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 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- 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 types (50%) (Schubbert et al., 2007). the human , 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). tion, autophagy, nutritional deprivation, oxygen stress, and cellular energy status (Takei et al., 2004; Karassek et al., 2010). Rheb’s impact on growth and cell cycle pro- gression is mediated by the regulatory influence of growth Structure in solution of Rheb/GDP factors on the insulin/mTORC1/S6K signalling pathway via the mammalian target of rapamycin (mTOR) (Manning and its dynamics on the pico- to and Cantley, 2003). A complex that consist of the tuber- nanosecond time scale ous sclerosis gene products, hamartin (Tsc1) and tuberin (Tsc2) serves as a GTPase activating protein (GAP) for Based on the NMR assignments of Rheb bound to both Rheb, which implies that Rheb plays an important role in GDP and GppNHp (Berghaus et al., 2007; Schwarten et al., (Manning and Cantley, 2003; Aspuria 2007), a non-hydrolysable analogue of GTP, we were and Tamanoi, 2004; Karassek et al., 2010). Patients suffer- able to determine the structure in solution of the Rheb/ ing from this genetic disorder characteristically develop GDP complex by multidimensional heteronuclear NMR benign hamartomatous tumours in the brain, kidneys, spectroscopy. The complex adopts the typical canonical heart, lungs, skin or eyes and cannot be medically cured fold (Figure 3) of Ras-GTPases (Karassek et al., 2010). In to date. Interestingly, activation of the Rheb-mTORC1 the NMR structure, switch I and, in part, switch II of Rheb- pathway may have opposite cellular effects depending on GDP are ill-defined in solution because of fluctuations on 580 M. Schöpel et al.: NMR studies on Rheb and Ras

spectrum (Schwarten et al., 2007). This observation sug- gests that switch I and II can adopt several (two or more) different slowly interconverting conformations (Karassek et al., 2010). It is important to note that a very similar dynamic behaviour also been observed for Ras (Kraulis et al., 1994; Ito et al., 1997; Spoerner et al., 2001; Vetter and Wittinghofer, 2001). This conformational flexibility might be of functional relevance not only for the GTP/GDP cycle but also for the selectivity of small GTPase towards GAPs and GEFs (Kraulis et al., 1994; Ito et al., 1997; Spoerner­ et al., 2001; Vetter and Wittinghofer, 2001). Based on these cumulative findings, the interaction between Rheb (or Ras) and its GAP and/or GEF is obviously character- ised by conformational selection, a feature also observed in numerous other protein ligand complexes (Karassek et al., 2010; Weikl and Paul, 2014).

Figure 3: Backbone representation of the structural ensemble of Rheb’s GAP and GEF Rheb in solution (PDB data bank code 2L0X). The unstructured N-terminal residues S1–K5 and C-terminal residues Whereas a protein complex formed by the tuberous S175–M184 have been omitted for clarity. α-helical secondary struc- ­sclerosis gene products, the tumour suppressors TSC1 tural elements are shown in red, β-sheets in green, and loops in (or hamartin) and TSC2 (or tuberin), has been shown to yellow. The dynamic switch I and II regions are shown in orange. The GDP is depicted as a stick model and the magnesium ion is shown function as a GTPase activating protein (GAP) for Rheb in magenta. (Manning and Cantley, 2003; Aspuria and Tamanoi, 2004; Karassek et al., 2010), the GEF for Rheb remains elusive to date. In fact, Rheb may not possess a genuine GEF but the pico- to nanosecond time scale, as observed in the het- instead basal nucleotide exchange rates may suffice to eronuclear NOE experiment (Karassek et al., 2010). This load Rheb with GTP given the fact that hormones down- seems to be a common feature of small GTPases, as this regulate its GAP, TSC (Wang and Proud, 2011). Amino has also been observed for the structure of Ras in solution acids mediate mTOR/raptor