direct attack on ras: intramolecular communication and ... · terms of hydrolysis rates and...

Direct Attack on RAS: IntramolecularCommunication and Mutation-Specific EffectsKendra Marcus and Carla Mattos

Abstract

The crystal structure of RAS was first solved 25 years ago. Inspite of tremendous and sustained efforts, there are still nodrugs in the clinic that directly target this major driver of humancancers. Recent success in the discovery of compounds that bindRAS and inhibit signaling has fueled renewed enthusiasm, andin-depth understanding of the structure and function of RAShas opened new avenues for direct targeting. To succeed, wemust focus on the molecular details of the RAS structure andunderstand at a high-resolution level how the oncogenicmutants impair function. Structural networks of intramolecularcommunication between the RAS active site and membrane-

interacting regions on the G-domain are disrupted in oncogenicmutants. Although conserved across the isoforms, these net-works are near hot spots of protein–ligand interactions withamino acid composition that varies among RAS proteins. Thesedifferences could have an effect on stabilization of conforma-tional states of interest in attenuating signaling through RAS.The development of strategies to target these novel sites will adda fresh direction in the quest to conquer RAS-driven cancers.Clin Cancer Res; 21(8); 1810–8. �2015 AACR.

See all articles in this CCR Focus section, "Targeting RAS-Driven Cancers."

IntroductionRAS GTPase is a master signaling protein at the hub of

numerous signal transduction pathways, responding toupstream signals from a variety of sources and interacting witha wide range of effector proteins to regulate cell proliferation,survival, migration, and apoptosis (1). Signal transductionpathways involving RAS are illustrated in McCormick's over-view of this CCR Focus series (2). The three major isoforms ofRAS, KRAS, NRAS, and HRAS together are mutated in about20% of human cancers, primarily in the active site at residuesG12, G13, and Q61 near the g-phosphate of the guanosinetriphosphate (GTP) substrate (Fig. 1). RAS GTPases are, there-fore, among the most urgent targets for the treatment of cancer.Yet, to date, there are no drugs in the clinic that directly targetRAS, and thus, these proteins have come to be thought of asundruggable. The current state of directly drugging the G-domain of RAS is the subject of this review. Targeting post-translational modifications at the C-terminus is discussed byCox and colleagues (3) in this CCR Focus series, while alterna-tive strategies focused on altered metabolic pathways driven byRAS mutants and synthetic lethality approaches are reviewed byKimmelman (4) and Downward (5), respectively.

RAS functions as an on/off switch regulated by guanine nucle-otide exchange factors (GEF), such as SOS, to facilitate loading ofGTP turning the signal on, and by GTPase-activating proteins

(GAP), such as p120GAP and neurofibromin (NF1), that increasethe very slow RAS intrinsic GTP hydrolysis rate to turn the signaloff (6). This cycle is represented in Fig. 1 in the article by Cox andcolleagues (3). Although this canonical cycle is the regulatory holygrail for RAS and related GTPases, a high-resolution view revealsvariations that embellish and complement this cycle based ondetailed structural features. RAS is composed of the core G-domain containing the catalytic machinery and a lipidated C-terminal hypervariable region with low homology between iso-forms, which tethers the G-domain to the membrane and driveslocalization to specific microdomains (7). The G-domain of RASis composed of a 6-stranded b-sheet flanked by 5a-helices and 10connecting loops with two switch regions, switch I and switch II,that have different conformations in theGDP (guanosine diphos-phate) andGTP-bound states (Fig. 1 and Fig. 2; ref. 8). Residues intheP-loop interact closelywith the nucleotide.Within the range ofconformations accessed by the switch regions in RAS–GTP, switchI has two major states: One in which it is open, away from thenucleotide (state 1) and the other where it is closed over thenucleotide (state 2; Fig. 2A; ref. 9). Switch I must be in state 2 tointeract with effector proteins (10). Switch II contains the catalyticresidueQ61 and is normally highly disordered (11), resulting in avery slow hydrolysis rate in the absence of GAPs (12). We haveshown an association between allostericmodulation and intrinsichydrolysis in the presence of RAF that would be expected to takeplace independent of GAPs (13–16). In our current model, theGTP hydrolysis rate is expected to be increased in the RAS/RAFcomplex by binding of Ca2þ at a remote allosteric site, resulting ina conformational shift from a catalytically impaired T state(disordered active site) to the competent R state in which thecatalytic residue Q61 is ordered in the active site (Fig. 2B). Notethat accompanying this disorder to order transition is a shift inhelix3/loop 7 toward helix 4. Given the canonical GTPase cycle,major points of interference expected to result in signal attenu-ation through RAS are the inhibition of exchange of GDP toGTP catalyzed by GEFs and the inhibition of interaction between

