[CANCER RESEARCH54. 5438-5444, October 15, 1994]

ABSTRACT

The bovine complementary DNA encoding the catalytic domain of RasGTPase activating protein was mutagenized semirandomly using a vanatlon of the polymerase chain reaction. Sixty-four mutated codons wereidentified with seventeen of the mutations deleterious to Ras GTPaseactivating function. All of the inactivating single mutations affected thestructure of the catalytic fragment as assessed by large decreases insoluble protein when expressed in Escherichia coli. Upon exnmlnation ofthe Ran binding properties of 10 of the mutants, only 1 was measurablyImpaired for I(as binding and 4 appeared to have increased affinity forRan. These results demonstrate that Ras binding and GTPase activationare two distinct properties of GTPase activating protein. Additionally, thecatalytic mechanism of GTPase activating protein Is much more sensitiveto structural perturbation than is Ras binding.

INTRODUCTION

Mutations in the ras proto-oncogenes have been implicated in theinitiation and proliferation of tumors in both animal carcinogenesismodels and human cancer (1). Oncogenic mutations result in a modified form of the Ras protein, which is refractory to negative regulation, resulting in a constant proliferative signal (2, 3). Ras is a smallMr 21,000 membrane-associated protein which shares many biochemical characteristics with the a subunits of the prototypical heterotrimeric 0-proteins (4). Ras binds both GDP and GTP with high affinity,possesses an intrinsic GTPase, and requires membrane association fornormal function. Current evidence suggests that the primary mitogenic effector of Ras is the Raf kinase. Ras-GTP binds to the Rafserine/threonine kinase and is believed to assist in its activation (5—8).GAP3 is known to function as a negative regulator of Ras through thedramatic stimulation of the slow intrinsic GTPase of the Ras protein.

In addition to being a negative Ras regulator, GAP has also beenimplicated as a component necessary for some Ras-dependent effects inmammalian cells and amphibian oocytes. In vitro evidence for a Ras

dependent effector role for GAP has been demonstrated in at least threeseparate model systems. Ras and GAP cooperate in an SH2/5H3 domaindependent manner to inhibit muscarinic gated potassium channel activation in patch-clamped atrial membranes (9, 10). Activation of Xenopus1@zevisp34 kinase (maturation promoting factor) by Ras has also been

suggested to be GAP-dependent (11), with Ras induction of germinalvesicle breakdown requiring the GAP SH3 domain (12). GAP has also

been implicated by expression-competition experiments as having posifive transcriptional effects on the Ras-dependent polyoma enhancer (13).The most conclusive evidence for a signaling role for GAP comes froma series of experiments that showed involvement of the NH2-terminal

Received 6/2/94; accepted 8/17/94.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

I This work was supported in part by the Project Development Program, Research and

Sponsored Programs, Indiana University at Indianapolis.2 To whom requests for reprints should be addressed, at Walther Oncology Center,

lndiana University School of Medicine, 975 West Walnut Street, Room 501, Indianapolis,1N 46202-5121.

3 The abbreviations used are: GAP, GTPase activating protein; PCR, polymerase chain

reaction.

region of GAP in the regulation of cytoskeletal structure and cell adhesion to the extracellular matrix (14). Expression of the GAP NH2-terminal SH2-SH3-SH2 region in Rat-2 fibroblasts resulted in a disruptionofactinstressfibersandfocalcontacts.Thiseffectisprobablycausedby the action of the GAP-associated protein, p190, and Rho (15-17).Changes in the cytoskeleton and loss of adhesion are important components of cell growth.

In addition to its potential as a Ras effector molecule, GAP iscurrently the best model for examining GTP-dependent protein interactions with Ras. Ras-GTP has been shown to bind to GAP, whileRas-GDP does not (18). In addition, many mutations within theso-called Ras effector region, which block cellular transformation,also reduces the GAP sensitivity of the GTPase of the mutant or theability of the protein to bind GAP (19, 20). The primary Ras bindingsite of GAP has been localized to the carboxy-terminal 343 aminoacids and is referred to as the GAP catalytic domain (21). This regioncan be expressed as a stable truncated protein in Escherichia coli,which can bind Ras-GTP and activate the Ras GTPase. The Rasbinding domain of GAP is conserved in other eukaryotic proteinspossessing Ras-GTPase activating function. It is not known if Rasbinds a smallregionof GAPanalogousto the Ras effectorregionora larger area of the surface typical of most protein-protein interactions.