signalling through activation (Kraulis et al., 1994). In sharp contrast to the GDP-loaded of class 3 phosphatidylinositol 3OH-kinase (Nobukuni inactive state, the 1H-15N HSQC spectrum of activated et al., 2005). It could be shown that TCTP is essential Rheb bound to Gpp(NH)p is characterised by extensive for growth and proliferation through regulation of Rheb line broadening for numerous resonances of the switch GTPase in Drosophila­ (Hsu et al., 2007) as Rheb acts on the regions. Obviously, switch I adopts multiple conforma- PI3 kinase/mTOR pathway and it was suggested that TCTP tions in solution that interconvert on an intermediate time could act as a GEF for Rheb. scale (Schwarten et al., 2007). This dynamic feature could TCTP regulates apoptosis and is involved in malig- be of functional relevance as switch I apparently adopts nant transformation (Hsu et al., 2007). However, several different conformations and some of these conformations studies suggest that TCTP is very unlikely to act as a might be of catalytical importance for GTP hydrolysis. GEF for Rheb, because neither in vitro nor in vivo experi- The crucial difference between small GTPases affects ments could show any nucleotide exchange activity the switch II region. In the crystal structures of Rheb, of TCTP towards Rheb bound to GDP (Rehmann et al., switch II is structurally very similar in the GDP- and GTP- 2008; Wang et al., 2008). In particular, 1H-15N HSQC-NMR- bound states (Vetter and Wittinghofer, 2001; Yu et al., based binding studies did not reveal any significant NMR 2005). Unlike the crystal structure however, switch II like chemical shift perturbation for Rheb backbone amide switch I of Rheb-Gpp(NH)p presumably adopts multiple resonances up to a threefold molar excess of TCTP. This conformations in solution. These multiple conformations is in sharp contrast to 1H-15N HSQC-NMR spectra of Ras alternate on the micro- to millisecond time scale, which in complex with SOS, a well-­established GEF, which leads to extensive line broadening in the 1H-15N HSQC are characterised by substantial line broadening. This M. Schöpel et al.: NMR studies on Rheb and Ras 581 suggests that the interaction between Rheb and TCTP is atom between the two phenyl rings (Schoepel et al., – if at all – extremely weak and not of any physiologi- 2013). In the case of Rheb, the most significant chemi- cal relevance (Rehmann et al., 2008). TCTP is structurally cal shifts were found for Bisphenol A, 4,4′-biphenol, related to Mss4, a known GEF for the family (Thaw and 4,4′-dihydroxybenzophenone. The identified et al., 2001). However, a detailed comparison of TCTP binding sites were then used as experimental restraints proteins and the MSS4Rab3 complex clearly shows that in a molecular docking procedure, using the HADDOCK while the core of TCTP and Mss4 can be superimposed, a programme suite (High Ambiguity Driven biomolecular sequence insertion in TCTP sterically clashes with Rab3 DOCKing) (Dominguez et al., 2003). Structures obtained (Rehmann et al., 2008). This excludes a mode of interac- from these docking calculations were refined using tion for TCTP and Rheb similar to that of Mss4 and Rab. OPLS_2005 force field minimisations in explicit water Obviously, more studies in the future are still required in as solvent (Schoepel et al., 2013). In the refined docking order to fully understand the relationship between TCTP model, the complex between Rheb and 4,4′-biphenol and the TSC/Rheb/mTOR pathway. is stabilised by three hydrogen bridges between I69 and I78 to one 4,4′-biphenol hydroxyl group and by another hydrogen bond between the second 4,4′-biphe- Low molecular weight compounds nol hydroxyl group and K109, which correlate well with the observed chemical shift perturbation. The hydrogen as functional modulators of small bonds stabilise the 4,4′-biphenol horizontally at the top GTPases of a deep lipophilic binding pocket and the two aromatic rings are twisted by approximately 30°. As the switch II Antagonising