
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Figure 1
GDF
P~
inactive
~
GTP
active
GDP
signalling & transport pathway CurrentOpinionin StructuralBiology Function and regulation of Ras-related GTP-binding proteins (Rax). Except for Ran, which is unmodified, all superfamily members undergo prenylation, which is catalysed by different forms of PTases. The isoprenylated proteins bind to GDI, which keeps the hydrophobic tail protected from solvent and is considered to be a transport factor. Interaction with GDF promotes membrane association via the isoprenyl and other hydrophobic moieties. For their function, Rax proteins cycle between an inactive, GDP-bound and an active, GTP-bound form. Conversion between these two forms is via guanine nucleotide dissociation catalysed by GEFs and via GTP hydrolysis catalysed by GAPs. Ran is not prenylated and inserted into membranes but rather cycles between the cytoplasm and the nucleus and regulates both nuclear import and export.
minal end and to serve as a cytosolic pool for Rab and Rho/Rac. T h e dissociation of the complex between G D I and the GTP-binding protein is catalysed by a protein named G D I dissociation factor (GDF), which has recently been identified for the Rab cycle [5].
Protein structures of the Ras superfamily T h e first 3D structure of a small GTP-binding protein in the Ras superfamily to be solved was that of Ras itself. It was solved in both the active triphosphate-bound conformation and the inactive diphosphate-bound conformation I6-8]. T h e structure, consisting of six 13-strands and five ot helices, led to the prediction that all GTP-binding domains have a common topology, known as the 'G domain'. By comparison of the two states, two highly flexible regions surrounding the y-phosphate have been established: the switch I region, from residues 30-37 within loop L2 and 132 (the 'effector region'); and the switch II region, from residues 60-76 within loop L4 and helix a2.
T h e crystal structures of human and rat ADP-ribosylation factor-1 (Arfl) complexed to GDP [9,10] showed the fold for a member of another subfamily. Irrespective of the presence of an additional N-terminal cx helix and a difference in Mg e+ coordination, these structures confirmed the idea of a common G domain topology. T h e structure of the nuclear import protein Ran in its GDP-bound form also shows the same overall fold, with differences in the conformation of the effector loop and in some aspects of the nucleotide-binding site [11]. Remarkably, both the structures of Arfl and Ran have a common feature, which is an additional 13 strand in the effector loop located antiparallel to strand 132, that has not been found in other Ras-related GTP-binding proteins. T h e structure of human Racl, a member of the Rho/Rac subfamily, has been solved in its active conformation using X-ray crystallography [12"]. Racl, with an overall sequence identity of 30% to Ras, has a 13-residue insertion between
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Catalysis and regulation
[35 and c~4 (residues 123-135) which forms an additional o~ helix. Excluding the insertion, the main-chain atoms of both molecules have an rmsd deviation of 1.2 ~. As in some other crystal structures of GTP-binding proteins, part of the effector loop, including the region of the magnesium ion binding by Thr35, is poorly defined in the electron density map. The switch II region, which is usually one of the most flexible elements in GTP-binding proteins, also differs between Ras and R a c l - - t h e latter of which has two short 310 helices after the DTAGQ "/-phosphate binding motif. Switch II is again different in the structures of two other members of the Rho family, Cdc42.GDP [13] and RhoA-GDP [14]. In Cdc42, no evidence has been found for an c~ helix in the switch II region, whereas RhoA has a regular a helix. The structures of the five major GTPase subfamilies are completed, with the crystal structure of Rab7 which has been recently announced [15] and is reported to be very similar to that of Ras (P MetcalE personal communication).
Dynamic aspects of the structure of GTP-binding proteins have been observed in Ras and its isoforms using NMR spectroscopy [16-18] and electron paramagnetic resonance spectroscopy [16,19]. A two-state model for the movement of the effector loop in triphosphate-bound Ras has been established; the flexibility of the loop can conveniently be monitored by a large shift of Tyr32 relative to the phosphate groups [16]. Binding of the effector kinase c-Raf-1 or the GTPase-activating protein (GAP) stabilizes the effector loop in either of the two conformations. The conformational transition in the uncomplexed protein demands an activation enthalpy of 90 kJ mo1-1. Whereas both conformations are almost equally populated in the GppNHp-bound form, one state predominates in the GTP-bound form. This internal fexibility is observed also for N-Ras-GppNHp, whereas it seems to be reduced for the GTPTS-bound form [17]. The conformational flexibihy is confirmed by a detailed NMR study on 13C-/15N-labelled protein bound to different nucleotides that shows the combination of three flexible regions in the triphosphate bound form, L1 L2 and L4, termed a regional polysterism [18].
The conformational changes of the effector loop in solution arc extended by the crystal structure of Rap2A, a homologue to Ras, which is the first structure of a small GTP-binding protein with its natural ligand GTP [20] besides the structures of wildtype and mutant Ras that have been obtained from Ras.caged GTP after photolysis in the crystal [21]. It shows that the hydroxyl group of Tyr32 forms hydrogen bonds with the y-phosphate of GTP and with Glyl3, which are not present either in Ras or in the Rap2A.GTPyS complex. A comparable change has been reported for elongation factor-Tu, which undergoes a major conformational transition in the effector loop upon GTP hydrolysis [22].
GEFs Guanine nucleotide exchange factors for the Ras, Rho/Rac, Ran, Arf subfamilies have been cloned together with mammalian suppressor of Sec4 (Mss4)--a presumed GEF for Rab proteins [3]. Mss4 is not a bona fide GEE as it has a very low activity and acts only stoichiometrically by forming a complex with nucleotide free Rab thereby replacing bound nucleotide [23], whereas other GEFs have been shown to act catalytically by increasing the rate of equilibrium formation between the GDP- and GTP-bound states [24]. The structure of human Mss4, a Rab isoform, has been determined using NMR spectroscopy [25°]. The 14kDa protein can be described as a three-layered [3-protein, with a central seven-stranded antiparallel sheet flanked by a small three-stranded [3 sheet on one face and a [3 hairpin on the other. Mss4 also binds a Zn2+ ion, which is tetrahedrally coordinated by two CxxC motifs arranged in the so-called rubredoxin 'knuckle' structure. The Zn2+-binding region and a neighboring loop define the proposed active site of the protein. The Rab-interacting surface of Mss4 has been determined using NMR exchange experiments with a nucleotidc-free form of Sec4. The ZnY+-binding motif of Mss4, whether or not it is required for its GEF activity; is not found in other GEFs such as RCC1, Sos or Cdc25 [3].