The goal of this study has been to use a structurally unbiasedapproach in order to identify specific amino acid residues involved inthe binding of GAP to Ras. Identification of discrete sites of proteininteraction on the Ras protein suggests that analogous sites may existon GAP. The DNA encoding the catalytic fragment of GAP wasmutagenized using a semirandom method; mutant proteins were identified and biochemically characterized. Inactivating mutations werefound in 17 distinct codons, while mutations in 47 other codons hadlittle effect. All of the inactivating single codon mutations were foundto significantly reduce the quantity of soluble protein produced inE. coli, suggesting that gross conformational changes had occurred.Only one of these mutants was found to have reduced binding affinityfor Ras. These results suggest that GAP interacts with Ras, eitherthrough a few specific contacts scattered throughout the primaryamino acid sequence or through a large surface which is unaffected bypoint substitutions. The activation of Ras GTPase by GAP is mucheasier to disrupt than binding alone, indicating that GTPase stimulation requires a more conformationally defined structure which is notrequired for binding.

MATERIALS AND METHODS

Mutagenesis of the GAP gene Semirandom base misincorporations wereintroduced into the bovine GAP complementary DNA by PCR using Taqpolymerase (Perkin-Elmer; Ref. 22). The reaction conditions used were asrecommended by the manufacturer except that the concentration of dATP waslimiting (40 @LMversus 200 pM dGTP, dTFP, and dCTP). Reduction of dATPresulted in limited random misincorporation of deoxyribonucleotides whenever an “A―(deoxyadenosine) was required during primer extension of theDNA. The addition of 0.1 mMMnCl2to the reaction further increased the rateof base misincorporation. Use of the oligonucleotide primers 5'-GCCCATAAACFCCCAGTAAAG-3' and 5'-CGCO@GCAGAAUAGCfCACACAT

5438

Structural Analysis of the Ras GTPase Activating Protein Catalytic Domain bySemirandom Mutagenesis: Implications for a Mechanism ofInteraction with Ras-GTP1

Lisa Hettich and Mark Marshall2

Department of Medicine, Division of Hematology/Oncology. Indiana UniversüySchool of Medicine and Waither Oncology Center, Indianapolis. indiana 46202

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RANDOM MUTAGENESIS OF RASGAP

(Qiagen) for 1 h at 4°C.The agarose was then washed twice in batch with 50ml of buffer A, packed into a small column, and connected to a PharmaciaFPLC pump and detector. After further washing, bound proteins were elutedwith a 0—0.250 M imidazole gradient. The A@-absothing peak was concentrated in a Centricon-lO concentrator (Amicon), washed twice with TED,assayed for GAP activity, and immunoblotted for GAP protein. Althoughwild-type GAP[701—1044Jwas typically purified to 90—95%homogeneity, theyield and purity of the mutant proteins were more variable.

Binding of Ran to Mutant GAP PrOteins. The binding of Ras to catalytically impaired GAP mutants was measured using a kinetic competition assay(19) which has been previously adapted for comparing the relative Ras bindingaff@mityof normal and reduced activity NFl mutants (24—26). Basically, asmall quantity of [‘y-32P]GTP-boundRas protein (0.2 nM) was incubated in thepresence of sufficient GAP or GAP mutant to stimulate hydrolysis of[y-32P]GTP bound to Ras (approximately 50—200 @g/mlfor most GAPs,including the wild-type control; 400—1,200 @g/mlfor mutants 4-33, 4-150,4-119, and 4-16). Increasing concentrations of nonradiolabeled Ras[L61]-GTP(0.006—200 ELM)were added to the assay mixture described above. All reagents, except for the purified GAP proteins, were premixed and incubated for2 mist at 30°C.The reaction was initiated by the addition of GAP[701—1044]or mutant GAP. After a b-mm incubation (30 mm for the 4-16 and 4-119mutants), the reaction was terminated with 0.2 ml of 5% activated charcoal(Sigma Chemical Co.) in 50 mMNaH2PO4.Ras GTPase activity was measuredby quantitating the amount of free [32Pjorthophosphate in the reaction super

natant following centrifugation to remove the charcoal. Competition wasexpressed as the percentage of activity detected relative to the valueobtained in the absence of competitor. Background counts measured in theabsence of both Ras competitor and GAP were subtracted from each samplevalue.

RESULTS

Semirandom Mutagenesis The DNA encoding GAP[701—1044]

was rnutagenized by a modified PCR. Using different reaction conditions, two different pUC8 expression libraries of GAP[701—1044Jmutants were constructed with different mutational frequencies. Onelibrary designated “numberfour―averaged one point mutation every266 base pairs in the 1029-base pair GAP insert, while library “number five―had approximately one point mutation every 114 base pairs.Library four was used primarily for the identification of inactivatingmutations, while library five was used to identify mutations which didnot affect GAP activity. Mutant proteins were initially characterizedin crude extracts following induction.