biomolecular interactions by means of region of Rheb has been shown to exhibit an increased small molecules is pivotal to medicinal chemistry and is flexibility on the pico- to nanosecond time scale, con- regarded a crucial strategy in cancer therapy. Yet, despite formational selection might indeed play a role in ligand some progress in recent years, the inhibition of protein- binding to Rheb (Karassek et al., 2010). Based on the protein interactions by low molecular weight compounds multidimensional NMR data on biphenol and Rheb, the still represents a major challenge in numerous research affinity is only approximately 1500 ± 200 μm (Schoepel endeavours. Biophysical interactions between biomol- et al., 2013). Such affinities are typical for fragment- ecules are usually mediated by rather large surfaces, based screening approaches and imply further optimi- which is why each residue of the biomolecular interface sation through chemical synthesis in order to increase only minimally contributes to the overall binding free binding affinity (Maurer et al., 2012; Sun et al., 2012). If energy. the two phenol moieties of 4,4′-biphenol are separated Initially, we set out to search for low molecular weight by introducing an sp2 carbon, like in benzophenone, or compounds that target Rheb and might provide novel can- by a quaternary sp3 carbon with limited conformational didate scaffolds that could, once chemically optimised, freedom, like in 4-[2-(4-Hydroxyphenyl)propan-2-yl] be used for treatment of tuberous sclerosis-mediated phenol known as Bisphenol A, the bent structure directs tumour growth. Inspired by previous studies, which, for the aromatic rings deeply into the binding pocket. The example, performed an NMR-based screening of a large KD value was extracted from multidimensional NMR 11 000-member library (Sun et al., 2012), we designed a spectra for a Bisphenol A/Rheb complex and is approxi- small fragment library based mainly on the privileged mately only 1800 ± 500 μm (Schoepel et al., 2013). In the structure concept and the ‘rule of five’ (Choy and Praus- refined docking model, Bisphenol A is stabilised in the nitz, 2011). This focused library that contained not more lipophilic pocket by three hydrogen bridges, similar to than 100 compounds was screened by multidimensional 4,4′-biphenol (Figures 4 and 5). In Rheb, S68 and Q72 NMR spectroscopy to identify structures that selectively form hydrogen bonds with one hydroxyl group of Bis- bind to Rheb. In detail, we have applied multidimen- phenol A. Y67, which is located at the bottom of the sional heteronuclear NMR spectroscopy and chemi- pocket, participates in another hydrogen bond with cal shift perturbation analysis in order to characterise the second hydroxyl group of Bisphenol A. In addi- the interactions between low molecular weight com- tion, π-cation interaction between the side-chain amino pounds and Rheb. It is interesting to note that most of group of K109 and the phenol group of Bisphenol A the molecular hits contained structural elements that located deep inside the pocket contribute to the overall had either a linear shape with a biphenyl scaffold or binding energy. A similar binding geometry is found were bent, induced by an sp2- or sp3-hybridised carbon for 4,4′-dihydroxybenzophenone. The highly flexible 582 M. Schöpel et al.: NMR studies on Rheb and Ras

Figure 4: The observed weighted chemical shift differences projected onto the molecular surface of K-Ras and Rheb, respectively. On the left, weighted chemical shift perturbation of K-Ras4B bound to GDP upon titration with Bisphenol A are projected onto the molecular surface of K-Ras4B. On the right, weighted chemical shift perturbation of Rheb bound to GDP upon titration with Bisphenol A are projected onto the molecular surface of Rheb. The colour code is yellow for small, orange for medium, and red for large NMR chemical shift perturba- tions upon ligand binding. Grey colour indicates no significant changes in the 2D 1H-15N NMR spectra.