Effectors Ras is the only small GTP-binding protein for which detailed structural information is available concerning its interaction with cffectors. The serine/threonine kinasc c-Raf-1 is a downstream target in the signalling cascade of Ras. Its Ras-binding domain (RBD) binds with high affinity (KD =20nM) to Ras [26] and with somewhat lower affinity to R a p l A - - a Ras-like protein. The structure of Raf-RBD alone has been solved using NMR spectroscopy [27], and the complex of RaplA with a GTP analogue has been solved using X-ray crystallography [28]. A detailed analysis has been carried out for the binding interface and its specificity, which turns out to be crucial for a molecular understanding of signalling pathways [29,30].
The cysteine-rich domain of c-Raf-1, residues 139-184, has been proposed as a second Ras-binding site [31]. Its structure determination using NMR spectroscopy has shown it to have a double ZnZ+-binding motif [32"]. The structure is slightly different from that of two related cysteine-rich domains from protein kinase C which correlates with its inability to bind diacylglycerol and phorbol esters. Another Ras-binding domain (RBD) that has been solved recently using both NMR spectroscopy and X-ray crystallography is the RBD of GEF RalGEF [33",34"]. RalGEF is an effector molecule for Ras and has GEF activiry for R a l - - a n o t h e r GTP-binding protein of the Ras subfamily. As Ral interacts with its effector RalBP1, which is a GAP
GEFs, GAPs, GDIs and effectors Geyer and Wittinghofer
for Cdc42/Rac, RalGEF has been proposed to couple the Ras to the Cdc42/Rac pathway. The overall structure of the RBD of RalGEF adopts the same ubiquitin superfold as the RBD of Raf, although the two domains have only 13% sequence identity. NMR titration experiments with Ras suggest that complex formation occurs via formation of an intermolecular 13sheet [33"], similar to that observed for the RBD of Raf and RaplA. The same global structure is also adopted by Rgl, a protein with 55% sequence identity to RalGEF (S Yokoyama, personal communication), and by Rlf, another RalGEF like factor with 30% sequence identity (D Esser, P Bayer, personal communication). GAPs As with GEFs, a large number of GTPase-activating proteins (GAPs) have been identified, and each subfamily of Ras-related proteins, such as Ras, Rac/Rho, Rab and Aft, have their own version of a specific GAP with its own conserved sequence elements [3]. Rap, a member of the Ras subfamily with four isoforms closely related to Ras, is noteworthy, as it is the only Ras-related protein that does not have an otherwise invariant glutamine in switch II region that is involved in GTP hydrolysis. Correspondingly, it has its own type of Rap-specific GAP (Rap GAP) which does not react with Ras, just as RasGAP does react with Rap proteins [35].
Five mammalian RasGAPs have been cloned, two of which - - pl 20-GAP and neurofibromin--have been extensively characterized [36]. Recently, the structure of the catalytic domain of pl20-GAP was solved [37°]. It is a purely helical protein which contains a shallow groove that has been identified as the active site because it contains most of the conserved residues and Ras can be modelled to fit into the groove, using biochemical and mutagenesis data that had been accumulated over the years. The structures of two members of the RhoGAP family; the catalytic domain of the p85 regulatory subunit of phosphoinositide 3-kinasc [38 °] and from p50Rho-GAP [39*], have also been determined recently. As expected, these two structures are very similar; however, they are not homologous to the RasGAP structure even though they are both purely ot helical. The mechanism of the GAP-mediated GTP hydrolysis on Ras has been a matter of considerable debate in recent years and has centered around the issue of whether Ras itself is an efficient GTPase device and GAP acts catalytically to push it into the active state or whether GAP actively participates in the reaction to make it efficient [40,41]. Evidence for the latter option came from experiments with aluminium fluoride, which binds in the y-phosphate binding site of most ATP/GTP-utilizing phosphoryl transfer enzymes where it mimics the transitions state of the reaction, as has been most clearly shown for the cc subunits of heterotrimeric G proteins [42,43]. Contrary to results on Gc~ proteins, Ras itself does not bind aluminium fluoride but rather
789
needs stoichiomctric amounts of RasGAPs to do so [44], implying that the catalytic centre of Ras is incomplete unless GAP is present. This was revealed by the 3D structure of the complex between RasGDP and the catalytic domain of pl20-GAP crystallized in the presence of aluminium fluoride. The structure shows that GAP supplies an arginine finger to the active site of Ras, which apparently stabilizes negative charges in the transition state of the GTPase reaction via its guanidinium sidechain believed to proceed by a mostly associative mechanism [45"]. The structure also shows that Gin61 of Ras, which in uncomplexed Ras is very mobile, becomes fixed by the presence of GAP and also participates in the stabilization of the transition state and also explains a structural recluirement for a glycine in position 12 for GAP catalysis to work. The 3D structure of the complex between Cdc42 in the GTP-bound state and the catalytic domain of p50Rho-GAP has also been solved recently [46"']. As in the Ras-RasGAP complex, the switch regions of Cdc42 are mostly responsible for the interaction. As the Cdc42.RhoGAP complex represents the ground state of the GTPase reaction, it was interesting to find that the arginine which is also believed to be involved in the GTPase reaction in RhoGAP, is not contacting the y-phosphate, which implies that it may do so only in the transition state of the reaction, as has been found for Gic~l [43]. The transition state of the latter is also stabilized by its specific GAP called RGS (for regulator of G protein signalling) using similar contact sites on the G domain of this large (42 kDa) GTP-binding protein [47"]. GDIs Recently, the structures of two types of GDIs have been determined. Rab-GDI functions in vesicular transport to recycle and regulate Rab family GTPases; it forms a complex with the GDP-bound, prenylated form of Rab and can interact with and retrieve a broad range of Rab proteins from the membrane bilayer. The bovine o~-isoform of Rab-GDI is a 50kDa protein, and the structure of the intact protein has been elucidated [48"']. GDI is constructed of two main structural u n i t s - - a large complex multisheet domain with two major [3 sheets (five parallel strands and seven mostly antiparallel strands nearly perpendicular to each other) and a smaller o~-helical domain. This domain is closely related to flavin adenine nucleotide containing monooxygenases and oxidases. From homology studies and site-directed mutagenesis experiments, the two most sequence-conserved regions, which form a compact structure at the apex of GDI, are shown to play a critical role in the binding of Rab proteins.