Department of Chemistry and Chemical Biology, Northeastern Uni-versity, Boston, Massachusetts.

Corresponding Author: Carla Mattos, Department of Chemistry and ChemicalBiology, Northeastern University, 102 Hurtig Hall, 360 Huntington Avenue,Boston, MA 02115. Phone: 617-373-6166; Fax: 617-373-8795; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-14-2148

�2015 American Association for Cancer Research.

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RAS–GTPand its effector proteins (Fig. 3). Stabilizationof distinctconformational states opens new opportunities for targeting RASdirectly. Switch I stabilized in state 1 would have greatly dimin-ished interaction with effectors and switch II stabilized in theordered R state would promote intrinsic hydrolysis (Fig. 3). Thisreview first focuses on the accomplishments achieved so far interms of small-molecule inhibitors that directly interact with RAS.We then turn to an analysis of the structural details associatedwithRAS–GTP conformational states and communication betweenremote areas of the G-domain, as well as the effect of oncogenicmutations. This structural understanding suggests unexploredstrategies to move forward in new directions.

The State of Small Molecules That DirectlyBind RAS

The problemof drugging RAS for treatment of human cancers isa major challenge for the academic and industry research com-munities, and the limited success attained so far has recentlyconverged in a call for renewed efforts to conquer this seeminglyinsurmountable problem (17, 18). Numerous reviews on thistopic have been published in the last 2 years (19–27). Thesearticles outline the diverse strategies used to target RAS and relatedsignaling pathways, including direct targeting. The picomolaraffinity of RAS for GDP and GTP, coupled with sub-millimolarconcentrations of these nucleotides in the cell, make the prospectof developing drugs that effectively compete with the nucleotidegenerally unrealistic. Furthermore, the lack of other well-definedactive site pockets have left us with limited options for directlytargeting RAS. The exchange of GDP for GTP, catalyzed by GEFs,turns on signaling through Ras, promoting interaction witheffector proteins (1). Hydrolysis of GTP toGDP is the key reactionthrough which RAS signaling is turned off and is the center of

action for oncogenicmutations at G12, G13, andQ61 that impairboth intrinsic and GAP-catalyzed hydrolysis (28). The challengeof directly targeting the RAS G-domain, thus, amounts to threeapproaches, all of which have been attempted with little ormoderate success: increasing hydrolysis rates of mutant RAS(29, 30), inhibiting the exchange of GDP for GTP (31–39), orinterfering with effector binding and activation (40–49). Recentefforts based on high-throughput screening of fragments to iden-tify compounds that affect downstream signaling, accompaniedby crystal structures that allowed structure-based optimization insome cases, have for the first time provided a high-resolution viewof how small molecules are accommodated in binding pocketsnear the active site to interfere with RAS signaling (33–35, 39).Within this cohort of structures are covalent inhibitors ofKRASG12C (33, 35), a mutant commonly found in lung adeno-carcinoma (28), bringing to the forefront the prospect of selec-tively targeting this specific mutant with minimal effect on wild-type RAS. This kind of specificity will be difficult to achieve withother oncogenicmutants due to their less reactive side chains.Oneof the two covalent series of inhibitors consists of nucleotideanalogues that bind in place of GDP (33), and the other series ofinhibitors bind in a pocket between switch II and helix 3 (35).Other compounds for which there are crystal structures are notselective for particular mutants and bind at a site on the outer sideof switch II (34, 39). All of these compounds with high-resolutioncrystal structures bind to the GDP-bound form of RAS on eitherside of switch II, inhibiting nucleotide exchange catalyzedbyGEFs(Fig. 4A).Given that oncogenic RASmutants retainRAS in itsGTP-bound form or promote rapid exchange in the absence of GEFs(50), it has been proposed that nucleotide exchange inhibitorsmay be best used in cancers bearingwild-type RAS (22).However,an increasing understanding of the role of thewild-type protein inpromoting oncogenic RAS-driven cancers (51, 52) suggests that

