Proc. Natl. Acad. Sci. USA Vol. 83, pp. 907-911, February 1986 Biochemistry ATP-binding site of adenylate kinase: Mechanistic implications of its homology with ras-encoded p21, Fl-ATPase, and other nucleotide-binding proteins DAVID C. FRY*, STEPHEN A. KUBYt, AND ALBERT S. MILDVAN* *Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205; and tLaboratory for the Study of Hereditary and Metabolic Disorders and the Departments of Biological Chemistry and Medicine, University of Utah, Salt Lake City, UT 84108 Communicated by Albert L. Lehninger, October 7, 1985

ABSTRACT The MgATP binding site of adenylate kinase, homologies for which the binding site of the nucleotide located by a combination ofNMR and x-ray diffraction, is near substrate has been characterized. Only one of the three three protein segments, five to seven amino acids in length, that homologous segments is found in phosphofructokinase, for are homologous in sequence to segments found in other which an x-ray structure also exists (14). Such information on nucleotide-binding phosphotransferases, such as myosin and adenylate kinase reveals interactions between the homolo- Fl-ATPase, ras p21 and transducin GTPases, and cAMP- gous residues and the bound nucleotide substrate that permit dependent and src protein kinases, suggesting equivalent reasonable predictions to be made of the functions of such mechanistic roles of these segments in all of these proteins. residues and their near neighbors, not only in adenylate Segment 1 is a glycine-rich flexible loop that, on adenylate kinase but also in the other nucleotide-binding phosphotrans- kinase, may control access to the ATP-binding site by changing ferases, and that rationalize the effects of mutations on both its conformation. Segment 2 is an a-helix containing two the GTPase and transforming activities ofthe ras p21 protein. hydrophobic residues that interact with the adenine-ribose moiety of ATP, and a lysine that may bind to the j8- and Sequence Homologies Among Adenylate Kinase and Other y-phosphates of ATP. Segment 3 is a hydrophobic strand of Proteins parallel a6-pleated sheet, terminated by a carboxylate, that flanks the triphosphate binding site. The various reported mutations of ras p21 that convert it to a transforming agent all In Table 1 portions of the amino acid sequence of rabbit appear to involve segment 1, and such substitutions may alter muscle adenylate kinase are given, and homologous regions the properties of p21 by hindering a conformational change at in the sequences of other proteins are listed. Included are the this segment. In F1-ATPase, the flexible loop may, by its homologies noted by Walker and co-workers (5, 8) and others position, control both the accessibility and the ATP/ADP derived from computer searches and visual inspection of constant on the enzyme. available sequences. There are three segments of homology. equilibrium Clearly, some of the proteins listed in Table 1 show only one or two of the three homologous segments. Segment 1 (resi- Adenylate kinase catalyzes the reversible transfer of a dues 15-21 in adenylate kinase: Gly-Gly-Pro-Gly-Ser-Gly- phosphoryl group from MgATP to AMP. Lys) is shared by several ATPases and GTPases, as well as by DNA A protein, Epstein-Barr virus protein, thymidine MgATP + AMP MgADP + ADP. [1] kinase, cAMP-dependent protein kinase, phospholipase A2, glycogen phosphorylase, nitrogenase, and the biotin-contain- The enzyme has been purified from many sources, the x-ray ing subunit of transcarboxylase.t Slightly further along in the structure of the porcine muscle enzyme has been reported at sequence is segment 2 (residues 27-31: Lys-Ile-Val-His-Lys) 3 A resolution (1), and amino acid sequences have been that bears weak homology to corresponding portions ofmany determined for muscle adenylate kinase from pig (2), human of these proteins and strong homology to regions of the (3), calf, and rabbit (4). It was noted by Walker et al. (5) that protein kinases and transcarboxylase. Segment 3 (residues two portions of the adenylate kinase sequence had homolo- 114-119: Leu-Leu-Leu-Tyr-Val-Asp) has counterparts in the gous counterparts in the sequences of several ATPases. One sequences of F1-ATPase, ras p21, ATP/ADP translocase, of these