Rho-GDI is a 204-residue protein, which binds the C-terminal isoprene moiety of Rho family members to form a complex that represents the cytoplasmic pool of these proteins [49]. The NMR and X-ray crystallography derived structure of Rho-GDI by two independent groups
790
Catalysis and regulation
[ 5 0 " , 5 1 " ] shows two distinct regions: a C-terminal folded domain of 16kDa which strongly binds Cdc42; and an unstructured N-terminal region which is necessary to inhibit nucleotide dissociation. T h e structure is different from that of Rab-GDI, which supports the notion that whereas the GTP-binding proteins from the different subfamilies are very similar, their regulators and effectors are unrelated. T h e folded domain of R h o - G D I shows a 13-sandwich motif of two largely antiparallel [3 sheets that pack against each other in a parallel manner with a narrow hydrophobic cleft which binds isoprenes. T h e binding properties of this cleft have been determined using N M R titration experiments with a farnesylated peptide of seven residues which mimics the isoprenylated C terminus of Rho-family GTPases [51"]. Similarly, the exposed surface that interacts with the protein portion of Cdc42 has been determined. T h e structural and biochemical data suggest a model for the Rho-GDI.Cdc42 complex in which the G T P a s c is on top of the C-terminal domain of the regulator, with its attached isoprene buried in the G D I hydrophobic pocket. This interaction anchors the inhibitory N-terminal peptide to the GTPase, allowing it to block the exchange of GDP, and also, probably, the binding of GAPs and GEFs. Other
proteins-FTase,
NTF2
For a complete description of the switch cycle of GTP-binding proteins (Figure 1), other proteins involved in the regulation and function need to be mentioned. T h e protein farnesyhransferasc (FTase), a heterodimer consisting of an o~ and 13 subunit, catalyses the C-terminal lipidation of Ras and Ras-like proteins using a subset of CAAX motifs, but also modifies other cellular proteins such as nuclear lamins and the ¥-subunit of the heterotrimeric G protein transducin. T h e transfer of the farnesyl group proceeds from farnesyl pyrophosphate onto the cysteine of the CAAX box to create a thioether linkage. In the case of Ras, farnesylation is necessary for further post-translational modification and biological function. T h e crystal structure of FTasc shows the 48 kDa subunit to be a crescent-shaped seven helical hairpin domain, and the 4 6 k D a 13 subunit to be an o~-ot barrel domain [52"]. T h e active site is formed by two clefts that intersect at a bound zinc ion. T h e structure solved is in fact a complex with a nine-residue peptidc which binds to one cleft and which may mimic the binding of the Ras substrate. T h e other cleft is a hydrophobic pocket at the centre of the a - a barrel, which is lined with highly conserved aromatic residues appropriate for binding the farnesyl isoprenoid group with required specificity. Interestingly, the fourth residue of the nonapeptide is a proline and corresponds to the position of the cysteine to be farnesylated in the CAAX protein. It is exactly in register with the zinc ion, adjacent to the presumed position of the farnesyl a-phosphate. An expansion on the conventional switch cycle comes from studies with Ran, which is a Ras-related nuclear
protein. Although the deletion or mutation of the genc has many pleiotropic effects, its major role appears to be the regulation of both nuclear import and export. As the G E F of Ran, RCC1, is in the nucleus and the Ran-GAP is in the cytosol, it may turn out that Ran-GDP controls import, whereas R a n . G T P regulates export. Another protein, nuclear transport factor 2 (NTF2), which facilitates protein transport into the nucleus, interacts only with Ran.GDP [53]. This indicates that the dogma that only the G T P - b o u n d form of GTP-binding protein can bind to an effector needs to be revised and may mean that, at least in the case of Ran, N T F 2 is the specific effector of Ran-GDP, T h e structure of N T F 2 , solved using X-ray crystallography, shows an cx+ 13 barrel that is open at one end to form a distinctive hydrophobic cavity which is a potential binding site for Ran [54"]. T h e structure of a complex between Ran.GDP and N T F 2 has also been solved and shows the switch region of Ran to be involved in the interaction, as is anticipated because the binding to Ran is specifically dependent on the presence of G D P (M Stewart, personal communication). Conclusions
A number of Ras-related proteins are now known at structural level. T h e structures show that this class proteins have a c o m m o n fold, the G domain, which is involved in the basic functions of guanine nucleotidc and Mg 2+ binding and the G T P a s c reaction. Very recently, a number of structures have been solved that allow us to take a first look at the regulation of these proteins such that we can now begin to have an understanding of how GAPs stimulate G T P hydrolysis, G D I s inhibit nucleotide dissociation and bind the isoprenylated C-terminal end, cffectors are designed to specifically interact with the active or, and in the case of Ran, the 'inactive' state of the molecule, and PTases catalyse C-terminal modification. T h e s e first glimpses have been very exciting and have made us curious for more insight into the function of the molecular switches, and their modes of regulation. Acknowledgements
We thank all colleagues for sending prcprints of their papers in press and for private communications.
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Mott HR, Carpenter JW, Zhong S, Ghosh S, Bell RM, Campbell SL: The solution structure of the Raf-1 cysteine-rich domain: a novel Ras and phospholipid binding site. Proc Nat/Acad Sci USA 1996, 93:8312-8317. The NMR structure of the cysteine-rich domain of c-Raf-1, which is considered to be involved in Ras binding and kinase activation. •
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Geyer M, Herrmann C, Wohlgemuth S, Wittinghofer A, Kalbitzer HR: Structure of the Ras-binding domain of Ral guaninenucleotide exchange factor: implications for Ras binding and signalling. Nat Struct Bio11997, 4:694-699. The structure of the Ras-binding domain of the Ras effector RaIGEF is solved using NMR spectroscopy and is found to be homologous to the RBD of c-Raf-1, another Ras effector. •
34.