© 2015 American Association for Cancer Research

G12

Q61

KRAS NRAS HRASG12

G13 G13 G13

Q61 Q61 Q61

G12 G12G12DG12VG12CG12AG12SG12R

G12DG12SG12CG12VG12AG12R

G12VG12SG12DG12CG12RG12A

G13DG13CG13SG13RG13AG13V

G13DG13RG13VG13CG13AG13S

G13RG13DG13VG13SG13C

Q61HQ61LQ61RQ61KQ61PQ61E

Q61RQ61KQ61LQ61HQ61PQ61E

Q61RQ61LQ61KQ61HQ61PQ61E

G13

SwitchII

SwitchI

P-loop

GppNHp

α4

α2

α5

α1

α3

Figure 1.Location and identity of oncogenicmutations atG12, G13, andQ61 onRAS–GTP. The RAS structure is shown onthe left in ribbon diagram bound to theGTP analogue 50-guanylylimidodiphosphate (GppNHp). Thefocus is on the active site with theP-loop, switch I, and switch II labeled inRoman type. The 5 a-helices, a1-a5,are also labeled in Roman type.Residues G12 (cyan), G13 (magenta),and Q61 (light orange) are drawnexplicitly, with residue labels inboldface type and with N, Ca, C ¼ O,and side chain groups shown explicitly.N atoms are in blue, O atoms are in red,and the P atoms of the nucleotide arein dark orange. The identities of themutations found in G12, G13, and Q61 inKRAS, NRAS, and HRAS are listed onthe right in the order corresponding tohow often they occur in humancancers (modified from ref. 1 andcrosschecked with ref. 28).

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GEF inhibitors may have a role to play in these types of cancers aswell.

Targeting RAS–GTP has produced a few low-affinity inhibitorsof RAS–effector interactions accompanied by NMR informationon the general binding area for the compounds near switch I(Fig. 4B; refs. 44–46). One of the compounds has a secondbinding site near loop 7 and the C-terminus, visible in a crystalstructure of RAS (44). These compounds stabilize switch I in anopen conformation that disfavors interaction with effector pro-teins. This mode of binding illustrates a strategy associated withstabilization of conformational states that takes advantage ofcontrolling accessibility to functionally important conformers.In RAS–GTP, when switch I is open in state 1 Y32 is far from theactive site where it obstructs the binding of RAF–RBD (RAS-binding domain) and other effectors, while in the closed state 2Y32 becomes part of the active site, allowing interaction witheffector proteins (9, 10). A recent crystal structure of HRAS in theGTP-bound form in which switch I is in state 1 reveals a bindingpocket between switch I and the nucleotide that may be used forstructure-based ligand development (53). This is only one angleon the more general strategy of stabilizing conformational statesthat may offer windows of opportunity for direct targeting. RASis full of this type of opportunity throughout its small butcomplex structure, where intramolecular communication net-works between distant sites on the RAS G-domain have beendiscovered in the past few years (13, 54–56), but remain largelyunexplored. To open new opportunities for drugging RAS, wemust understand these structural features in depth, their relativeimportance in KRAS, NRAS, and HRAS, as well as the ways inwhich specific mutations at G12, G13, and Q61 affect them interms of hydrolysis rates and communication with themembrane.