homologous segments was later found to be present phosphofructokinase, and transcarboxylase. § in p21, the GTPase that is the product of the ras oncogene (6-8). The recent finding that a mutation in this segment is the basis of the transforming ability of this protein (9, 10) has The MgATP Binding Site of Adenylate Kinase amplified the importance ofdetermining the functional role of this segment. We have identified a third homologous region High-field proton NMR was used to study the interaction of in several ofthese proteins and have further expanded the list metal-ATP substrates with porcine (11) and rabbit muscle of proteins with sequence homologies to adenylate kinase. adenylate kinase (12), and with a globular peptide fragment of Through a series of NMR studies on porcine (11) and rabbit the latter enzyme consisting of residues 1-45 that binds muscle (12) adenylate kinase and on an ATP-binding peptide metal-ATP with comparable affinity (13). Paramagnetic ef- fragment of the latter (12, 13), we have located the MgATP fects of B,y-bidentate Cr3+-ATP on the relaxation rates of binding site within the x-ray structure ofthe enzyme and have protons of the enzyme and the peptide were measured and obtained a detailed description of the environment of the provided a total ofeight distances from Cr3+ to the side chains bound nucleotide. Adenylate kinase is the only protein of specific amino acid residues. Time-dependent nuclear among those sharing the aforementioned extensive sequence tIn some of the proteins of Table 1 this homology extends beyond residue 21 to residue 23. The publication costs of this article were defrayed in part by page charge §Regions of near homology to segments 1 and 2 are found in payment. This article must therefore be hereby marked "advertisement" phosphoglycerate kinase (30, 31) and to segment 3 in creatine kinase in accordance with 18 U.S.C. §1734 solely to indicate this fact. (32). 907 Downloaded by guest on September 29, 2021 908 Biochemistry: Fry et al. Proc. Natl. Acad. Sci. USA 83 (1986) Table 1. Sequence homologies among the ATP-binding region of adenylate kinase and segments of other proteins Sequence Protein 15 20 25 30 110 115 120 Adenylate kinasea G-G-P -G-S-G-K-G-T-Q-C-E-K -I-V-H-K G-Q-P-T-L-L-L-Y-V-D-A-G Fl-ATPaseb a(E. coli) G-D-R -Q- -G-K-T- -A-I- G- -A-L-I-I-Y-D-D- f3(E. coli) G-G-A -G- -G-K-T- -L-I- G- -V-L-L-F-V-D- p(bovine) G-G-A -G- -G-K-T- -L-I- G-Q- -V-L-L-F-I-D- Myosinc nematode -G-G -G-G-G-K- -V- G- -G- -G-K-T- -K -V-I- rabbit G- -G- -G-K-T- -R -V-I- Thymidine kinased G- -G- -G-K-T-T- -L-V- RecA protein G- -S-G-K-T-T- -V-I- Transducin ae G- -G- -S-G-K- -T- -K- Go protein ae G- -G- -S-G-K- -T- -K- ras p21f G- -Gg-G- -G-K- -L-I G- -L-L-D-I-L-D-T-Ah DnaA proteini G-G- -G- -G-K-T- -V- Epstein-Barr virus proteins G-G- -G-K-G- -A- Glycogen phosphorylasek G- -G -G- -G-R- -C- Phospholipase A2' G- -G -G- -G-R- Protein kinasem cAMP-dependent G- -G- -G-R- -/10/-Kn-I-L- -K cGMP-dependent -Kn-I-L- -K src protein G- -G- -G- -/10/-Kn-T-L-K- Nitrogenase (Fe protein)0 G- -G -G- -G-K- -T- -/11/-K -I-L- Transcarboxylase biotin subunitP G-G- -G- -G-K- -/10/-K -I-L- -K G-Q-T-V-L-V-L-E -Bct- ATP/ADP translocase G- -V-L-V-L-Y-D Phosphofructokinase G- -L-V-V-I- -D- aThe sequence of rabbit muscle adenylate kinase is from Kuby et al. (4). bComparisons involving Fl-ATPases, myosin, RecA protein, translocase, and phosphofructokinase are from Walker et al. (5). cThe upper homologous sequence of nematode myosin is an alternative region to that (lower) presented by Walker et al. (5). dSequence of the thymidine kinase from herpes simplex virus is from McKnight (15). ePartial sequences of the subunits of transducin from bovine retina and G. protein from bovine brain are from Hurley et al. (16). 'the sequence of a ras gene product is typified by that from the c-has/bas human protooncogene (17). Some homology involving this protein has been noted by Gay and Walker (8). gMutations at this position alter the transforming ability (9, 10, 18-21) and GTPase activity (18-20) of the protein. hSubstitution of a threonine at this position apparently results in autophosphorvlation (18). Sequence from Hansen et al. (22). Sequence from Bankier et al. (23). kSequence from Titani et al. (24). 'Sequence from Joubert and Haylett (25). mATP-binding site sequence for cAMP-dependent protein kinase, and partial sequence for cGMP-dependent protein kinase, were obtained from Hashimoto et al. (26). Comparison between these sequences and that of p6osrc, the transforming tyrosine kinase of Rous sarcoma virus, is from Kamps et al. (27). nThis lysine is the residue labeled during inactivation with p-fluorosulfonylbenzoyl 5'-adenosine (27). °Sequence of the Fe-protein of nitrogenase and some of the homology involving this protein is from Robson (28). PSequence from Maloy et al. (29). Bct is the biocytin residue.