Huang L, Weng X, Hofer F, Martin GS, Kim SH: Three dimensional structure of the Ras-interacting domain of RalGDS, a guanine nucleotide stimulator of the Ral protein. Nat Struct Biol 1997, 4:609-615. The X-ray crystallography structure of the Ras-binding domain of the Ras effector RalGEF, which is homologous to the RBD of c-Raf-1, another Ras effector. (See [33"]). •
35.
36.
Rubinfeld B, Munemitsu S, Clark R, Conroy L, Watt K, Crosier WJ, McCormick I=, Polakis P: Molecular cloning of a GTPase activating protein specific for the Krev-1 protein p21rap I. Cell 1991, 65:1033-1042. Ahmadian MR, Wiesm(Jller L, Lautwein A, Bischoff FR, Wittinghofer A: Structural differences in the minimal catalytic domains of the GTPase-activating Protein p21(GAP) and neurofibromin. J
B/o/Chem 1996, 271:16409-16415. 37.
Scheffzek K, Lautwein A, Kabsch W, Ahmadian MR, Wittinghofer • A: Crystal structure of the GTPase-activating domain of human p120GAP and implications for the interaction with Ras. Nature 1996, 384:591-596. This paper shows the structure of RasGAP with the conserved residues centered around a shallow groove and shows how Ras may bind to it. 38.
Musacchio A, Cantley LC, Harrison SC: Crystal structure of the breakpoint cluster region-homology domain from phosphoinositide 3-kinase p85(x subuniL Proc Nat/Acad Sci USA 1996, 93:14373-14378. This paper shows the structure of the RhoGAP domain of a subunit of PI(3) kinase, which is inactive as a RhoGAP. •
39. •
Barrett T, Xiao B, Dodson EJ, Dodson G, Ludbrook SB, Nurmahomed K, Gamblin SJ, Musacchio A, Smerdon SJ, Eccleston
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JF: The structure of the GTPase-acUvating domain from p5OrhoGAP. Nature 1997, 385:458-461. The authors show the helical nature of RhoGAP and suggest a binding site for Rho and residues of GAP involved in the activation. 40.
Maegley KA, Admiraal SJ, Herschlag D: Ras-catalyzed hydrolysis of GTP: a new perspective from model studies. Proc Nat/Acad Sci USA 1996, 93:8160-8166.
41.
Schweins T, Geyer M, Kalbitzer HR, Wittinghofer A, Warshel A: Linear free energy relationships in the intrinsic and GTPase activating protein-stimulated guanosine 5'-triphosphate hydrolysis of p21 ras. Biochemistry 1996, 35:14225-14231.
42.
Sondek J, Lambright DG, Noel JP, Hamm .HE, Sigler PB: GTPase mechanism of G proteins from the 1.7-A crystal structure of transducin ccGDP.AIF4-, Nature 1994, 372:276-279.
43.
Coleman DE, Berghuis AM, Lee E, Linder ME, Gilman AG, Sprang SR: Structures of active conformations of Gi~1 and the mechanism of GTP hydrolysis. Science 1994, 265:1405-1412.
44.
Mittal R, Ahmadian MR, Goody RS, Wittinghofer A: Formation af a transition-state analog of the Ras GTPase reaction by RasGDP, tetrafluoroaluminate, and GTPase-activaUng proteins. Science 1996, 273:115-117.
45. ••
Scheffzek K, Ahmadian MR, Kabsch W, Wiesm~ller L, Lautwein A, Schmitz F, Wittinghofer A: The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants. Science 1997, 277:333-338. The structure of the complex between Ras and RasGAP in the transition state shows how RasGAP catalyzes GTP hydrolysis on Ras, and why oncogenic Ras is insensitive to its activation. 46. **
Rittinger K, Walker PA, Eccleston JF, Nurmahomed K, Laue E, Owen D, Gamblin SJ, Smerdon SJ: Crystal structure of a small G protein in complex with the GTPase-activating protein rhoGAP. Nature 1997, 388:693-69Z The crystal structure of the complex between RhoGAP and Cdc42 in the ground GppNHp-bound state shows that they interact mainly via the switch regions of Cdc42. 4'7. ••
Tesmer JJG, Berman DM, Gilman AG, Sprang SR: Structure of RGS4 bound to AIF4--activated Gkxl: stabilization of the transition state for GTP hydrolysis. Ceil 1997, 89:251-261.
This 3D structure shows RGS, the G~ version of a GAP, stabilizes the transition state of GocGDP.AIF4-, such that the resulting structure is similar to the Ras.Ras.GAP complex. 48. **
Schalk I, Zeng K, Wu SK, Stura EA, Matteson J, Huang M, Tandon A, Wilson IA, Balch WE: Structure and mutational analysis of Rab GDP-dissociation inhibitor. Nature 1996, 381:42-48. The paper shows RabGDI to be a two-domain structure with three conserved sequence regions clustered at the tip of the molecule and explores the interaction with Rab using structure-based mutational analysis. 49.
Isomura M, Kikuchi A, Ohga N, Takai Y: Regulation of binding of rhoB p20 to membranes by its specific regulatory protein, GDP dissociation inhibitor. Oncogene 1991, 6:119-124.
50. ,*
Keep NH, Barnes M, Barsukov I, Badii R, Lian LY, Segal AW, Moody PCE, Roberts GCK: A modulator of rho family G proteins, rhoGDI, binds these G proteins via an immunoglobulin-like domain and a flexible N-terminal arm. Structure 1997, 5:623-633. A combination of crystal and NMR structure determinations show the mode of binding of RhoGDI to Rac (see [51°°]). 51. •*
Gosser YQ, Nomanbhoy TK, Aghazadeh B, Manor D, Combs C, Cerione RA, Rosen MK: C-terminal binding domain of Rho GDP-dissociation inhibitor directs N-terminal inhibitory peptide to GTPases. Nature 1997, 387:814-819. The NMR structure of RhoGDI, supported by fluorescence studies, shows how the protein binds to Cdc42 and inhibits its GDP dissociation via two different regions of the molecule and also shows the lipid-binding pocket. (See also [50"°]). 52. .*
Park H-W, Boduluri SR, Moomaw JF, Casey PJ, Beese LS: Crystal structure of protein farnesyltransferase at 2.25 A resolution. Science 1997, 275:1800-1804. The only structure of a prenyltransferase known to date. It shows how this pharmacologically very important protein might bind substrates. 53.