Inactivating Mutations in the GAP Catalytic Domain. Approximately 200 potentially mutant GAP[701—1044]proteins were examinedfor the ability to stimulate Ras GTPase. Twenty-three clones were foundto be inactive in the primary screen. Immunoblots of the induced E. colilysates confirmed protein expression for each of the inactive clones.Plasmid DNA was prepared from each inactive GAP clone positive forexpression and was completely sequenced to identify every mutationpresent in each gene. Twelve clones were found to contain only onecodon change (not counting silent changes), and six were found tocontain two codon changes (Table 1). Of the six double mutants, fivewere shown to have one codon change which alone was nonphenotypic,indicative that the remaining mutated codon was the inactivating lesion.Of the eighteen inactivating mutants, nine had mutations effecting residues highly conserved among the RasGAP-related proteins (Fig. 1).Mutants V893A, F898S, L9O1P, I902N, and I906T were all localized inthe most highly conserved region of GAP. A variety of amino acidsubstitutions were Obtained, ranging from conservative (N841S, V853A,V893A, and H1O18R), loss or change in charge (E8260, E947K, andD991G), and introduction of potential helix breakers (E826G, L845P,5877P, L9O1P, and D991G) to the most common class, loss of a hydrophobic sidechain (L732H, Y798H, L845P, F898S, I902N, 1906T, 195Th,and L995S).

CACI'G-3' amplified only the portion of the bovine GAP gene, which encodesthe catalytic domain from codon 701 to 1044. The mixture of mutant GAPDNA fragmentswas subclonedfrom the PCR reactioninto the BamHI-PstIsites ofpU@8 in-frame with the lacZ gene to yield an E. coli expression libraryof GAP[701—1044]mutants. Approximately 80% of the codons in this GAPgene fragment had an “A―or a “r'in the first or second position, making themsusceptible to amino acid substitution. To enable affinity purification ofselected mutant proteins, mutated genes were subcloned as BamHI-PstI fragments into the pQE-30 expression plasmid (Qiagen). The pQE-30 plasmidprovided for high-level expression of proteins fused to the 6x(histidine) affinity tag. The F898S mutation was separated from a second mutation in the 4-26mutant by subcloning a restriction fragment containing the point change intothe wild-type pQE-30-GAP[701-1044] plasmid. Standard recombinant DNAmanipulations were followed throughout this study (23). Plasmid DNAS werecompletely sequenced using Sequenase (United States Biochemical).

Isolation and Preparation of GAP Mutants Single-colony isolates wereobtained from the mutated pUC8-GAP[701—1044]expression libraries bytransformation of RRllaci―E. coli. Individual colonies were transferred into 5ml of LBA (Luria broth with 100 mg/ml ampidillin; Ref. 21) and grownovernight at 37°C.Each culture was split into 4.5 ml of stock culture forplasmid preparation and 0.5 ml for protein expression. GAP protein was madeby diluting the 0.5 ml sample into 4.5 ml of fresh LBA. After 1 hour ofincubation at 37°C,protein expression was induced by the addition of 0.5 mr@iisopropylthio-@-D-galactoside, followed by 4 more h of incubation. The cellsin each culture were pelleted and stored at —80°C.Bacterial pellets werethawed and lysed on ice by the addition of 0.25 ml of TED buffer [50 mMTris-HC1 (pH 73)-i mM EDTA-5 mM dithiothreitol] with 0.2 mg/mI lysozyme,1 ,.@g/mleach of pepstatinA andleupeptin,and200 p.Mphenylmethylsulfonylfluoride. Cell lysis was completed by the addition of 0.1% Triton X-100.Chromosomal DNA was degraded by the addition of 10 mM MgCl2 and 10@.&g/mlDNaseI (Boehringer Mannheim). Cell debris was removed by highspeed centrifugation in a chilled microcentrifuge, and the clarified extractswere used in GAP activity assays and immunoblots.

Direct GAP Assays. The ability of each GAP mutant to stimulate RasGTPase activity was measured by the nitrocellulose filter binding procedure(18, 19). Each 0.05 ml reaction contained approximately 0.1 ass of[‘y-32P]GTP-charged Ras protein, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.5), 1 mM MgCI2, and 1 mg/ml bovine serum

albumin. The reaction mixture was prewarmed for 2 mm at 30°Cprior to theaddition of 10 pi of clarified lysate. Samples were incubated an additional5 mm at 30°Cand then quenched by the addition of ice-cold 20 m@i4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and 250 @MMgC12.Samples were collected onto nitrocellulose filters (Schleicher and Schuell;BA 85), washed extensively with quench buffer, and quantitatedby Cerenkov counting. Samples were done in duplicate. Lysates were preparedfrom cells transformed with pUCS, and pUC8-GAP[701—1044Jwere included as controls in each assay series. Lysates, which were negative forGAP activity, were reconfirmed for authenticityby two additional sets ofassays performed on lysates prepared from cells which had been retransformed with the purified mutant plasmid.