Overhauser effects were utilized to measure interproton to this region selectively binds 1,N6-ethenoadenosine distances, the analysis of which yielded very similar confor- monophosphate (EAMP) with substantial affinity (13). mations of bound MgATP on both the enzyme and the Our new position of MgATP places it in close contact with peptide (12). Such studies also provided a total of 14 close the five strands of parallel 1-sheet structure and with one of distances (-4 A) from protons of amino acid residues of the the interconnecting helices, more closely analogous to the enzyme and peptide to those of the bound substrate. binding ofpyridine nucleotide coenzymes to dehydrogenases Intermolecular distances obtained for both the enzyme and (35). As also found in dehydrogenases, the phosphate groups the peptide were in agreement (12). This information was of ATP on adenylate kinase are bound near the amino used to fit MgATP into the x-ray structure of the enzyme (1, terminus of an a-helix begun by a glycine (36). Despite this 12), correcting the proposed binding site that had been based similarity in three-dimensional structure and function be- tween the nucleotide binding sites of adenylate kinase and on the crystallographic location of salicylate (33). dehydrogenases, sometimes involving similar amino acid The MgATP binding site ofadenylate kinase as determined residues (35), the interesting paradox exists that these func- by NMR is shown in Figs. 1-3. The adenine-ribose moiety is tional residues often come from regions that differ with in a hydrophobic pocket formed by residues Ile-28, Val-29, respect to their protein sequences and differ in their second- His-36, Leu-37, and Leu-91. The triphosphate moiety is ary structures. Accordingly, little primary sequence homol- located between the side chains of Lys-21, Gln-24, and ogy exists between adenylate kinase and dehydrogenases (5, Lys-27, and a hydrophobic sequence, residues 114-118, 35). While alignments based on both structure and sequence terminated by an anionic residue, Asp-119. In this orientation have been made with dehydrogenases (35), only the latter the y-phosphoryl group points toward the C-terminal 23- alignments are currently possible with the proteins of Table amino acid residues of the enzyme; a peptide corresponding 1. Downloaded by guest on September 29, 2021 Biochemistry: Fry et al. Proc. Natl. Acad. Sci. USA 83 (1986) 909

FIG. 1. Computer graphics representation of rabbit muscle ade- nylate kinase showing the location of bound metal-ATP. The three segments of the enzyme exhibiting sequence homology to other proteins (Table 1) are shown in pink, and the ATP molecule is shown in red. The x-ray coordinates ofconformation A ofporcine adenylate kinase (1) were used, with substitution of a histidine residue for N glutamine at position 30. Metal-ATP was fit into the enzyme structure using a set of distances obtained by NMR (12). Segments FIG. 3. The metal-ATP binding site of adenylate kinase is shown 1, 2, and 3 and MgATP are identified. with respect to the two crystal forms of the enzyme: A (solid) and B (open). The binding of Mn2+ATP changes the conformation from B to A (33, 34). The three segments of homology (residues 15-21, Functional Roles of Homologous Regions 27-31, and 114-119) are depicted by stippling in conformation B. The drawings ofthe enzyme are based on the x-ray structures (1, 34). The The three segments of adenylate kinase exhibiting sequence position of metal-ATP was determined by NMR (12). homology to those of other proteins are all located at or near We will consider each of these homologous segments in the (within 11 A from) the