54. •
Moore MS, Blobel G: Purification of a Ran-interacting protein that is required for protein import into the nucleus. Proc Nat/ Acad Sci USA 1994, 91:10212-10216.
Bullock TL, Clarkson WD, Kent HM, Stewart M: The 1.6 A resolution crystal structure of nuclear transport factor 2 (NTF2). J Mo/ Bio/1996, 260:422-431. The structure of an effector that specifically recognizes the GDP-bound form of Ran.
GEFs, GAPs, GDIs and effectors: taking a closer (3D) look at the regulation of Ras-related GTP-binding proteins Matthias Geyer* and Alfred Wittinghofert Cell biology depends on the interactions of macromolecules, such as protein-DNA, protein-protein or protein-nucleotide interactions. GTP-binding proteins are no exception to the rule. They regulate cellular processes as diverse as protein biosynthesis and intracellular membrane trafficking. Recently, a large number of genes encoding GTP-binding proteins and the proteins that interact with these molecular switches have been cloned and expressed. The 3D structures of some of these have also been elucidated.
Addresses *Max-Planck-lnstitut fLir medizinische Forschung, Abteilung Biophysik, JahnstraBe 29, 69120 Heidelberg, Germany; e-mail: [email protected] tMax-Planck-lnstitut f~ir rnolekulare Physiotogie, Abteilung Strukturelle Biologic, Rheinlanddarnm 201, Postfach 10 26 64 44139, Dortmund, Germany; e-maih [email protected] Current Opinion in Structural Biology 1997, 7:786-792
http://biomednet.corn/elecref/O959440XO0700786 © Current Biology Ltd ISSN 0959-440X Abbreviations FTase GAP GDF GDI GEF NTF2 PTase RBD rmsd
farnesyl transferase GTPase-activating protein GDI dissociation factor GDP-dissociation inhibitor guanine nucleotide exchange factor nuclear transport factor 2 prenyl transferase Ras-binding domain root mean square deviation
Introduction A mammalian cell contains an estimated 50-100 different GTP-binding proteins, which function as molecular switches involved in regulating many different biological processes ranging from protein biosynthesis and membrane trafficking to communicating signals from the outside of the cell to its interior. The Ras superfamily of GTP-binding proteins are small, 20-25 kDa proteins, which bind guanine nucleotides very tightly and cycle between a presumed inactive GDP-bound and an active GTP-bound state [1,2]. GTP-binding proteins contain a set of five conserved sequence elements by which they can be easily identified as being a member of this class of proteins. These elements are necessary for guanine nucleotide and Mg 2+ binding, for GTPase activity and for the conformational switch. In addition, almost all Ras-related proteins contain a C-terminal cysteine-containing motif such as the CXC or the CAAX ('CAAX box' using single-letter amino acid code, where X represents any amino acid) motifs which
serve as attachment points for one or two prenyl groups such as farnesyl or geranylgeranyl. This post-translational modification is necessary for membrane insertion and biological activity. The enzymes required for post-translational modifications, such as farnesyl transfcrase, are therefore important regulators of functions. On the basis of sequence similarities, the superfamily can be subdivided further into the Ras, Rho/Rac, Rab, Ran, Rad and Arf subfamilies, and these subfamilies can be correlated very well with the different biological functions of their members [3]. Atf is myristoylated at the N terminus and Ran is not at all post-translationally modified. Three-dimensional structures of GTP-binding proteins and some of their regulators and effectors have been elucidated recently. The implications for the molecular mechanisms of their action are the focus of this review. The GTPase
regulation
cycle
The Ras-rclated proteins bind guanine nucleotidcs very tightly--with binding constants of up to 1011 M - l - a n d the dissociation rate constants for bound nucleotides are very low. Guanine nucleotide exchange factors (GEFs) increase the rate of nucleotide dissociation by several orders of magnitude and thus facilitate loading with GTP. During the process, protein-bound GDP is released and GTP, which is more abundant in the cell than GDP, binds instead. In the GTP-bound state, Ras-related proteins interact with so-called downstream targets or effectors, which in turn communicate with other partners located further downstream in the signalling cascade. Effectors are defined as proteins that interact much more tightly with the GTP-bound form than with the GDP-bound form of the Ras-related proteins. This interaction is terminated by hydrolysis of protein-bound GTP to GDP, which restores the GDP-bound form and terminates interaction with the effector. The intrinsic GTPase reaction is usually very slow (the half life is in the order of minutes to hours), but it can be stimulated by several orders of magnitude (half life milliseconds to seconds) by GTPase-activating proteins (GAPs) [4]. The schematic GTPase cycle is shown in Figure 1. Before GTP-binding proteins can enter the GTPasc cycle, their localisation at the cell membrane is facilitated by post-translational prenylation. This is mediated by prenyl transferases (PTases) as shown in Figure 1. Certain Ras-related proteins such as Rab and Rho/Rac are additionally regulated by guanine nucleotide dissociation inhibitors (GDIs). GDIs have been found to inhibit GDP dissociation; their true function is to extract the membrane-inserted, post-translationally modified protein from the membrane by binding to the prenylated C-ter-
GEFs, GAPs, GDIs and effectors Geyer and Wittinghofer
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Figure 1
GDF
P~
inactive
~
GTP
active
GDP
signalling & transport pathway CurrentOpinionin StructuralBiology Function and regulation of Ras-related GTP-binding proteins (Rax). Except for Ran, which is unmodified, all superfamily members undergo prenylation, which is catalysed by different forms of PTases. The isoprenylated proteins bind to GDI, which keeps the hydrophobic tail protected from solvent and is considered to be a transport factor. Interaction with GDF promotes membrane association via the isoprenyl and other hydrophobic moieties. For their function, Rax proteins cycle between an inactive, GDP-bound and an active, GTP-bound form. Conversion between these two forms is via guanine nucleotide dissociation catalysed by GEFs and via GTP hydrolysis catalysed by GAPs. Ran is not prenylated and inserted into membranes but rather cycles between the cytoplasm and the nucleus and regulates both nuclear import and export.
minal end and to serve as a cytosolic pool for Rab and Rho/Rac. T h e dissociation of the complex between G D I and the GTP-binding protein is catalysed by a protein named G D I dissociation factor (GDF), which has recently been identified for the Rab cycle [5].