Intramolecular Communication in theStructure of Wild-type Isoforms of RASBound to GTP

The Ras G-domain is subdivided in two lobes (57). The first(residues 1-86), identical in sequence between the Ras isoforms,contains the nucleotide-binding site, including the P-loop (resi-dues 10-17), as well as the switch I (residues 30-40) and switch II(residues 60-76) regions that recognize effector and regulatoryproteins, and thus is referred to as the effector lobe (58). Thesecond contains several membrane-interacting regions that linkelements of allosteric communication between the active site andthemembrane (13, 55, 59), and thus is referred to as the allostericlobe (58). This lobe is 90% identical between the isoforms, withmost differences residing in sites of protein–membrane interac-tions, thus conferring specificity to the way in which the G-domain of KRAS, NRAS, and HRAS interacts and communicateswith the membrane (60).

A challenging aspect of directly targeting RAS in its GTP-bound form is the fact that switch I and switch II are disorderedin solution (11, 58, 61), as is also the case in RAS–GDP (62),making it difficult for small molecules to bind. When bound toRAF–RBD, switch I is in state 2 and Y32 interacts with theg-phosphate of GTP via a bridging water molecule (Fig. 5). Thisbrings Y32 into reach of a water-mediated H-bonding networkthat leads to N85 at the N-terminal end of helix 3 (56) andonto nucleotide-sensing residues R161 and R164 in helix 5 andD47 and E49 in the effector lobe loop L3 (55). This water-mediated H-bonding network is highly conserved in HRAS–GTP, but not present in HRAS–GDP, with switch I providing astructural venue for communication between the g-phosphateof GTP and the membrane through the allosteric lobe (56).


L7

State 1

State 1 vs. state 2 T state vs. R state

Allostericsite

L7

T state

State 2

SwitchII

SwitchII

Switch I Switch IGppNHp GppNHp

a2

a3

a4

a1

a4a3

a2

A B R stateFigure 2.Conformational states associated withRAS–GTP. The RAS structures areshown in ribbon diagram with switch Iand switch II labeled. The elements ofsecondary structure discussed in thetext are also labeled. All structures arebound to the GTP analogue GppNHp,shown explicitly. A, switch I in state 1 isrepresented by the HRAS structurewith PDB code 4EFL (pink), and state2 is represented by the HRAS structurewith PDB code 3K8Y (green). B, switchII in the T state is represented by theHRAS structure with PDB code 2RGE(blue), and the R state is representedby the HRAS structure with PDB code3K8Y (green). Note that the model inthe T state (blue) does not contain theN-terminal end of switch II (includingQ61) due to disorder in that part of thestructure. Also note the Ca2þ andacetate bound in the allosteric site inthe R state structure (green), withaccompanying shift in helix 3/loop7 (a3/L7) toward helix 4 (a4).

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The GTP-bound orientation of RAS with respect to the mem-brane is mediated through R128 and R135 in helix 4 inresponse to the nucleotide sensor residues (55, 59), and thusthis intramolecular network that connects switch I to theallosteric lobe may have an impact on the orientation of theG-domain.

Switch II also has been observed in distinct conformationsassociated with T and R states of allosteric modulation of theactive site, given a state 2 conformation of switch I (16). Althoughnormally disordered in crystals in the T state, switch II becomeshighly ordered by binding of small molecules at a remote allo-steric site found between helix 3, loop 7, and helix 4 (Fig. 2B;ref. 13). In crystals in which switch I has the conformationobserved in the RAS/RAF–RBD complex (15), binding of calciumat the allosteric site results in a shift of helix 3 toward helix 4 andaway from switch II, making room for the switch II helix tobecome ordered (13). This results in positioning of R68 at thecenter of an H-bonding network that facilitates placement ofcatalytic residue Q61 in the active site. Thus, in its effector-boundR state, switch II is connected to the membrane-interacting allo-steric lobe through an H-bonding network to the allosteric site.Residues in the intramolecular communication networks fromswitch I and switch II in the effector lobe to the membraneinteracting allosteric lobe described above are conserved in thethree isoforms (Fig. 5), implicating the interlobal connectivity ofthe G-domain in the fundamental mechanisms through whichRAS functions.