MgATP binding site, as is shown in order of increasing complexity of its presumed role: first Figs. 1-3. Spatial relationships between residues in these segment 3, next segment 2, and finally segment 1. segments and bound MgATP can be used to help discern their Segment 3, a hydrophobic strand ofparallel P-pleated sheet mechanistic roles, which may reasonably be extended to the terminated by an aspartate, flanks the triphosphate chain of other nucleotide-binding proteins sharing those sequences. MgATP, including the reaction center. It probably serves to exclude water and minimize hydrolysis. Asp-119, at the end of segment 3, may accept a hydrogen bond from a water ligand of Mg2> on MgATP, a reasonable role for a carboxyl- ate residue at the reaction center of adenylate kinase, since general base catalysis (i.e., deprotonation of the substrate by an amino acid residue of the enzyme) is not necessary in the case of AMP. Alternatively, by directly coordinating Mg2+, the carboxylate group of aspartate-119 might facilitate the migration of Mg2+ from P3,y-coordination in MgATP to a,,-coordination in MgADP as reaction 1 proceeds from left to right. An analogous role has been proposed for Asp-372 in phosphoglycerate kinase, another case in which general base catalysis is probably unnecessary (30). The aspartate residue in segment 3 of phosphofructokinase may be near the Mg atom of bound MgATP (14), although the homology with phosphofructokinase is otherwise not detectable. Proximity to the triphosphate chain of GTP is suggested for the comparable segment ofras p21, particularly since mutation of the alanine, which concludes this segment, into a threonine results in autophosphorylation of the protein at this position (37, 38). Segment 2 is mainly an a-helix consisting of two lysines separated by three residues, two of which are hydrophobic and a third that is variable, even among adenylate kinases from different species. In adenylate kinase the hydrophobic FIG. 2. ORTEP (computer graphics program) representation residues form part of the pocket in which the adenine-ribose showing MgATP and the three homologous segments of adenylate moiety of MgATP is located. The first lysine, Lys-27, can be kinase. positioned such that its NH' nitrogen is 5.7 ± 2.0 A from the Downloaded by guest on September 29, 2021 910 Biochemistry: Fry et A Proc. Natl. Acad. Sci. USA 83 (1986) f3- and y-phosphorus atoms of MgATP and may interact with x-ray and model-building studies (40) to control the access of them. Although no x-ray structures are available for the other oligosaccharide substrates to the catalytic site. proteins that share segment 2, affinity labeling studies of the The second possible function of segment 1 is modification cyclic nucleotide-dependent and src protein kinases with of the affinity of the binding site. Current theories on the p-fluorosulfonylbenzoyl 5'-adenosine have shown that in mechanisms of myosin (42) and F1-ATPase (43-45) involve these enzymes the lysine residue homologous to Lys-27 is alternating levels of affinity for ATP and ADP at the nucle- near the y-phosphate of bound ATP (27). otide binding sites, as controlled through conformational Segment 1 is a glycine-rich flexible loop that is terminated interactions with actin in the case of myosin and with by a cationic residue, Lys-21 on adenylate kinase and lysine He-channel proteins and/or a separate nucleotide binding or arginine on the others (Figs. 2 and 3). In adenylate kinase site in the case of F1-ATPase. Transducin functions by Lys-21 is near (i.e., 4 + 2 A) the a-phosphoryl group of varying its affinity for GTP vs. GDP in response to interaction MgATP and could easily interact with it. Pro-17 near the apex with rhodopsin (46). The G proteins and p21 are believed to of the loop appears to stabilize this loop by occupying act in a comparable manner. The mechanism of adenylate position 2 of a type IV P-turn, as is often seen in proteins (39). kinase may involve an alternating preference for MgATP vs. With the exception of Lys-21, the loop makes no direct MgADP at its MgATP site, possibly controlled by the interactions with the bound MgATP molecule. Segment 1, occupancy of the AMP site. Segment 1 is likely to undergo a therefore, though previously