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Gtpase-Activating Proteins and Their Complexes Steven J Gamblin* and Stephen J Smerdon

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Gtpase-Activating Proteins and Their Complexes Steven J Gamblin* and Stephen J Smerdon 195 GTPase-activating proteins and their complexes Steven J Gamblin* and Stephen J Smerdon In the past year, crystallographic structures for four complexes Figure 1 of GTPase-activating proteins (GAPs) with their target G proteins have been described and substantially enhance our understanding of how these proteins function. GAPs specific for the Rho and Ras families of small G proteins insert an GMPPNP arginine residue into the active site of the G protein, stabilise its switch regions and share an underlying topological relationship. The complex of a heterotrimeric G protein with its activating protein shows that the latter protein does not participate directly in the hydrolytic reaction and has a different structure to RhoGAP and RasGAP. P-loop Addresses Protein Structure Division, National Institute for Medical Research, The Ridgeway, London NW7 1AA, UK *e-mail: [email protected] I Correspondence: Steven J Gamblin Current Opinion in Structural Biology 1998, 8:195–201 http://biomednet.com/elecref/0959440X00800195 © Current Biology Ltd ISSN 0959-440X Abbreviations II BH BCR homology EF elongation factor Ga G protein α subunit GAP GTPase-activating protein Hs Homo sapiens Current Opinion in Structural Biology PH pleckstrin homology PI3-kinase phosphatidyl inositol 3-kinase RGS regulator of G-protein signalling The structure of the archetypal small G protein, Ras, bound to the SH src homology nonhydrolysable nucleotide analogue GMPPNP [6]. The switch I, switch II and P-loop regions (in black) are labelled. The small G proteins Ras, RhoA and Cdc42 are structurally closely related. An Introduction extra helical segment (residues 117–137) is present in the Rho family members [16•] and is located C-terminal to the switch and P-loop The G-protein family is made up of a diverse range of mol- regions. Thus, the Ras and Cdc42Hs residue numbers are directly ecules that control a complex array of biological processes comparable. Unfortunately, RhoA possess two additional N-terminal but have in common a structurally homologous GTP-bind- amino acids, so residues are numbered two higher than their structural ing domain. They act as molecular switches that cycle equivalents in Ras or Cdc42Hs. In the main text, residues will be between the active, GTP-bound form and the inactive, numbered and subscripted according to the molecule to which they belong, whilst secondary structural elements will be named according GDP-bound form [1–3]. In the active form, they are com- to the original publication. These terms will also be used for referring to petent to interact with a broad range of effector molecules. the heterotrimeric G proteins and their complexes, although the The lifetime of this active state is determined by the com- numbering of its residues is unrelated to that of the small G proteins. bination of slow intrinsic GTPase activity and the action of The Gα switch III region, which responds indirectly to GTP hydrolysis, is specific to these proteins and has no counterpart in the small G GTPase-activating proteins (GAPs), which can accelerate protein relatives. GTP hydrolysis by as many as five orders of magnitude [4,5]. GTP hydrolysis causes conformational changes in the G protein that are localised to two distinct regions of the molecule, switch I (residues 30–40 in Ras, also known subfamily of small G proteins (Rac, Cdc42 and RhoA) as the effector loop) and switch II (residues 60–67 in Ras). [16•,17,18]. G proteins also contain a conserved region The locations of the switch regions with respect to the known as the P-loop (residues 10–17 in Ras), which nucleotide are shown in Figure 1. The structural respons- forms a structural cradle for the β- and γ-phosphate es of the switch regions to the loss of the γ-phosphate have groups of the nucleotide and additionally supplies a lig- been comprehensively described for the archetypal G pro- and (serine or threonine) to the octahedrally coordinated tein, Ras [6–8], Rap2A [9], elongation factor Tu (EF-Tu) magnesium ion [19•]. Mutations within this region of [10,11], the heterotrimeric G protein α subunit (Gα) Ras, result in the loss of GTPase activity and are [12–15], and more recently, members of the Rho oncogenic [20]. 