4,4′-methylenediphenol, however, only induces minor tumour growth (Schoepel et al., 2013). In order to investi- chemical shift changes in the NMR spectra of Rheb (Sch- gate as to whether there is a cellular effect of 4,4′-biphenol oepel et al., 2013). by Rheb we determined dose-response curves for cellular Finally, we could also show that 4,4′-biphenol selec- degeneration and signalling. Microscopic assessment of tively inhibits Rheb signalling and induces cell death, cells indicated maximal cellular degeneration and block- which suggests that this compound might be a novel ade of phospho-S6 at around 100 μm thus correlating with candidate for treatment of tuberous sclerosis-mediated Rheb’s binding affinities to 4,4′-biphenol while there was

Figure 5: Examples of HADDOCK-derived structures of K-Ras4B in complex with BPA and BPS. BPA (left) exhibits a higher degree of flexibility due to an sp3 hybridisation of its central carbon atom. BPS (right) lacks this flexibility and remains more rigid. Observed chemical shift perturbations are coloured in orange (medium) and red (large), respectively. M. Schöpel et al.: NMR studies on Rheb and Ras 583 no such effect of bisphenol A. Obviously, 4,4′-biphenol et al., 2014; Schoepel et al., 2016), GTP analogues that inhibits the mTOR downstream signalling without affect- bind tightly or even irreversibly to Ras could in principle ing Ras signalling (Schoepel et al., 2013). lead to severe side effects because of unselective cross- The switch-dependent heterogeneity of the Ras confor- reactivities towards other GTPases. Nonetheless, a prom- mations, especially in the GDP-bound form, is only poorly ising study recently reported on selectively targeting a Cys portrayed by structures that were obtained by crystallog- mutant of Ras (Ostrem et al., 2013; Hunter et al., 2014; Min raphy. Moreover, on the basis of these measurements only, Lim et al., 2014; Wiegandt et al., 2015; Ostrem and Shokat, the Ras protein was believed to be undruggable, since it 2016). These ligands bind irreversibly to a mutant version lacks hydrophobic cavities on its molecular surface (Cox of the Ras protein (G12C) (Ostrem et al., 2013). The syn- et al., 2014). Nevertheless, during the past years several thesised compounds rely on the mutant cysteine to be small molecules and zinc-chelating compounds have been present in the GTPase amino acid sequence for binding identified and characterised as Sos antagonists of K-Ras due to acrylamide and sulphonamide coupling groups (Palmioli et al., 2009; Rosnizeck et al., 2012). In 2012, and should therefore not affect the wild-type protein. This co-crystal structures of several ligands bound to the full- binding pocket for these compounds differs from the site length and truncated Ras were published (Maurer et al., previously described and is located between the central β- 2012; Sun et al., 2012). With the help of a NMR-based frag- sheet of Ras, and the α2-(switch II), and α3-helices (Maurer ment screening, Genentech identified DCAI (4,6-dichloro- et al., 2012; Rosnizeck et al., 2012; Sun et al., 2012; Ostrem 2-methyl-3-aminoethyl-indole) as a Ras ligand (Maurer et al., 2013; Schoepel et al., 2013). Like the aforementioned et al., 2012), which binds in a hydrophobic cavity located other binding pockets, this ligand binding cavity does not between helix α2 and the core β-sheet, β1–β3. The dimen- exist in the crystal structures of GDP-bound wildtype Ras sions of this binding pocket are of approximately 7 × 7 Å and hence is probably highly dynamic in solution. Inspired at the opening with a depth of 5 Å. The interacting amino by this work and the fact that we recently had found a acids include K5, L6, V7, I55, L56, and T74. The DCAI com- Rheb ligand (as described above) we also tested those pound has a KD value of 1.1 ± 0.5 mm, as derived from ligands on the K-Ras4B protein. We did not observe any NMR experiments. The same binding pocket was identi- chemical shift perturbation when we tested 4,4′- biphe- fied in another study (Sun et al., 2012). There, a indolic nol for its binding to K-Ras4B as judged from 1H-15N HSQC compound (S)-N-(2-((1H-indol-3-yl)methyl)-1H-benzo[d] spectra. Obviously, this ligand is quite selective for Rheb. imidazol-5-yl)pyrrolidine-2-carboxamide) (Sun et al., 2012) Bisphenol A however, binds to K-Ras4B with an affinity of was discovered as an interaction partner for Ras. This KD = 600 ± 200 μm, which is therefore significantly higher compound was found to bind to mutant (G12V and G12D, than for Rheb (Schoepel et al., 2013). The binding site in respectively) versions of the Ras protein. This compound K-Ras