Immunoblot Analysis of Mutant GAP[701-10441 PrOtein Expression.Mutant GAP clones which did not produce activity in cell extracts wereanalyzed for the expression of GAP protein. Lysates used for the primary GAPassays were immunoblotted using denaturing polyacrylamide gel electrophoresis as described previously. GAP[701—1044]was detected using the GAPCOOH-terminus-specific peptide antiserum 677 (21) and the ECL system(Amersham). Because of variability associated with the low-titer 677 antiserum, each immunoblot was repeated on fresh samples three times to confirmexpression.

Purification of 6x(histidlne)-GAP[701-1044] Proteins. E. coli strain@ was transformedwith 0.5 ,.@gof pQE-30-GAP[701—1044]or mutant

GAPplasmidandplatedon a single LEA-plate.After 16 h of 37°Cincubation,the confluent plate was washed into one liter of LBA and incubated for 1 h.Protein expression was induced by the addition of 0.5 mMisopropylthio-@3-D-galactoside, after which the culture was incubated an additional 4 h. Cellpellets were stored at —80°Cuntil lysis. The lysis of each sample was asdetailed above with the exception that 10 ml of 50 mMNaH2PO4(pH 7.0)-300mM NaG (buffer A) was used. Following DNA degradation and centrifugationof the lysate, the proteins were bound in batch to 1 ml of nickel-agarose beads

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RANDOM MUTAGENESISOF RASGAP

Table1 Descriptionof randomlyderivedinactivatingmutationsin GAP(701—1044J

No. ofMutant Mutation mutations Activity―

GAP[701—1044] None 1.004-42 L732H 1 <0.024-53 Y798H 1 0.024-137 E826G 1 <0.024-113 N841S 1 0.024-35 L845P 1 <0.024-117 V853A 1 <0.024-87 S877P 2― <0.024-33 V893A 1 <0.024-26 F898S 2b <0.024-98 L9O1P 2@' <0.024-141 1902N 2@' <0.024-150 1906T 1 0.024-177 E947K 1 <0.024-119 I957T 1 <0.024-16 D991G 1 <0.024-125 L995S 2b <0.024-44 H1O18R 1 <0.024-68 L732P 2 <0.02

C873R4-111 E836K 3 ND

1862MM950T

4-124 T804A 3 NDL1O2OP01032R

4-194 V934A 3 NDF943SL703P

4-23 Y720H 4 NDE722RK724RK812R

4-127 M715T 4 NDM795VS877P191ST

(2BaseduponserialdilutionsofexpressingcellextractsandnormalizedtoGAP[701—10441.

b Second mutation probably not responsiblefor inactivatingphenotypebaseduponanalysis of nonphenotypic mutations. Second mutations: 4-26, I752T; 4-87, D987G; 4-98,I856M; 4-141, F943V; 4-125, K855E. The I752T mutation in 4-26 was later reverted towild-type without affecting the inactivated phenotype; ND, not done.

In order to more accurately assess the degree of loss of catalyticactivity for each mutant GAP protein, dilutions of each lysate weremade and assayed for GAP activity. The amount of activity relative tothe wild-type GAP[701—1044] control lysate was determined (Table1). In most cases, there was no activity observed with each mutantunder the assay conditions. Only mutants Y798H, N841S, and 1906Thad weak amounts of detectable activity. Loss of GAP function couldbe accounted for by any of three reasons: (a) protein expression orstability was significantly reduced, resulting in a corresponding dropin detectable GAP activity; (b) the catalytic activity of the mutantswas reduced; or (c) the ability to bind Ras was affected. To determinethe relative amounts of each mutant GAP protein in the lysates, a GAPcarboxy-terminal antibody was used to immunologically quantitateeach mutant. The epitope recognized by the 677 antibody consists ofresidues 968—981,which were unaffected by inactivating mutations.By immunoblot, each of the mutant GAP proteins was observed to bepresent in low levels relative to the wild-type control (Fig. 2). OnlyN841S (4-113) was found to be expressed at a level approachingwild-type GAP, although over the course of four separate experiments, reduced levels of N841S were routinely observed. Variationsin expression levels were observed with many of the mutants. Sinceno activity and little protein was detected for many mutants, only acrude estimate of catalytic function could be made using cell lysatesnecessitating purification of the mutant proteins.