believed to be a sequence conformational change that affects binding site affinity, due diagnostic of a nucleotide-binding site, appears to have a to the fact that it is conserved in so many of these similarly function other than simply holding a nucleotide. In fact, its functioning proteins. Other ATPases that unlike F1 form presence in the biotin subunit of transcarboxylase, which covalent phosphoenzyme intermediates, such as the Na'-K' does not bind nucleotides, and in glycogen phosphorylase at ATPase and the Ca2+-ATPase, do not possess this homolo- a site remote from the AMP-binding site (40) emphasizes this gous region suggesting that they function by a different point. The function of this loop most likely involves its ability detailed mechanism. If modification of binding site affinity is to undergo a conformational change, in response either to the role of segment 1, then flexibility must not be the only substrate binding or to an interaction with another protein or structural requirement of this loop. Flexible glycine-rich domain. The ability of this loop to change its conformation is regions are found in the sequences of many proteins, includ- evidenced by comparison of the two crystal forms of porcine ing such unrelated examples as Ig X chain, keratin, collagen, adenylate kinase (A and B) (34). A transition from B to A, ribosomal proteins, enolase, and heat shock protein. Table 1 corresponding to the binding of Mn2+ATP, involves struc- lists only those which share a distinct homology at segment tural changes in four regions, with the largest displacement (6 1 and the characteristic termination of this sequence by a A) occurring at the glycine-rich loop (Fig. 3). lysine or arginine. The lysine common to the functionally The proteins sharing segment 1, whose mechanisms are similar proteins is positioned in adenylate kinase to interact known but are not described in detail, all participate in with the a-phosphoryl group of bound MgATP. A different transfer of a phosphoryl group from ATP. The exception is orientation of this lysine could favor MgADP at this site, the transcarboxylase biotin subunit. The presence of segment since the negative charge distribution among the phosphoryl 1 near the biotin in this enzyme and inhibition at the propionyl groups is different for the two substrates. Such a change in CoA site by the binding of oxalate to the pyruvate site (41) the orientation of lysine-21 represents a plausible method by suggest that this flexible loop may differentially control which a conformational change affecting segment 1 could access to biotin at the two reactive sites. Together with alter binding site affinity. segment 3, it may also limit the access of water to bound An additional or alternative result of the sensitivity of carboxybiotin, thereby minimizing the decarboxylation of segment 1 to the identity of the bound substrate might be the this labile intermediate, analogous to the roles proposed for transmission of this structural information to a different the ATP and GTP enzymes. Similarly, segment 2 may section of the protein through conformational means. For provide a catalytic lysine to facilitate the enolization and example, the site on transducin that interacts with cGMP carboxylation of biotin. Alternatively, the homology of the phosphodiesterase is modified according to whether the sequence of the biotin subunit of transcarboxylase may be an transducin molecule has GTP or GDP bound (46). ras p21 is evolutionary artifact, since other biotin-containing enzymes believed to function in an analogous manner (16), as will be utilize ATP to activate bicarbonate in the carboxylation of discussed below. The presence of segment 1 in nitrogenase biotin. may indicate a similar function, since binding of MgATP or Our study of adenylate kinase (12) and the information MgADP to this enzyme causes conformational changes that available on the structure and mechanisms of the other differentially affect the accessibility of the Fe4S4 cluster to homologous proteins suggest three possible roles for a chelating agents and to the MoFe protein that accepts conformational change at segment 1. These are: (i) control of electrons from it (47). modification of The third possible role of a conformational