Binding Pockets on the Allosteric Lobe ofRAS Differ between the Isoforms

Screening of large compound libraries resulted in the iden-tification of molecules that inhibit RAS signaling, as summa-rized above. In a very different approach, a smaller group ofgeneric compounds or probe molecules have been used withboth experimental and computational methods to identify thelocation of binding site hot spots on RAS–GTP in search ofpockets that can be explored in drug discovery (58, 63). Inaddition to identifying known sites on the effector lobe of RAS–GTP, this work identified hot spots of protein–ligand interac-tions on the allosteric lobe that are not far from the elementsinvolved in the interlobal communication networks discussedabove. These hot spots are key targets of opportunity, as theyare not identical between the isoforms (60) and to date mosthave been unexplored.

The locations of identified hot spots on the RAS–GTP effectorlobe include the binding pocket near switch I known as thebinding site for RAF–RBD (site 1) and the binding site on theouter side of switch II (site 2), while the binding site betweenswitch II and helix 3 is found in the interlobal region (site 3; Fig.5A). Sites 2 and 3 are also found to bind small molecules in RAS–GDP (Fig. 4A). These three sites, as well as the site near loop 7mentioned above (Fig. 4B), are identified as high consensus sitesbecause they are found repeatedly in several studies (25). Site 3between switch II and helix 3 is of note because, in spite of it being


Covalentinhibitors

RAS/GEF

RAS/RAFinhibitors

Cell proliferation,survival

State 1inhibitors

RAS/RAF

RAS/GAP

Intrinsichydrolysis

Hydrolysisstimulation,

R state

RAS-GDP

RAS-GTP, state 2

RAS-GTP, state 1

GEFinhibitors

Figure 3.The GTPase cycle, including thenuances of conformational states. RASis shown in cyan, with switch I in lightblue and switch II in yellow. The GEFSOS is in a dark brick color and GAP isin pink-gray. Black crosses indicateopportunities for blocking interactionsthat result in signal propagationthrough RAS, and the thick blackarrow associated with stabilization ofthe R state indicates enhancedhydrolysis of GTP. GEF and covalentinhibitors target RAS–GDP, while RAFinhibitors, inhibitors that stabilizestate 1 (state 1 inhibitors), or potentialinhibitors that stabilize the R state tostimulate hydrolysis, target RAS–GTP.Note the distinction betweeninhibitors that prevent binding of RAFto RAS in state 2 and the state 1inhibitors that stabilize a RASconformation that intrinsically doesnot bind RAF.





a strong hot spot of protein–ligand interaction, every smallmolecule known to bind at this site in RAS–GTP is observed tostabilize an anticatalytic conformation associated with the T stateof the allosteric switch (58, 64). Thus, targeting the site betweenswitch II and helix 3 might inadvertently result in prolongedsignaling through RAS–RAF in some situations. Because RAF doesnot bind switch II (15), a compound at this site would be unlikelyto have a significant effect on RAF binding while abrogatinghydrolysis of GTP, working against the goal of inhibiting RASsignaling. Although no natural binding partners have been iden-tified to interact with RAS at site 3, importin-b occupies it in RANGTPase to halt hydrolysis during transport of cargo into thenucleus (65).

All of the sites identified on the allosteric lobe other than thealready mentioned site near loop 7 (site 5) have been classifiedas low consensus sites because they have been found in a singlestudy (58) and no compounds from large library screens havebeen found to bind at those sites. Interestingly, however, theamino acid composition of these sites varies among the isoforms(60) and they are found conspicuously close to the elements of theinterlobal communication networks in RAS–GTP. Figure 5 showsthe sites of interest in the allosteric lobe and their relationship tothe H-bonding networks that connect the two lobes. One of thesites is between helices 3 and 4, adjacent to the allosteric site (site6; Fig. 5A), and compounds that bind there interact with the sidechain hydroxyl group of Y137 (58), whose main chain carbonylgroup coordinates the Ca2þ in the R state of the allosteric switch(13). Twoother sites are between helices 4 and 5 and contain helix4 residues R128 (site 7) and R135 (site 8), respectively (Fig. 5B).Residues R128 and R135 helpmaintain an orientation of the RASG-domain with respect to the membrane conducive to effectorbinding and were shown to abrogate signaling when mutated incells (59). Another low consensus hot spot identified by the samestudy with an experimental approach (58) is at the interfacebetween the effector and allosteric lobes and is known to bind

RAF–CRD (cystein-rich domain), the second RAS-bindingdomain found in RAF (site 4; Fig. 5B; ref. 66). Binding at thissite can potentially have an effect on the switch I to helix 5communication network associated with nucleotide sensing resi-dues in helix 5 near the membrane or affect the orientation of theG-domain at the membrane surface.

