Biol. Chem. 2017; 398(5-6): 577–588 Review Open Access Miriam Schöpel, Veena Nambiar Potheraveedu, Thuraya Al-Harthy, Raid Abdel-Jalil, Rolf Heumann and Raphael Stoll* The small GTPases Ras and Rheb studied by multidimensional NMR spectroscopy: structure and function DOI 10.1515/hsz-2016-0276 Keywords: ligand binding; nuclear magnetic resonance Received September 1, 2016; accepted January 23, 2017; previously (NMR); Ras; Rheb. published online February 15, 2017 Abstract: Ras GTPases are key players in cellular signal- ling because they act as binary switches. These states Introduction: the small GTPases Ras manifest through toggling between an active (GTP-loaded) and an inactive (GDP-loaded) form. The hydrolysis and and Rheb replenishing of GTP is controlled by two additional pro- Cells are constantly sending messages and are checking tein classes: GAP (GTPase-activating)- and GEF (Guanine nutrient levels and growth rates within the cell as well as nucleotide exchange factors)-proteins. The complex inter- with other cells. These messages need to be explicit and play of the proteins is known as the GTPase-cycle. Several one way to amplify signals is to link them to a process that point mutations of the Ras protein deregulate this cycle. is chemically irreversible, like the cleavage of ATP or GTP. Mutations in Ras are associated with up to one-third of Adenosine triphosphate (ATP) and guanosine triphos- human cancers. The three isoforms of Ras (H, N, K) exhibit phate (GTP) are used in living cells as cofactors for various high sequence similarity and mainly differ in a region biochemical transformation reactions. called HVR (hypervariable region). The HVR governs the Whereas ATP is regarded as a ‘molecular unit of cur- differential action and cellular distribution of the three rency’ in intracellular energy metabolism, the hydroly- isoforms. Rheb is a Ras-like GTPase that is conserved from sis of GTP to GDP (guanosine diphosphate) mainly plays yeast to mammals. Rheb is mainly involved in activation a regulatory role in biochemical processes, including of cell growth through stimulation of mTORC1 activity. In cell growth, cell differentiation, as well as vesicular and this review, we summarise multidimensional NMR studies nuclear transport. Proteins capable of binding guanosine on Rheb and Ras carried out to characterise their struc- nucleotides are called guanosine binding proteins (or ture-function relationship and explain how the activity of guanine nucleotide-binding proteins, GNBPs). The super- these small GTPases can be modulated by low molecular family of monomeric small GTPases includes proteins weight compounds. These might help to design GTPase- with a size of 20–25 kDa and a common fold that consists selective antagonists for treatment of cancer and brain of a central six-stranded mixed β-sheet surrounded by a disease. total of five α-helices. The regulatory role of the small GTPases is fulfilled through their capability to act as binary switches by tog- gling between an ‘on’ and ‘off’ state (Figure 1). These *Corresponding author: Raphael Stoll, Biomolecular NMR, Ruhr University of Bochum, D-44780 Bochum, Germany, states are chemically characterised by the bound nucle- e-mail: [email protected] otides: GTP ( = on) and GDP ( = off). Since the intrinsic Miriam Schöpel: Biomolecular NMR, Ruhr University of Bochum, GTP hydrolysis, performed by the small GTPases, is D-44780 Bochum, Germany very slow (10−6 1/s), it is accelerated by GTPase activat- Molecular Veena Nambiar Potheraveedu and Rolf Heumann: ing proteins (GAPs) by several magnitudes (Gibbs et al., Neurobiochemistry, Ruhr University of Bochum, D-44780 Bochum, Germany 1984). The hydrolysis products GDP and Pi are not auto- Thuraya Al-Harthy and Raid Abdel-Jalil: Chemistry Department, matically released, because the protein exhibits the College of Science, Sultan Qaboos University, Muscat, Oman same affinity for GTP and GDP (Renault et al., 2003; ©2017, Miriam Schöpel et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License. 