Protein structures of the Ras superfamily T h e first 3D structure of a small GTP-binding protein in the Ras superfamily to be solved was that of Ras itself. It was solved in both the active triphosphate-bound conformation and the inactive diphosphate-bound conformation I6-8]. T h e structure, consisting of six 13-strands and five ot helices, led to the prediction that all GTP-binding domains have a common topology, known as the 'G domain'. By comparison of the two states, two highly flexible regions surrounding the y-phosphate have been established: the switch I region, from residues 30-37 within loop L2 and 132 (the 'effector region'); and the switch II region, from residues 60-76 within loop L4 and helix a2.
T h e crystal structures of human and rat ADP-ribosylation factor-1 (Arfl) complexed to GDP [9,10] showed the fold for a member of another subfamily. Irrespective of the presence of an additional N-terminal cx helix and a difference in Mg e+ coordination, these structures confirmed the idea of a common G domain topology. T h e structure of the nuclear import protein Ran in its GDP-bound form also shows the same overall fold, with differences in the conformation of the effector loop and in some aspects of the nucleotide-binding site [11]. Remarkably, both the structures of Arfl and Ran have a common feature, which is an additional 13 strand in the effector loop located antiparallel to strand 132, that has not been found in other Ras-related GTP-binding proteins. T h e structure of human Racl, a member of the Rho/Rac subfamily, has been solved in its active conformation using X-ray crystallography [12"]. Racl, with an overall sequence identity of 30% to Ras, has a 13-residue insertion between
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Catalysis and regulation
[35 and c~4 (residues 123-135) which forms an additional o~ helix. Excluding the insertion, the main-chain atoms of both molecules have an rmsd deviation of 1.2 ~. As in some other crystal structures of GTP-binding proteins, part of the effector loop, including the region of the magnesium ion binding by Thr35, is poorly defined in the electron density map. The switch II region, which is usually one of the most flexible elements in GTP-binding proteins, also differs between Ras and R a c l - - t h e latter of which has two short 310 helices after the DTAGQ "/-phosphate binding motif. Switch II is again different in the structures of two other members of the Rho family, Cdc42.GDP [13] and RhoA-GDP [14]. In Cdc42, no evidence has been found for an c~ helix in the switch II region, whereas RhoA has a regular a helix. The structures of the five major GTPase subfamilies are completed, with the crystal structure of Rab7 which has been recently announced [15] and is reported to be very similar to that of Ras (P MetcalE personal communication).
Dynamic aspects of the structure of GTP-binding proteins have been observed in Ras and its isoforms using NMR spectroscopy [16-18] and electron paramagnetic resonance spectroscopy [16,19]. A two-state model for the movement of the effector loop in triphosphate-bound Ras has been established; the flexibility of the loop can conveniently be monitored by a large shift of Tyr32 relative to the phosphate groups [16]. Binding of the effector kinase c-Raf-1 or the GTPase-activating protein (GAP) stabilizes the effector loop in either of the two conformations. The conformational transition in the uncomplexed protein demands an activation enthalpy of 90 kJ mo1-1. Whereas both conformations are almost equally populated in the GppNHp-bound form, one state predominates in the GTP-bound form. This internal fexibility is observed also for N-Ras-GppNHp, whereas it seems to be reduced for the GTPTS-bound form [17]. The conformational flexibihy is confirmed by a detailed NMR study on 13C-/15N-labelled protein bound to different nucleotides that shows the combination of three flexible regions in the triphosphate bound form, L1 L2 and L4, termed a regional polysterism [18].
The conformational changes of the effector loop in solution arc extended by the crystal structure of Rap2A, a homologue to Ras, which is the first structure of a small GTP-binding protein with its natural ligand GTP [20] besides the structures of wildtype and mutant Ras that have been obtained from Ras.caged GTP after photolysis in the crystal [21]. It shows that the hydroxyl group of Tyr32 forms hydrogen bonds with the y-phosphate of GTP and with Glyl3, which are not present either in Ras or in the Rap2A.GTPyS complex. A comparable change has been reported for elongation factor-Tu, which undergoes a major conformational transition in the effector loop upon GTP hydrolysis [22].
GEFs Guanine nucleotide exchange factors for the Ras, Rho/Rac, Ran, Arf subfamilies have been cloned together with mammalian suppressor of Sec4 (Mss4)--a presumed GEF for Rab proteins [3]. Mss4 is not a bona fide GEE as it has a very low activity and acts only stoichiometrically by forming a complex with nucleotide free Rab thereby replacing bound nucleotide [23], whereas other GEFs have been shown to act catalytically by increasing the rate of equilibrium formation between the GDP- and GTP-bound states [24]. The structure of human Mss4, a Rab isoform, has been determined using NMR spectroscopy [25°]. The 14kDa protein can be described as a three-layered [3-protein, with a central seven-stranded antiparallel sheet flanked by a small three-stranded [3 sheet on one face and a [3 hairpin on the other. Mss4 also binds a Zn2+ ion, which is tetrahedrally coordinated by two CxxC motifs arranged in the so-called rubredoxin 'knuckle' structure. The Zn2+-binding region and a neighboring loop define the proposed active site of the protein. The Rab-interacting surface of Mss4 has been determined using NMR exchange experiments with a nucleotidc-free form of Sec4. The ZnY+-binding motif of Mss4, whether or not it is required for its GEF activity; is not found in other GEFs such as RCC1, Sos or Cdc25 [3].