Effects of Oncogenic Mutations onIntramolecular Communication Networksin RAS–GTP

It is well known that oncogenic RAS mutants are insensitive toGAPs (67–70). This is a difficult problem to overcome, as themutations themselves severely perturb the active site and impairthe ability of GAPs to enhance GTP hydrolysis even upon bindingRAS (50, 71). If it is indeed the case that the duration of the RAS–RAF interaction is regulated by intrinsic hydrolysis activated bymembrane interaction and calcium binding at the allosteric site(13, 14, 16, 56), there an excitingwindowofopportunity exists fortargeting signaling through RAS–RAF enhanced by some onco-genicmutants. This is of interest because the RAS–RAF–MEK–ERKpathway is a potentmitogenic pathway and an aggressive driver ofseveral types of cancers (72). Intrinsic hydrolysis is dependent onan ordered active site with an intact communication network tothe allosteric site that requires the R state positioning of helix 3/loop 7 and correct placement of the R68 link to Q61 throughwater-mediated H-bonds (Fig. 5A). It has been shown that bothG12V and Q61L can attain the R and T states of the allostericswitch under circumstances where switch I is in state 2 (14), as it iswhen bound to RAF, and it is likely that other oncogenic mutantscan as well.

In structures available for the G12 and Q61 mutants, those inwhich switch I is in state 2 and the allosteric switch is in the R state(14, 34, 35, 67, 73, 74), with the exception of G12D, the bridgingwatermolecule seen in thewild-type protein is not present and Y32


State 1inhibitor

Rafinhibitors

GDP

L7L7

SwitchII

SwitchII

Switch I

Covalentinhibitor

(35)

GEF inhibitors(34, 39)

State 1 inhibitor(44)

Switch I

GppNHp

a5

a4

a3

a1

a4

a3

a2

a5

a2

A B Figure 4.Binding sites on RAS for currentlypublished inhibitors. RAS is shown inribbon diagram with labeledsecondary structural elements. A,binding sites for inhibitors that bindRAS–GDP with representativemolecules on either side of switch II.Protein residues that interact withinhibitors between switch II and helix 3(a3) are colored blue, and those thatinteract on the outer side of switch IIand the b-sheet are colored orange. B,binding sites for inhibitors that bind toRAS–GTP. The one inhibitor for whichthere is a crystal structure is shownnear loop 7 (L7) and the C-terminalend of helix 5 (a5) with associatedresidues in green. There are no crystalstructures for molecules bound toswitch I, but residues with which theseinhibitors are known to interact areshown in red. Numbers in parenthesesare references to articles in which therepresentative inhibitor moleculesshown in this figure were published.