Mutant Purification and Binding Determinations. In order topurify the GAP proteins, ten of the single codon mutants were

subcloned into the poly-histidine expression plasmid pQE-30. ThepQE-30 expression vehicle provided for the fusion of six histidineresidues to the NH2-terminus of each GAP mutant, allowing purification of each protein using nickel-agarose affinity chromatography.Initially, each construct was induced and evaluated for protein cxpression relative to wild-type GAP[701—1044]subcloned into pOE30. In every case, mutant expression was 10% or less than thewild-type GAP, consistent with the results obtained by immunoblot.The mutant proteins were purified and concentrated; protein concentration was determined by Bradford assay and polyacrylamide gelelectrophoresis, and GAP activity measured. Only the N841S,V893A, 1906T, 195Th, and D991G GAP mutants were expressed insufficient quantity to purify to near homogeneity. The catalytic activities of these five mutants were compared to the wild-type GAP[701—1044] control by measuring the stimulation of Ras GTPase by increasingconcentrations of each protein. As can be seen in Fig. 3, all five GAPmutants were significantly impaired in their catalytic activity when cornpared to equal concentrations of wild-type GAP[701-1044]. Extendedincubation (30 ruin) of concentrated D991G and 195Th revealed weakstimulation of Ras GTPase, although the activity of 195Th was extremelydifficult to detect. The partially purified Y798H, E826G, V853A, F858S,and E947K mutants were all weakly active, although insufficient proteinwas Obtained to compare activity as a function of concentration.

For mutants with measurable Ras-GTPase stimulating activity, itwas possible to determine if the reduction in activity was due to adecrease in Ras binding affinity. Using a kinetic competition assay,relative binding affinities for Ras were estimated for each of thepurified mutants (Table 2). Only one of the mutations, V853A, wasactually impaired for Ras binding. Surprisingly, the NM1S, F898S,E947K, and D991G mutations actually increased the affinity of GAPfor Ras. Binding affinity was increased approximately 6-fold withN841S and E947K and 16—80-fold with F898S and D991G. Themutant 195Th was measurably inhibited by a range of Ras[L61]-GTPconcentrations (1 to 100 p,M), but a reliable dissociation constant wasnot obtained due to the extreme reduction of GAP activity associatedwith this mutation. Complete inhibition of 195Th activity was reproducibly observed with 1 to 10 @LMcompetitor, suggesting an increasein Ras binding affinity over wild-type GAP[701—1044J.

Noninactivating Mutations. Identifying nonessential amino acidsis an excellent method for locating nonbinding regions of a protein.An opposing data set of nonessential amino acids in the GAP catalyticdomain was generated by identifying clones from library number 5,which were fully active. Due to the high rate of mutation in thisparticular library, over 47 distinct codon changes were identified in 33clones, which had little or no effect upon GAP activity. A number offalse negatives originally identified in the number 4 library search alsocontributed to the identification of nonessential amino acids. Thesemutations are summarized in Table 3. The majority of nonessentialresidue changes were substitutions into positions which are not wellconserved among the GAP family of proteins (32 nonconservedcodons, 15 semiconserved) and are shown in Fig. 1. As with theinactivating mutations, each mutant was tested multiple times toconfirm its phenotype and to calculate the activity relative to wildtype GAP[701—1044] (Table 3). Most of the mutants had near to

wild-type activity, with only four of the mutations resulting in proteinswith less than 10% wild-type activity. Immunoblot analysis of selected noninactivating mutant proteins showed expression levels cornparable to wild-type GAP[701—1044]as shown in Fig. 2.

DISCUSSION

Prior studies of GAP and neurofibromin have identified GAPresidues E774, A791, L899, R900, K932, 0935, K946, and G944 as

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RANDOM MUTAGENESIS OF RASGAP

mutationsphenotypebOAP&mGAP1NFlIRA1IRA2sari

H RL@ 0

EFXELIL.QKE LHWYALSH- -VC--GQ- --dlmnLlLesv dqrpitvSa- -VsilGelvstnmg-d-QgE LpiamALanv vpC--sQ-- -dPlkyliknp ilaffg-S1- -aC--sp--ary--.tlehpq L-ssfg-aa- -vC-pas---

A/SP R00 0

DRTLLASILL KIFLH-EKLEgkTevAqpLv rlFtHtEria-wdeLArvLv tlFds-rhLlDvdLyAggfL naFdt-rnasDidayAagLi rtaFet-rnat

EhL1

P T GE0 0 00

.GSLR----V RARYSM---EK IMPEEEYS-

.GSLR----l nlnYta--dh vfPlatYd-

.dtL a etvla---dr ferlvElv-

.afLRvfidi vtnYpvnpEK hemdkmlaid

. Aktdl--gK leaadkf 1--

mutationsphenotypebGAPdGAP1NFlIRA1IRA2sarihomologyblock

mutationsphenotypebOAPdGAP1NFlIRA1IRA2sarihomologyblock

mutationsphenotypebOAPdGAP1NFlIRA1IRA2sari

G PYG0 0OL@

DSILRI-MES KQ----SCEpvlsql-vae Kk-----*p(@DplLRIvitS sdwqhv*SfEpvlqgl-.vdn Ke--- -*sfEplikkI-iqn rd----ffEqclndv-aih pdlqld*ryl