identified in our study for Bisphenol A is identical to binds to K-Ras(G12D) with an affinity of KD = 190 μm. Since the one described by Genentech and Sun et al. in 2012 and the binding pocket of both compounds is located in the is located between α-helix and the core β-sheet, β1–β3. Ras-GDP/SOS interaction interface, it is not surprising Accordingly, we could show that amino acids L6, V7, L56, that the interaction between both proteins is disturbed T74, and G75 contact Bisphenol A. In addition, our HAD- after ligand binding. The prolonged indole compound DOCK-based docking models show that the aromatic rings showed an inhibition of the SOS-meditated nucleotide must adopt an almost right angle for optimal binding. Our exchange of 58 ± 8% and the DCAI molecule blocked both structural analyses reveal that the binding site in Rheb is nucleotide exchange and release reactions with an IC50 of considerably bigger than in K-Ras so that stretched, linear 342 ± 22 μm and 155 ± 36 μm, respectively (Maurer et al., molecules such as 4,4′-biphenol cannot bind into the 2012; Sun et al., 2012). Interestingly, both authors high- latter pocket (see Figures 4 and 5) (Schoepel et al., 2013). light tyrosine 71, which is located in switch II. This residue We also addressed the question as to whether our plays an important role as it occupies the hydrophobic ligands could interfere with the SOS-meditated nucleo- pocket in the apo-crystal structure. Upon ligand binding tide exchange, since the SOS protein mainly binds both this residue is replaced by the ligand and it is this replace- switch-regions of the GTPase, which is close to the binding ment that exposes the hydrophobic cavity. pocket of the afforested ligands. Indeed, we could detect a Although several (fragment-based) screenings for reduced of the Sos-mediated nucleotide exchange of both small molecular antagonists of protein-protein interac- H- and K-Ras by a factor of 2.5-fold and 1.6-fold, respec- tions between Ras and its effectors have been proven to tively. This is again in accordance with recent studies, in be difficult as affinities remain often rather low (Ganguly which a comparable stoichiometric excess of small mole- et al., 1998; Maurer et al., 2012; Rosnizeck et al., 2012; Cox cular compounds was used (Maurer et al., 2012; Sun et al., 584 M. Schöpel et al.: NMR studies on Rheb and Ras

2012). Judged from our HADDOCK docking models and As judged from the NMR titration data, BPS and BPA the available co-crystal structures, it seems highly likely, bind K-Ras4B. However, we did not observe any reduction that these different ligands, including Bisphenol A, bind in the exchange rate for Bisphenol S in the SOS-nucleotide to GDP-Ras and interfere with SOS complex formation due exchange assay. We therefore conclude, that only ligands to steric reasons. Interestingly, the observed affinity of Bis- with a KD value in the micromolar range, such as BPA, are phenol A is in the same micromolar range as the affinity able to interfere with the nucleotide exchange because the between Ras-GDP and the SOS molecule itself (Palmioli affinity between the Ras and SOS is in a similar affinity et al., 2009). regime (Palmioli et al., 2009). Taken together, BPS is a The HADDOCK-based docking studies suggest that the ligand for K- Ras4B, whose affinity is ten times lower than Bisphenol A binding site on K-Ras is smaller than the cor- that of BPA. Thereby, BPS apparently loses its antagonis- responding Rheb site. It seems like that rigid, linear mole- ing function in the Sos-meditated nucleotide exchange of cules such as 4,4′-biphenol cannot bind in this pocket, K-Ras4B. whereas the more flexible bisphenolic compound does. Overall, numerous groups were able to show that low Bisphenol A (BPA) is one of the most-produced chemicals molecular weight compounds can selectively bind to Ras worldwide with approximately 10 billion tonnes in 2011 and interfere with the Sos-mediated nucleotide exchange (Vom Saal et al., 2012). BPA, in which central carbon is in H- and K-Ras (Taveras et al., 1997; Spoerner et al., 2001; accompanied by two methyl and two phenolic groups, is Gorfe et al., 2008; Araki et al., 2011; Patgiri et al., 2011; used in food can linings, thermal papers, and other daily Maurer et al., 2012; Sun et al., 2012; Hocker et al., 2013; life plastic products. The main source for human exposal Ostrem et al., 2013; Shima et al., 2013; Min Lim et al., 2014; to bisphenols is based on the uptake through food and Leshchiner et al., 2015; Winter et al., 2015). Apparently, the drinks (Vandenberg et al., 2007). Therefore, bisphenols activity of small GTPases can be