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makes a direct H-bond with the g-phosphate of GTP (Fig. 6). Thearrangement in wild-type RAS is shown in Fig. 6, with the bridgingwater molecule within H-bonding distance of Y32, the g-phos-phate, and of Q61. RASG12V and RASQ61L, superimposed on thewild-type, are representative examples of the direct H-bondbetween Y32 and the g-phosphate in most oncogenic mutants(Fig. 6A). This results in a severed connection between switch I andthe helix 5 nucleotide sensor residues associated with membraneorientation (54). In the G12D mutant, for which there are twocrystal structures, the bridging water molecule is either replacedwith one of the side-chain oxygen atoms of the D12 residue or ispresent and D12 replaces Q61 in its interaction with the bridgingwater molecule (Fig. 6B). Contrary to what is observed in all otheroncogenic mutants, the water-mediated network from the g-phos-phate to the nucleotide sensing residues in helix 5 is intact in bothstructures ofRASG12D, and thismay contribute to the effectivenessand frequency of this mutant in human cancers (20, 28). The twostructures differ in their effects on switch II. When D12 replaces thebridging water molecule, the side chain of Q61 is rotated towardthe solvent. The network from the allosteric site to R68 is intact, butthe connection to Q61 is impaired because of steric hindrance inthe active site.WhenD12 takes the positionofQ61, theN-terminusof switch IImovesoutofplace, completely severing the connectivityto the allosteric site. All of the mutants in structures where helix 3/loop7 is in theR state affect switch II inoneof these twoways.G12VandG12Caffect the connectivity to the active sitewith respect to theQ61 rotamer, and could potentially benefit from stabilization ofthe R state by ligand binding at the allosteric site or possibly in one

of the nearby allosteric lobe sites topromote the shift inhelix 3. Fullwild-type rates will not be achieved because of the perturbed activesite.However, it has been argued that a 30- to50-fold enhancementof the hydrolysis rate may result in significant anticancer activity(75), and it is worth testing whether this is possible for the G12VandG12Cmutants. TheG12R andQ61Hmutants displace residue61 from the active site, abrogating connectivity to the allostericlobe, and are less likely to respond to ligand binding aimed atpromoting the R state. The G12D andQ61mutants directly impairthe chemistry of GTP hydrolysis by either placing a negative chargeright over the nucleotide or removing an important catalyticresidue. Furthermore, Q61L is also unique in that it has a strongtendency to stabilize the T state in the anticatalytic conformationwhen bound to RAF, abolishing even the slow hydrolysis rateobserved in the uncomplexed protein (14, 15, 73).

In the absence of binding partners, both switch I and switch IIare disordered in solution (11, 61), and they become stabilized byinteractions with other proteins (15, 30, 76, 77). In the crystalstructures we have discussed, switch I is stabilized in state 2 bycrystal contacts in the conformation found in the RAS/RAF–RBDcomplex (15) or in the RAS/NORE1A complex (78) and switch IIis less constrained by crystal contacts. This may not always be thecase. Therefore, in general one must be vigilant in taking intoaccount the effects of crystal contacts. Remarkably for RAS, mostof the switch I conformations stabilized by crystal contacts are alsoobserved in a biologically relevant complex (64). Given a carefulstructural analysis, the kind of information discussed herein willbe invaluable in obtaining insights leading to specific targeting of


L7A B

L3

L7 D107

6

N86

7

8

1

E47R161

4

R135

R128

E49

R164

5D107

Allostericsite

Allostericsite

6

8

3H94

R97R68

2

SW II

SW I

1

N86

Y32

Q61

Y137

α4

α5

α1

β2

α4

α2

α35

Figure 5.Interlobal communication networks and ligand-binding hot spots on RAS in its GTP-bound form. RAS is shown in ribbon diagram (PDB code 3K8Y), withresidues conserved in the KRAS, NRAS, andHRAS isoforms shown in gray, those appearing in two of the isoforms in yellow, and those that are distinct in each isoformin red. The GTP analogue GppNHp is shown in the active site near switch I (SWI) and switch II (SWII). Calcium and acetate are green and shown in the allosteric site.Residues involved in the communication networks are shown explicitly. Two orientations are shown in A and B, rotated approximately 180 degrees relativeto each other. The communication network from SWII to the allosteric site and from SWI to the nucleotide sensor residues in helix 5/loop 3 (a5/L3) isshown in black dashed lines. Binding site hot spots are shown in spheres. Two sites in the effector lobe are shown in olive green: the RAF-RBD–binding pocket nearSWI (site 1) and the site on the outer side of SWII (site 2). Two sites in the interlobal region are shown in cyan: the site between SWII and helix 3 (a3; site 3)and the RAF-CRD–binding site between helix 1 (a1), beta sheet 2 (b2), and helix 5 (a5; site 4). Four sites in the allosteric lobe are shown in violet: the site near loop 7(L7) opposite the allosteric site (site 5), the site between helices a3 and a4 near Y137 and H94 (site 6), and two sites between helices a4 and a5 near R128and R135, respectively (sites 7 and 8). Amino acid residues are in boldface type, and secondary structural elements of proteins are in Roman type.