Effectors Ras is the only small GTP-binding protein for which detailed structural information is available concerning its interaction with cffectors. The serine/threonine kinasc c-Raf-1 is a downstream target in the signalling cascade of Ras. Its Ras-binding domain (RBD) binds with high affinity (KD =20nM) to Ras [26] and with somewhat lower affinity to R a p l A - - a Ras-like protein. The structure of Raf-RBD alone has been solved using NMR spectroscopy [27], and the complex of RaplA with a GTP analogue has been solved using X-ray crystallography [28]. A detailed analysis has been carried out for the binding interface and its specificity, which turns out to be crucial for a molecular understanding of signalling pathways [29,30].
The cysteine-rich domain of c-Raf-1, residues 139-184, has been proposed as a second Ras-binding site [31]. Its structure determination using NMR spectroscopy has shown it to have a double ZnZ+-binding motif [32"]. The structure is slightly different from that of two related cysteine-rich domains from protein kinase C which correlates with its inability to bind diacylglycerol and phorbol esters. Another Ras-binding domain (RBD) that has been solved recently using both NMR spectroscopy and X-ray crystallography is the RBD of GEF RalGEF [33",34"]. RalGEF is an effector molecule for Ras and has GEF activiry for R a l - - a n o t h e r GTP-binding protein of the Ras subfamily. As Ral interacts with its effector RalBP1, which is a GAP
GEFs, GAPs, GDIs and effectors Geyer and Wittinghofer
for Cdc42/Rac, RalGEF has been proposed to couple the Ras to the Cdc42/Rac pathway. The overall structure of the RBD of RalGEF adopts the same ubiquitin superfold as the RBD of Raf, although the two domains have only 13% sequence identity. NMR titration experiments with Ras suggest that complex formation occurs via formation of an intermolecular 13sheet [33"], similar to that observed for the RBD of Raf and RaplA. The same global structure is also adopted by Rgl, a protein with 55% sequence identity to RalGEF (S Yokoyama, personal communication), and by Rlf, another RalGEF like factor with 30% sequence identity (D Esser, P Bayer, personal communication). GAPs As with GEFs, a large number of GTPase-activating proteins (GAPs) have been identified, and each subfamily of Ras-related proteins, such as Ras, Rac/Rho, Rab and Aft, have their own version of a specific GAP with its own conserved sequence elements [3]. Rap, a member of the Ras subfamily with four isoforms closely related to Ras, is noteworthy, as it is the only Ras-related protein that does not have an otherwise invariant glutamine in switch II region that is involved in GTP hydrolysis. Correspondingly, it has its own type of Rap-specific GAP (Rap GAP) which does not react with Ras, just as RasGAP does react with Rap proteins [35].
Five mammalian RasGAPs have been cloned, two of which - - pl 20-GAP and neurofibromin--have been extensively characterized [36]. Recently, the structure of the catalytic domain of pl20-GAP was solved [37°]. It is a purely helical protein which contains a shallow groove that has been identified as the active site because it contains most of the conserved residues and Ras can be modelled to fit into the groove, using biochemical and mutagenesis data that had been accumulated over the years. The structures of two members of the RhoGAP family; the catalytic domain of the p85 regulatory subunit of phosphoinositide 3-kinasc [38 °] and from p50Rho-GAP [39*], have also been determined recently. As expected, these two structures are very similar; however, they are not homologous to the RasGAP structure even though they are both purely ot helical. The mechanism of the GAP-mediated GTP hydrolysis on Ras has been a matter of considerable debate in recent years and has centered around the issue of whether Ras itself is an efficient GTPase device and GAP acts catalytically to push it into the active state or whether GAP actively participates in the reaction to make it efficient [40,41]. Evidence for the latter option came from experiments with aluminium fluoride, which binds in the y-phosphate binding site of most ATP/GTP-utilizing phosphoryl transfer enzymes where it mimics the transitions state of the reaction, as has been most clearly shown for the cc subunits of heterotrimeric G proteins [42,43]. Contrary to results on Gc~ proteins, Ras itself does not bind aluminium fluoride but rather
789
needs stoichiomctric amounts of RasGAPs to do so [44], implying that the catalytic centre of Ras is incomplete unless GAP is present. This was revealed by the 3D structure of the complex between RasGDP and the catalytic domain of pl20-GAP crystallized in the presence of aluminium fluoride. The structure shows that GAP supplies an arginine finger to the active site of Ras, which apparently stabilizes negative charges in the transition state of the GTPase reaction via its guanidinium sidechain believed to proceed by a mostly associative mechanism [45"]. The structure also shows that Gin61 of Ras, which in uncomplexed Ras is very mobile, becomes fixed by the presence of GAP and also participates in the stabilization of the transition state and also explains a structural recluirement for a glycine in position 12 for GAP catalysis to work. The 3D structure of the complex between Cdc42 in the GTP-bound state and the catalytic domain of p50Rho-GAP has also been solved recently [46"']. As in the Ras-RasGAP complex, the switch regions of Cdc42 are mostly responsible for the interaction. As the Cdc42.RhoGAP complex represents the ground state of the GTPase reaction, it was interesting to find that the arginine which is also believed to be involved in the GTPase reaction in RhoGAP, is not contacting the y-phosphate, which implies that it may do so only in the transition state of the reaction, as has been found for Gic~l [43]. The transition state of the latter is also stabilized by its specific GAP called RGS (for regulator of G protein signalling) using similar contact sites on the G domain of this large (42 kDa) GTP-binding protein [47"]. GDIs Recently, the structures of two types of GDIs have been determined. Rab-GDI functions in vesicular transport to recycle and regulate Rab family GTPases; it forms a complex with the GDP-bound, prenylated form of Rab and can interact with and retrieve a broad range of Rab proteins from the membrane bilayer. The bovine o~-isoform of Rab-GDI is a 50kDa protein, and the structure of the intact protein has been elucidated [48"']. GDI is constructed of two main structural u n i t s - - a large complex multisheet domain with two major [3 sheets (five parallel strands and seven mostly antiparallel strands nearly perpendicular to each other) and a smaller o~-helical domain. This domain is closely related to flavin adenine nucleotide containing monooxygenases and oxidases. From homology studies and site-directed mutagenesis experiments, the two most sequence-conserved regions, which form a compact structure at the apex of GDI, are shown to play a critical role in the binding of Rab proteins.