196 Macromolecular assemblages GAPs also form a structurally and functionally diverse fam- As mentioned before, the GAPs for Ras and Rho are unre- ily of molecules that appear to be specific for defined sub- lated at the protein sequence level but it is apparent that families of G proteins. At the time of writing, three crystal there is some similarity in terms of the arrangement of sec- structures of isolated GAP molecules [21–23] and four ondary structure elements in the G-protein binding sites of complexes of GAPs with their cognate G protein these GAPs. In RasGAP the G-protein binding site is gen- [24••–27••] have been solved. The purpose of this review erated from two adjacent helices and two associated loops is to assess the current state of our structural understand- [25••]. In the RhoGAP complexes, although there are ing of GAP-mediated G-protein regulation. some detailed differences in the interface between the ground and transition states, the binding surface again GAPs for small G proteins involves two adjacent helices and an adjoining loop GAP activity domains occur in many proteins, usually in [26••,27••]. combination with other signalling modules that may include src homology (SH) 2 and SH3 domains, and pleck- Comparison of the ground and transition state strin homology (PH) domains together with proline-rich G-protein complexes of RhoGAP regions [3,5]. Crystal structures have been solved for GAP The Cdc42Hs–RhoGAP complex contains the nonhy- domains from the p85α subunit of phosphatidyl inositol 3- drolysable GTP analogue GMPPNP, which can be regard- kinase (PI3-kinase) [21], p120RasGAP [22] and ed as representing the nucleotide substrate in its ground p50RhoGAP [23], which will be referred to hereafter as state complex. Crystal structures of nucleotide complexes BH (BCR homology domain), RasGAP and RhoGAP, of Ras have demonstrated that GMPPNP is a good struc- respectively. These structures reveal that all three GAP tural analogue of GTP [8]. The RhoA–RhoGAP complex α – domains are extensively -helical in composition. BH has contains GDP·AlF4 , which is thought to be an analogue of sequence and structural homology with RhoGAP and both the transition state of the phosphoryl transfer reaction. bind to members of the Rho family of small G proteins. Indeed adenylate cyclase was first identified 40 years ago BH does not enhance GTPase activity, however, [21,28,29] because aluminofluorides are potent activators of het- and this has led to the suggestion that Asn194 of RhoGAP erotrimeric G proteins [31–33]. These compounds – is important in GAP function since it is conserved in all (GMPPNP and GDP·AlF4 ) have subsequently been used RhoGAP domains that possess GAP activity, but not in BH to great effect in establishing the molecular basis of het- [23]. There is essentially no sequence homology between erotrimeric G-protein function [13,14]. Using these two RasGAPs and RhoGAPs and the occurrence of 15 helical compounds, structural snapshots of the RhoGAP–Rho pro- segments in RasGAP compared with nine in the smaller tein complex at two different points along the reaction RhoGAP fragment gives the two types of molecule quite pathway have been obtained. different appearances. Both RasGAP and RhoGAP, howev- er, have a pair of conserved basic residues important for A comparison of the ground [26••] and transition-state their function (Arg789 and Arg903 in RasGAP [22], and complex [27••] structures reveals a substantial rearrange- Arg85 and Lys122 in RhoGAP [23]) and it is now apparent ment that can largely be described as a 20° rigid-body rota- that they share a common fold [30•] as shown in Figure 2. tion of the G protein with respect to RhoGAP about an axis that passes close to the phenolic sidechain of Tyr66RhoA. In Three small G protein–GAP complexes the transition state, but not the ground-state complex, Crystal structures have now been described for the follow- helix 3 of the G protein interacts with the C-terminal end •• ing complexes: Ras·GDP·AlF3–RasGAP [25 ], of the catalytic loop of RhoGAP (A–A1) and accounts for – •• 2 RhoA·GDP·AlF4 –RhoGAP [27 ] and Cdc42Hs·GMPP- an additional 460 Å of interaction surface largely consist- NP–RhoGAP [26••], which are shown in Figure 3a–c. ing of direct and solvent-mediated hydrogen bonds. Some Knowledge of these structures enables us to address a limited, local changes in the conformation of the A–A1 number of important issues. Firstly, since Cdc42Hs and loop of RhoGAP occur during its relocation such that RhoA both belong to the Rho family of small G proteins, Arg85 can now interact with the GTP and assist in cataly- what insights into the structural changes that take place sis. Both switch I and II are better ordered in the transi- during the GAP-assisted GTP hydrolysis reaction are evi- tion-state complex and this change probably constitutes an dent from comparing these two structures? Secondly, how important contribution to GAP activity. A significant do the two distinct Ras–RasGAP and Rho–RhoGAP fami- amount of the increased order of the switch I region is lies compare at the structural and functional level? These achieved through Tyr34RhoA. The mainchain carbonyl of questions will be addressed in the following two sections. this residue hydrogen bonds with the essentially conserved Asn194RhoGAP, whilst the aromatic ring of the sidechain Since all three small G proteins involved in these com- forms an electrostatic interaction with the repositioned plexes are closely related, it is not surprising that all three Arg85RhoGAP guanidinum group.
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