578 M. Schöpel et al.: NMR studies on Rheb and Ras Figure 1: The GTPase cycle involves the exchange of GDP by guanine nucleotide exchange factors (GEFs), which control the nucleotide exchange by increasing the dissociation rate. The GEF protein directly inserts certain amino acids into the nucleotide binding domain (NBD). Thereby, the affinity of the GTPase towards the nucleotide is reduced. Once activated, the GTP-bound state enables GTPases to interact with various effectors. GTPase activating pro- teins (GAPs) catalyse GTP hydrolysis and return GTPases to their GDP-bound ‘OFF’-state, thereby completing the GTPase cycle. Pasqualato and Cherfils, 2005; Cherfils, 2014). There- Our project mainly focused on two small GTPases, fore, the exchange process is also catalysed by a group Rheb and K-Ras4B. Ras is probably the most famous small of different proteins, which are structurally totally unre- GTPase, because of its importance for cell growth, differ- lated: guanine exchange factors (GEFs). Ultimately, entiation, and survival. In the human proteome, three Ras GTPases bind and hydrolyse GTP and thereby switch isoforms (or splice versions) and one splice variants are from an active (GTP-loaded) to an inactive (GDP-loaded) found: N-, H-Ras, K-Ras4A, and K-Ras4B. These amino form. Thus they play a crucial role in cellular signal- acid sequences share a high similarity ( >95%) and differ ling. The communication between the G-Domain and only in a ‘hyper variable region’ (HVR), that comprises the its various effector proteins is mediated by two regions, carboxy-terminal 25 amino acids. The final four residues switch I and switch II, to transmit external signals from of the HVR region are known as the CAAX-box, which is growth factors to intracellular signalling cascades. Both the target of posttranslational modification (Figure 2). regions undergo dramatic structural changes, depend- The Ras proteins undergo four steps of modification to ing on which nucleotide is bound. The underlying mediate membrane binding: isoprenylation, proteolysis, molecular mechanism is known as the ‘loaded-spring methylation, and palmitoylation (Aronheim et al., 1994). mechanism’, introduced by Vetter and Wittinghofer Additionally, K-Ras4B possesses a polybasic stretch of six (2001). This universal mechanism involves two amino lysines that is capable of binding to negatively charged acids, a conserved threonine (T35), located in switch I, phospholipids of distinct cell membranes. The Ras protein and a glycine (G60), which adopt distinct conformations is a known proto-oncogene and approximately up to one- when GTP is bound. During the process of GTP-hydroly- third of all human cancers are caused by a mutation in sis, the ɣ-phosphate is released and the switch regions this particular protein. The highest frequency of Ras muta- are allowed to relax into different conformations. Hith- tions are mostly found in pancreatic (90%), lung (40%), erto, 167 proteins have been identified that are part of and colorectal cancer types (50%) (Schubbert et al., 2007). the human Ras superfamily, of which 39 proteins belong Three mutations hotspots have been identified, which to the Ras family (Rojas et al., 2012). mainly occur at codons 12, 13, and 61. In total, these three M. Schöpel et al.: NMR studies on Rheb and Ras 579 Figure 2: Schematic presentation of the four Ras isoforms. H-Ras, N-Ras, K-Ras4A and K-Ras4B are highly homologous throughout the conserved G domain (amino acids 1–166). The C-terminal hypervariable domain (amino acids 166–188/189) specifies membrane localisation through post-translational modifications that include the farnesylation of each isoform on the C-terminal CAAX motif and palmitoylation of cysteines on H-Ras, N-Ras, and K-Ras4A (highlighted in yellow). Membrane localisation of K-Ras4B is also facilitated by a stretch of lysine residues in support of the farnesyl moiety. mutation sites occur in 97–99% of all Ras mutations in the cell stress state. While in cancer cells Rheb promotes cancer (Cox and Der, 2010). Interestingly, most mutations cell cycle progression, there is an enhancement of apopto- are found in K-Ras (85%), followed by N-Ras (12%), and sis by Rheb observed after cellular application of various rarely in H-Ras (3%) (Cox et al., 2014). toxic stimuli such as UV stress or ER-stress (Ozcan et al., Rheb (Ras homologue enriched in brain) is a small 2008; Karassek et al., 2010). Furthermore, Rheb synthesis GTPase that is related to Ras, Rap, and Ral (Tee et al., is rapidly up-regulated as an immediate-early response 2003, 2005; Ehrkamp et al., 2013). Rheb, is part of the gene after injury (Yamagata et al., 1994; Potheraveedu mTOR pathway that integrates intra- and extracellular et al., 2017). Over-expression of Rheb enhances cellular signals and thereby regulates cell metabolism and growth degeneration, which can be prevented by rapamycin or by proliferation. Unlike Ras, Rheb harbours an Arg and knock down of apoptosis signal-regulating kinase (ASK-1) Ser instead of Gly residues in its P-loop that binds the β (Karassek et al., 2010). Thus, clinical application of rapa- phosphate of either GDP or GTP (Karassek et al., 2010). mycin should take into consideration the cellular stress As a molecular switch, Rheb regulates cell volume, cell state which could change its therapeutic effects (Karassek growth, cell cycle progression, neuronal axon regenera- et al., 2010).