PG00

LS PSKLEXNEidPSKikdrsvdPt rLEpsEidkmK-pgsEieklKpE-dsvntgqLspsE

G STLP ART VV H P AA S PN TP C0 £@0OA Aoo oo o A o@ AM @o oDVN*ThLAHLL NILSELVEKI FMASEIIJPPT LRYIYGCLQK -SVQHK--WPT -N'flIIRTRVVSGFVFLRLIC PAILNPRMFNaVd*TNLhnLq dyvervfEal tksadrcPkv LcqlfhdL-r ecageh-fPs -NrevRysW SGF1FLRffa PAILgPk1Fdsle*eNqrnLL qmtekffhal issSsefPPq LRsvchCLyq -vVsqr-fP- -qnsi-gaVg Sa-mFLRfIn PAlvsPyeagnsekntbdife kymtrL@idaI tssiddfPie Lvdlcktiyri -aasvn-fPe -yayi-a--V gsFVFLRfIg PAlvsPdseNDae*rqielfv kymnELlEsI snsvsyfPPp LfYlcqniyK -vaceK-fPd -haii-a--a gsFVFLRffC PAlvsPdseN

@J*ersAqLL lltkrfldav insideiPyg iRwvck-Ldr -nltnrlfPs isdsticsli gGFfFLRfvn PAlisPqtsm

Block 2 Block 3A

TR Rtb o

NPFIKSN-KH

NdFvKSNfda arrfFLDiaseeFlKtc-sd ki fnFL.sELckdFlKec-sd RifrFLaELcqPmlKey-ee kvhnlLrkLG

G TRHI A0 ooAo o

SLLIJCTLNDR EISMEDEAT7 LFRATTLAST LMEQYMK-ATATQFVHHALKpiik-aLaDh EIShltdpTr iFRgnTLvSk ntlldeaNr-lsglhylHqtLryqLLwnlnfsk EvelaDsmqT LFRgnsLASk iMtfcfK-vy gatylqklLhiLvteLlkq Elkraarsdd ilRrnscAtr alslYtr—srgnkyliktLrhivvaqLikn Elekssrp'1\5ilRrnscAtr slsmlar-sk gneylirtLqlsL,fqmvltt Efeatsdvls L1RAnTpvSr mlttYtrrgp gqaylrsiLy

Block 1

mutationsphenotypebGAPdOAP1NFlIRA1IRA2sarihomologyblock

T P V A M L K0 0 0 0 0 0 AIISDSPSPIA ARTLTLVAKS VQNLANLIVEFGAKEPYMEGVltterldaqt sRTLTLisKt iQsLgNLVssildkkPkPri eRgLkL,rnsKi 1Qs1ANhV1F -tKEehMrpfIlivthah-d rkpfitlAlv iQsLANgrEn ifKkdilvskIl-Dishise kRTf1s1AKV iQNiANgsEn fsrwPalcsqlldscPSdnv rkThatiAKi iQsvAN—gtsstKthldvsf

Block 3B

TS GPG P N0 0 000 0 0

RMIMFLDELIG NVPEIJPD'VFEHSR-TDLC-R

dcPt-sDavn HS1-sfis-dkiPt—nnfTv nvR-eD. ...r-td-rtidi qvR—TD....NvgdffealE ldayiaLskk

G SYA Ao

-DLA---ALH

gnvl---ALH

RA

EICVAHSDEL -RTLSNERGA QQHVLKKIJLA

rllwnnqeki gayLSsnRdh kavgrrpfdk

G G0 0

ITELLQQKQN QYTKTNDVR

matLL

-sLAlemtvn EIyltHeiiL -enLdNlydp dsHVhliLqe lgE

Fig. 1. Mutational summary of the conserved Ras GAP catalytic domain. Bovine GAP (residues 701—1044)was aligned with the five known Ras GAP homologues using the methoddescribed by Wilbur and Lipman (33). Amino acid substitutions are listed above the location in the sequence where they were introduced. Below each substitution is a symbol denotingthe in vitro activity of the mutation: o, active; i@,catalytically impaired. Below the sequences are shown blocks of significant homology among the RasGAP proteins (29). Proteindesignations: bGAP, bovine RasGAP residues 701—1044;dmGAP1, Drosophila Gapi residues 455 to 701; NFl, human neurofibromin residues 1194—1521;IRA1, Saccharomycescerevisiae Iraip residues 1503—1791;IRA2, S. cerevisiae Ira2p residues 1666—1936;sari, Schizosaccharomycespombe GAP residues 169—472(34—37).

being essential for catalytic activity (26—29).Although Ras bindingdata has not been reportedfor most of these mutations, residues L899 andK932 have been shown to decrease catalytic activity without reducing theaffinity for Ras-GTP. However, a recent report suggested that mutationsat the position analogous to K932 in NFl (K1423) might prevent bindingto Ras (25). The reason for the discrepancy is unclear but could be related

to the instability of NFl protein mutated at this position. Similarly,catalytically impaired neurofibromin mutants with substitutions corresponding to GAP residues K946 and G944 reverted Ras-transformedfibroblasts, suggesting that the mutant NFl proteins were competing withthe Ras effector for binding to Ran (28).