RAS mutants. Although focusing on the allosteric site and nearbypockets on the allosteric lobe (sites 5 and 6) may target RAS in amutant-specific fashion, targeting the sites near R128 and R135between helices 4 and 5 (sites 7 and 8) is likely to work moregenerally by disrupting the functional orientation of the G-domain relative to the membrane, which is specific for each ofthe isoforms (54, 55, 79, 80), possibly superseding the effects ofparticular mutations.

ConclusionsTargeting RAS against cancers has been a challenge for the past

25 years. Yet recent progress in the discovery of small moleculesthat bind directly to the G-domain with inhibitory effects on itsfunction is exciting and encouraging. For RAS–GTP, focusing onthe RAS–effector interaction at the active site by stabilizing state 1is particularly promising, as this is the most direct way to inhibitRAS signaling, although nonspecific with respect to particularisoforms. In-depth understanding of the structure and functionof RAS and its interaction with themembrane opens new avenuesfor targeting isoforms or specific mutations that are in need offurther development. Targeting the allosteric lobe will requireassays that detect binding at those sites in high-throughputscreens, and the hypotheses derived from structural analysis ofspecific mutants regarding the activation of the allosteric switchneed to be confirmed. Another unexplored area of opportunity is

interaction between the C-terminal hypervariable region andswitch II in K-RAS4B with calmodulin, which results in signalattenuation (81–84). Although promotion of protein–proteininteraction is not trivial, it is not unprecedented (85) and shouldbe explored in the future. Although treatment of RAS-drivencancerswill continue to require a cocktail of inhibitors ofmultiplepathways (86), the rapidly increasing knowledge of RAS structureand function fuels the vision that among available inhibitors inthe future will be a drug that directly binds to RAS.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: K. Marcus, C. MattosAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K. Marcus, C. MattosWriting, review, and/or revision of the manuscript: K. Marcus, C. MattosAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): K. Marcus

Grant SupportThis work was supported by a grant from the National Science Foundation

(MCB-1244203; to C. Mattos).

Received December 12, 2014; revised February 17, 2015; accepted February24, 2015; published online April 15, 2015.


SW II SW II

SW I

SW IG/V12

Bridgingwater

Bridgingwater

Y32

L61

Y32

K117

N85

N86

Q61Q61

Q61

G12D

Q61Q61

GppNHp K117

N85

N86

GppNHp

α3

α3

A B

Figure 6.The active site of wild-type and mutant RAS proteins with switch I in the effector-bound conformation (state 2). Wild-type HRAS and its associated watermolecules in the network are in green (PDB code 3K8Y). The network fromN85 to the g-phosphate of GTP is shown in black dashed lines. Note that this network alsoextends to Q61 in wild-type RAS. A, the oncogenic mutants other than G12D, represented by HRASG12V shown in pink (PDB code 3OIW) and HRASQ61Lin yellow (PDB code 3OID). Note the direct H-bond between Y32 and the g-phosphate of the nucleotide shown in red dashed lines. The bridging water molecule ismissing in the mutant structures. B, the G12D mutant in HRAS and KRAS. HRASG12D is in cyan (PDB code 1AGP). The D12 residue takes the place of thebridging water molecule in this structure. KRASG12D is in orange (PDB code 4DSN). The bridging water molecule is present in this structure, and the D12 side chaintakes the place of the Q61 side chain inmaking an H-bond to the bridgingwatermolecule, displacing the N-terminal end of SWII near the active site. Red dashed linesare shown for the active site H-bonding interactions in the two G12D structures. In both structures Y32 is in a position to connect to the water network toN85,which in turn continues on to the nucleotide sensor residues in helix 5, R161, andR164 (shown in Fig. 5B). Amino acid residues are in boldface type, and secondarystructural elements of proteins are in Roman type.

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