indirectly modulated by are found in human serum (Ribeiro-Varandas et al., 2013), through disrupting the Ras/SOS guanine urine (Matsumoto et al., 2003), adipose (Ben-Jonathan nucleotide exchange complex. After decades in search of et al., 2009), and placental tissues (Arbuckle et al., 2015). low molecular weight ligands for small GTPases, recent Outside the human body, significant amounts of bisphe- progress now paves the way for future design of much- nols can be found in drinking and wastewater, air, as well needed GTPase-selective antagonists with higher affinity as dust (Vandenberg et al., 2007). The second main source to benefit the treatment of Rheb-mediated brain disease is thermal paper, used, e.g. cashier receipts, in which the and Ras-driven tumours. BPA is not polymerised. This renders it more available for For membrane-associated proteins such as Rheb exposure than polymerised BPA in resins or plastics (Piv- and Ras, a completely different strategy from the ones nenko et al., 2015). described above aims at masking the crucial C-terminal Lately, BPS has been adopted as a BPA substitute CaaX-box with peptidomimetic receptors. These will because of public pressure and new governmental restric- interfere with or even prevent the membrane insertion tions (Glausiusz, 2014). For example, BPS can be found in and localisation of Ras-proteins that is a prerequisite for ‘BPA-free’-labelled thermal paper (Liao et al., 2012). Also, their normal biological function. Ras-proteins are post- BPS is a known endocrine disruptor and is believed to translationally lipidated by prenyl transferases at the have a comparable physiological effect (Hashimoto et al., C-terminal CaaX-box of Ras-proteins with farnesyl and/ 2001; Grignard et al., 2012). Lately, we turned our atten- or palmitoyl moieties (Figure 2) (Brunsveld et al., 2006; tion to this bisphenolic analogue of BPA. While the two Triola et al., 2012). For Rheb and Ras to be physiologically ring systems in BPA are connected via a sp3-hybridised active, proper membrane attachment and localisation carbon atom, these rings are connected via a sulphonyl is essential even though Rheb is only monofarnesylated group (SO2) in Bisphenol S, which leads to reduced flex- (Buerger et al., 2006). During the past decade, several ibility. Judged from the chemical shift perturbations, BPS inhibitors of these farnesyl transferases (FTIs) could be also binds to K-Ras4B, although with a lower affinity of developed, some of which even entered clinical trials 5.8 ± 0.7 mm (Schoepel et al., 2016). In addition to those (Appels et al., 2005). Although only one farnesyl trans- induced by BPA we observed a shift (twice the stand- ferase farnesylates all GTPases that accept the farnesyl ard deviation) for the amino acid S39, which is part of moiety, FTIs are likely to have side effects on related the switch I region. Obviously, BPS adopts a different, GTPases (Basso et al., 2006) K-Ras4B, one most impor- twisted orientation in concordance with our docking tant oncogenic Ras-proteins, can even be alternatively results (Dominguez et al., 2003), which showed a different prenylated by geranyl-geranyl transferases (Rowell bending angle for BPA and BPS, respectively. et al., 1997). It was also shown that antagonising the M. Schöpel et al.: NMR studies on Rheb and Ras 585 prenyl-binding protein PDEδ with small molecules effi- regarded as the basis for subsequent structure-activity ciently disturbs the spatial organisation and signalling relationship (SAR) studies in order to improve binding of farnesylated K-Ras4B (Zimmermann et al., 2013). To affinities of initial fragment hits in transit to lead struc- interfere with the Ras-prenylation via sequence-selective tures to ultimately achieve a significant therapeutic effect. recognition of the CaaX-box of a specific Ras-protein by This process further benefits from improved methods and a synthetic small receptor molecule has been largely protocols in structural biology, including both X-ray crys- undervalued. This complementary approach to develop tallography and biomolecular NMR spectroscopy. low molecular weight antagonists holds the promise of Among all strategies to target small GTPase like Ras GTPase selectivity as CaaX-boxes sequences of Ras-pro- (Ostrem and Shokat, 2016), the GEF reaction is regarded to teins are different. Like other CaaX-box regions present in be a prime target as it currently holds the greatest promise small GTPases, the C-terminus of Rheb is highly flexible for success within the (near) future. In addition, it now and completely solvent-exposed (Karassek et al., 2010). even seems possible to