Our primary goal was to identify, if possible, a localized bindingsurface for Ras by mutagenically eliminating the amino acid sidechains involved in the interaction. In searching for such mutations inGAP, we predicted that these residues would be exposed to the solventand not be important for maintaining the tertiary structure of thecatalytic domain. Such mutations would be analogous to those foundin the Ras effector loop, which are located on the surface of theprotein and are available for side chain interactions. Mutation of Rasin this region blocks biological activity and effects interaction withGAP without reducing protein stability in either E. coli or animal cells

(20). Since previous mutagenesis of conserved residues in GAP andneurofibromin has resulted only in catalytic impairment and not reductionin Ras binding, we chose to use a semirandom approach to identify aminoacid residues in the GAP catalytic domain essential for binding Ras.

In our analysis, 17 of 64 codon changes were deleterious to thenormal function of GAP. Without exception, every inactivated mutantfound in this study failed to match all the criteria predicted for aprotein binding site mutant. Each inactivating mutation resulted tosome degree in destabilization or insolubility of the GAP catalyticfragment when expressed in E. coli. Examination of every mutation,including silent changes, within each defective GAP mutant failed toidentify any suboptimal codon usage which might have resulted inreduced expression in E. coli. We conclude that these mutationsdisrupted intramolecular interactions required for maintaining thestability of the folded protein. The loss in catalytic activity observedwith many mutants may have been more a result of global distortionof the native conformation than targeting of a specific active site. Thenaturally occurring neurofibromin K1423E mutant (K932 in GAP) isvery similar to our inactivating GAP mutants in that it is catalytically

impaired, binds Ras, and is structurally destabilized (26). Randommutagenesis of the NFl/GAP-related domain has also resulted exclu

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0.0 0.1 0.2 0.3 0.4 0.5

Table 2 Binding affinity ofRas for different GAP(701—1044Jmutant proteinsRelative binding affinities for each @roteinare estimated by adding increasing con

centrations of Ras[L61]-GTP to a Ras- 2P-GTP hydrolysis assay containing sufficientmutant GAP protein to give measurable hydrolysis. The IC50 obtained for GAP[701—1044] is in the micromolar range due to its expression in E. coli. To confirm the potencyoftheRas[L611-GTPproteinused,acompetitionprofilewasmadewiththeneurofibrominGAP-relateddomain,whichhadan IC50of 40nat.Clone

# MutationIC50―GAP[701—1044]

None 80±11@4-53 Y798H 1754-137 E826G 140±72 p.M4-113 N8415 16±5p.at4-117 V853A >200 pM4-33 V893A 80 ±2 pat4-26 F898S 5±2pat4-150 I906T 200±204-177 E947K 12±2p.ai4-119 1957T <80 pM―4-16 D991G 1 ±0.6 pat

RANDOM MUTAGENESISOF RASOAP

no single residue is significantly responsible for the binding interaction with Ras or a few, as yet uncharacterized, residues mediate Rasbinding.

Many of the inactivating mutations identified affected residuespredicted to be important to structure/function based upon their conservation among the GAP homologues. L732H, E826G, L845P,F898S, L9O1P, 1902N, 1906T, and 195Th all had impaired catalyticactivity. These residues appear to be conserved because of their rolein defining the tertiary structure of the protein. The cluster of inactivating changes in the highly conserved and catalytically importantFLR...PA...P region (amino acids 898—909)demonstrates the efficacyof the randomized mutagenic method. We also found that mutations inthe neighboring residues V893, F898, and 1906 reduced catalyticfunction but not binding. A synthetic peptide corresponding to thisregion of bovine GAP was previously shown to inhibit the ability ofGAP to stimulate Ras GTPase (30). This peptide also promoted

Table3 DescriptionofrandomlyderivedmutationswithinGAP(701—1044Jwhichhavea negligible effect on activity.

L@

Fig. 2. InStabilityofthe mutated GAP catalytic domain in E. coli. Bacteria expressingGAP[701—10441,inactive mutants of GAP[701—1044),and a pUC8 vector control werelysed and an equal amount of soluble protein (—15 @g)separated on a sodium dodecylsulfate polyacrylamide geL Proteins were transferred to nitroceilulose and immunoblottedwith an antibody specific for a region of GAP carboxy-terminus not affected in the mutantproteins analysed.

2.@‘

IGAP concen@ation(mg/mi)

Fig. 3. Activity profiles for homogeneous purified GAP mutants. Purified mutantGAP[701—1044] proteins were serially diluted and tested for the ability to stimulate theintrinsic GTPase of H-Ras-GTP. A 100% value indicates complete hydrolysis of RasGTP. Each protein used in this experiment was purified to near (80% or better) homogeneity. •,GAP[701—1044];, N841S; 0, V893A; A, 1906T;0, I957T; +, D991G.