Rho-GDI is a 204-residue protein, which binds the C-terminal isoprene moiety of Rho family members to form a complex that represents the cytoplasmic pool of these proteins [49]. The NMR and X-ray crystallography derived structure of Rho-GDI by two independent groups
790
Catalysis and regulation
[ 5 0 " , 5 1 " ] shows two distinct regions: a C-terminal folded domain of 16kDa which strongly binds Cdc42; and an unstructured N-terminal region which is necessary to inhibit nucleotide dissociation. T h e structure is different from that of Rab-GDI, which supports the notion that whereas the GTP-binding proteins from the different subfamilies are very similar, their regulators and effectors are unrelated. T h e folded domain of R h o - G D I shows a 13-sandwich motif of two largely antiparallel [3 sheets that pack against each other in a parallel manner with a narrow hydrophobic cleft which binds isoprenes. T h e binding properties of this cleft have been determined using N M R titration experiments with a farnesylated peptide of seven residues which mimics the isoprenylated C terminus of Rho-family GTPases [51"]. Similarly, the exposed surface that interacts with the protein portion of Cdc42 has been determined. T h e structural and biochemical data suggest a model for the Rho-GDI.Cdc42 complex in which the G T P a s c is on top of the C-terminal domain of the regulator, with its attached isoprene buried in the G D I hydrophobic pocket. This interaction anchors the inhibitory N-terminal peptide to the GTPase, allowing it to block the exchange of GDP, and also, probably, the binding of GAPs and GEFs. Other
proteins-FTase,
NTF2
For a complete description of the switch cycle of GTP-binding proteins (Figure 1), other proteins involved in the regulation and function need to be mentioned. T h e protein farnesyhransferasc (FTase), a heterodimer consisting of an o~ and 13 subunit, catalyses the C-terminal lipidation of Ras and Ras-like proteins using a subset of CAAX motifs, but also modifies other cellular proteins such as nuclear lamins and the ¥-subunit of the heterotrimeric G protein transducin. T h e transfer of the farnesyl group proceeds from farnesyl pyrophosphate onto the cysteine of the CAAX box to create a thioether linkage. In the case of Ras, farnesylation is necessary for further post-translational modification and biological function. T h e crystal structure of FTasc shows the 48 kDa subunit to be a crescent-shaped seven helical hairpin domain, and the 4 6 k D a 13 subunit to be an o~-ot barrel domain [52"]. T h e active site is formed by two clefts that intersect at a bound zinc ion. T h e structure solved is in fact a complex with a nine-residue peptidc which binds to one cleft and which may mimic the binding of the Ras substrate. T h e other cleft is a hydrophobic pocket at the centre of the a - a barrel, which is lined with highly conserved aromatic residues appropriate for binding the farnesyl isoprenoid group with required specificity. Interestingly, the fourth residue of the nonapeptide is a proline and corresponds to the position of the cysteine to be farnesylated in the CAAX protein. It is exactly in register with the zinc ion, adjacent to the presumed position of the farnesyl a-phosphate. An expansion on the conventional switch cycle comes from studies with Ran, which is a Ras-related nuclear
protein. Although the deletion or mutation of the genc has many pleiotropic effects, its major role appears to be the regulation of both nuclear import and export. As the G E F of Ran, RCC1, is in the nucleus and the Ran-GAP is in the cytosol, it may turn out that Ran-GDP controls import, whereas R a n . G T P regulates export. Another protein, nuclear transport factor 2 (NTF2), which facilitates protein transport into the nucleus, interacts only with Ran.GDP [53]. This indicates that the dogma that only the G T P - b o u n d form of GTP-binding protein can bind to an effector needs to be revised and may mean that, at least in the case of Ran, N T F 2 is the specific effector of Ran-GDP, T h e structure of N T F 2 , solved using X-ray crystallography, shows an cx+ 13 barrel that is open at one end to form a distinctive hydrophobic cavity which is a potential binding site for Ran [54"]. T h e structure of a complex between Ran.GDP and N T F 2 has also been solved and shows the switch region of Ran to be involved in the interaction, as is anticipated because the binding to Ran is specifically dependent on the presence of G D P (M Stewart, personal communication). Conclusions
A number of Ras-related proteins are now known at structural level. T h e structures show that this class proteins have a c o m m o n fold, the G domain, which is involved in the basic functions of guanine nucleotidc and Mg 2+ binding and the G T P a s c reaction. Very recently, a number of structures have been solved that allow us to take a first look at the regulation of these proteins such that we can now begin to have an understanding of how GAPs stimulate G T P hydrolysis, G D I s inhibit nucleotide dissociation and bind the isoprenylated C-terminal end, cffectors are designed to specifically interact with the active or, and in the case of Ran, the 'inactive' state of the molecule, and PTases catalyse C-terminal modification. T h e s e first glimpses have been very exciting and have made us curious for more insight into the function of the molecular switches, and their modes of regulation. Acknowledgements
We thank all colleagues for sending prcprints of their papers in press and for private communications.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • *•
of special interest of outstanding interest
1.
Bourne HR, Sanders DA, McCormick F: The GTPase superfamily: a conserved switch for diverse cell functions. Nature 1990, 348:125-132.
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Bourne HR, Sanders DA, McCormick F: The GTPase superfamily: conserved structure and molecular mechanism. Nature 1991, 349:117-12Z
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Boguski MS, McCormick F: Proteins regulating Ras and its relatives. Nature 1993, 366:643-654.
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4.
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Gideon P, John J, Frech M, Lautwein A, Clark R, Scheffler JE, Wittinghofer A: Mutational and kinetic analysis of the GTPaseactivating protein (GAP)-p21 interaction: the C-terminal domain of GAP is not sufficient for full activity. Mol Cell Biol 1992, 12:2050-2056. Diracsvejstrup AB, Sumizawa T, Pfeffer SR: Identification of a GDI displacement factor that releases endosomal Rab GTPases from Rab-GDI. EMBO J 1997, 16:465-472.
6.
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