selectively target tumour-specific The latter feature is an important prerequisite for a suit- Ras mutants, such as G12C and G12D, or even Ras iso- able receptor molecule to recognise its target sequence forms (e.g. K- or H-Ras), which seemed an unachievable (Schmuck et al., 1999). Such potential CaaX-box antago- goal not so long ago (Ostrem and Shokat, 2016). Recently, nists might therefore be less toxic compared to known numerous small molecular compounds have emerged as farnesyl transferase inhibitors. We were able to develop low molecular weight GEF/Ras antagonists (Thaw et al., for the first time more drug-like CaaX-box receptors that 2001; Dominguez et al., 2003; Palmioli et al., 2009; Maurer contain d-, β-, and other non-proteinogenic amino acids. et al., 2012; Schoepel et al., 2013; Min Lim et al., 2014). All These receptors bind to the C-terminal CSVM sequence of these studies have focused on the GDP-loaded form as of Rheb in a sequence-selective manner. Indeed, satu- Ras as it is commonly accepted that this target will proba- ration transfer difference (STD) NMR spectra, a ligand- bly lead to an efficient (and therefore efficacious) antago- based approach, confirm that these receptors also bind nistic lead structure much faster. Allosteric low molecular to the full-length Rheb protein in an aqueous buffer weight compounds with high affinity and selectivity for (Düppe et al., 2014). These pincer-like receptors might certain GTPase or even isoforms thereof have not yet been thus constitute excellent molecular scaffolds for future described. But such compounds promise to perform much peptidomimetic sequence-selective receptors that exhibit better in modulating the interaction of GTPases with GEF higher affinities and may provide another promising new and/or their effectors, which are characterised by rather approach to selectively inactivate Ras proteins. large molecular contact interfaces. Thus, the low molecular compounds that have recently emerged as ligands of the hitherto ‘undrugga- ble’ Ras proteins will be instrumental in modulating the Conclusion and future perspectives GEF/GTPase-mediated signalling cascade that plays a fundamental role in conveying and amplifying biologi- Today – with ever increasing computer power for mole- cal signals. In the light of this progress, a Ras-targeted cular modelling of (protein) receptor complexes (using cancer therapy now does not appear an unreachable goal programmes such as HADDOCK) and the now wide- anymore and the supremacy of NMR spectroscopy com- spread application of fragment-based drug discovery and bined with fragment-based drug design shall substan- development – medicinal chemistry is progressing at an tially contribute to pharmacological and biochemical impressive pace. Together with additional biophysical studies of Rheb- and/or Ras-related malignancies in the techniques, such as surface plasmon resonance (SPR) (not too distant) future. spectroscopy, isothermal titration calorimetry (ITC), microscale thermophoresis technology (MST), the analy- Acknowledgements: We are extremely grateful to Gregor sis of small molecular weight fragments can complement Barchan, Martin Gartmann, and Hans-Jochen Hauswald classical high throughput screening endeavours, which for expert technical help. We are also indebted to all can be described as a fragment-assisted rather than former co-workers on this project, in particular Drs. fragment-based technology (Renaud et al., 2016). NMR Berghaus, Jockers, and Schwarten. This work was sup- spectroscopy uniquely contributes to this process in that ported by a DFG grant (SFB 642, project A6). In addi- in not only reports on the affinity of a ligand for a selected tion, R. S. gratefully recognises generous support from protein target but also reveals vital structural information the BMBF, FCI, Proteincenter (NRW Center of Excel- on this interaction at atomic resolution. This is (often) lence), RUB Protein Research Department, and Krebshilfe 586 M. Schöpel et al.: NMR studies on Rheb and Ras e.V. (109776, 109777). M.S. thanks the Ruhr University Düppe, P.M., Tran Thi Phuong, T., Autzen, J., Schöpel, M., Yip, K.T., Research School­ Plus funded by Germany’s Excellence Ini- Stoll, R., and Scherkenbeck, J. (2014). Sequence-selective molecular recognition of the C-terminal CaaX-boxes of Rheb tiative for financial support. and related Ras-proteins by synthetic receptors. ACS Chem. Biol. 9, 1755–1763. Ehrkamp, A., Herrmann, C., Stoll, R., and Heumann, R. (2013). Ras and Rheb signaling in survival and cell death. Cancers (Basel) References 5, 639–661. 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