No. ofmutations

5

23

131

2122

123

23

53232

23

522235

2222

3

22225

22

Mutant

5-744-1565-275-124-634-485-125-184-1435-264-1894-1684-695-405-564-1915-305-75-64-494-695-484-855-745-485-295-485-724-844-665-65-625-745-72

5-264-1735-65-744-1425-414-1734-304-1685-555-125-35-295-414-665-75-744-434-305-27

Mutation

L703PM711TE712GK713EH740RT747S1747AT747AL749PK755RR773GM795T0797KK800IT8O2AS821GS821G58210S824P

@25YS830PE833GD837GA843TH844LK855RK855RK855E1856TA859V1862VY869HT890AN908PF912C1914TS918PS918PA923VV930AV934MF943LS959RK961R1965TL968SE976GL977PD979GS984PD987NH996YE1028GR10440

Activity―

1.000.25ND0.151.001.000.152.000.330.670.500.071.000.332.001.001.000.150.671.001.002.001.001.002.002.002.002.001.001.000.670.671.002.001.000.500.671.001.000.330.501.000.071.000.150.092.000.331.000.151.00ND1.00ND

a IC5@,s represent the average of at least three separate experiments.b @eto extremelyweak GAP activity, anexactIC@ wasnotobtainedfor thismutant.

sively in the identification of inactivating mutants which destabilizedthe protein.4 The failure of this and other studies to identify mutationsin GAP which significantly affect Ras association suggests that either

a Based upon serial

GAP[701—1044].

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dilutions of expressing cell extracts and normalized to4 F. McCormick, personal communication.

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RANDOM MUTAGENESIS OF RASGAP

guanine nucleotide exchange, suggesting that the peptide interacteddirectly with the Ras protein. Since mutations within this region ofGAP do not disrupt Ras binding, it is possible that the conserved siteis not significantly involved in the primary association of Ras andGAP but in a secondary interaction which leads to Ras GTPaseactivation.

This study has identified a new subregion of GAP which is importantfor function. Four inactivating mutations (E8260, N841S, L845P, andV853A) were found flanking a section of GAP strongly predicted to bean external loop (data not shown). Each of these positions is moderatelyto highly conserved, although this area has not been designated assignificantly conserved among the members of the GAP family (29).These mutations effect catalysis much like the other three subregionsshown here to be important.The V853A mutation in this region was theonly one identified in this study which reduced affinity for Ras-GTP.However, it cannot be firmly concluded that these residues aredirectly involved in GTPase activation or Ras binding since theyalso destabilize the structure of the protein. The resulting changesin protein conformation could be significant enough to influenceinteraction between Ras and GAP mediated by distant sidechaininteractions.

While most of the GAP mutants examined had dissociation constants for Ras similar to the wild-type GAP[701—1044] control, fouractually bound Ras with higher affinity than wild-type GAP. However, due to the structural effects of these mutations, interpretation ofthe binding data is difficult. It is possible that the protein preparationsconsist of a mixture of active and inactive polypeptides. A highproportion of inactive molecules would require less Ras-GTP toinhibit the assay, giving an artifactually low 50% inhibitory concentration.

While many of the conserved GAP residues remain to be analyzedfor their role in GAP function, our data allow specific conclusions tobe made. The activation of Ras GTPase is a function distinct from theinitial binding to Ras-GTP. This is not surprising in light of theobservation that the Ras D33N and D38N/D38E effector mutationsalso block GTPase activation by GAP without significantly decreasing GAP binding (31). The activation of Ras GTPase by GAPwas much easier to disrupt than binding alone, indicating thatGTPase activation requires a smaller, more conformationally defined structure which is independent of binding. At least onekinetic study has suggested that GAP accelerates the isomerizationof Ras-GTPto a conformation where GTE can be rapidly hydrolyzed (32).

The major finding of this study is that binding of Ras to GAP is notmediated by a conserved, contiguous stretch of Ras-like effectorresidues. While our results might also suggest the existence of anextremely small and compact binding site which was missed by therandomized procedure, this and other mutagenic studies have ruledout most conserved residues as being involved in Ras binding. Proteinassociation occurring through many hydrophobic and hydrogen bondinteractions across a large surface region would explain why all of theknown Ras effector proteins, such as yeast adenylyl cyclase, Raf, andGAP, show little or no obvious sequence similarity within their Rasbinding domains.

ACKNOWLEDGMENTS

We thankPaul Polakis for helpfulcomments.

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1994;54:5438-5444. Cancer Res   Lisa Hettich and Mark Marshall  a Mechanism of Interaction with Ras-GTPCatalytic Domain by Semirandom Mutagenesis: Implications for Structural Analysis of the Ras GTPase Activating Protein

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