change at accessibility to the substrate binding site, (ii) reaction center. binding site affinities, and (iii) relocation of catalytic groups segment 1 is to bring catalytic groups into the If by a conformational change this loop were to approach the toward the reaction center of the bound substrate. Such triphosphate chain of bound MgATP in adenylate kinase, the actions are not mutually exclusive and could all be operative phosphoryl transfer reaction could be facilitated by the in varying combinations, possibly in a coordinated manner. of serine-19 and the amide NH of the with to hydroxyl protons Each will be discussed in turn respect adenylate backbone functioning as H-bond donors as has been pro- kinase and the other homologous proteins. posed for phosphoglycerate kinase (30). Control of access to the MgATP binding site by the The specific conformational changes required of segment 1 segment 1 loop is suggested by its location in adenylate to carry out its role may be analyzed more rigorously in the kinase. It is situated in the middle of a cleft that leads from special case of ras p21, since genetic variants of this protein the bound MgATP molecule to the surface of the enzyme differing only in the segment 1 sequence are available, (Fig. 1). The change in conformation of this loop observed in allowing the comparison of wild-type p21 with oncogenic the two crystal forms of the enzyme shows that it is capable mutants found in nature and induced by site-specific of alternately blocking and opening the cleft (Fig. 3). A mutagenesis. Replacement of one of the glycine residues in similar situation apparently exists for the comparable seg- segment 1 of p21 (corresponding to proline-17 of adenylate ment of glycogen phosphorylase that has been shown from kinase) by any residue other than proline converts the protein Downloaded by guest on September 29, 2021 Biochemistry: Fry et al. Proc. Natl. Acad. Sci. USA 83 (1986) 911 into a transforming agent (9, 10, 18-21). The substitution of 9. Tabin, C. J., Bradley, E., Bargmann, C., Weinberg, R., Papageorge, A., Scolnick, E. M., Dhar, R., Lowy, D. & Chang, E. (1982) Nature a valine at this position, which is oncogenic, has also been (London) 300, 143-148. shown to reduce the GTPase activity ofthe protein by afactor 10. Reddy, E. P., Reynolds, R. K., Santos, E. & Barbacid, M. (1982) of 6 to 10 without weakening the binding of GTP (18-20). By Nature (London) 300, 149-152. analogy with transducin, the transforming ability of p21 may 11. Smith, G. M. & Mildvan, A. S. (1982) Biochemistry 21, 6119-6123. 12. Fry, D. C., Kuby, S. A. & Mildvan, A. S. (1985) Biochemistry 24, be enhanced because it is locked into the GTP-bound mode 4680-4694. that activates processes necessary for cell proliferation (16, 13. Hamada, M., Palmieri, R. H., Russell, G. A. & Kuby, S. A. (1979) 46). From the previous arguments, the inability of mutant p21 Arch. Biochem. Biophys. 195, 155-177. to hydrolyze GTP and attain its GDP-bound form is probably 14. Evans, P. R., Farrants, G. W. & Hudson, P. J. (1981) Philos. Trans. R. Soc. London Ser. B 293, 53-62. due to hindrance ofthe conformational change in the segment 15. McKnight, S. L. (1980) Nucleic Acids Res. 8, 5949-5964. 1 loop that would normally be needed to switch the binding 16. Hurley, J. B., Simon, M. I., Teplow, D. B., Robishaw, J. D. & Gilman, preference of the site from GTP to GDP and/or to bring A. G. (1984) Science 226, 860-862. reactive residues into the catalytic area. Apparently, the 17. Yuasa, Y., Srivastava, S. K., Dunn, C. Y., Rhim, J. S., Reddy, E. P. & Aaronson, S. A. (1983) Nature (London) 303, 775-779. glycine in p21 that corresponds to proline-17 of adenylate 18. McGrath, J. P., Capon, D. J., Goeddel, D. V. & Levinson, A. D. (1984) kinase plays a critical role in the proper functioning of Nature (London) 310, 644-649. segment 1. Conversion of that glycine into any residue 19. Sweet, R. W., Yokoyama, S., Kamata, T., Feramisco, J. R., bulkier than proline diminishes the conformational mobility Rosenberg, M. & Gross, M. (1984) Nature (London) 311, 273-275. 20. Gibbs, J. B., Sigal, I. S., Poe, M. & Scolnick, E. M. (1984) Proc. Natl. of segment 1 or destabilizes the P-turn, changing the protein Acad. Sci. USA 81, 5704-5708. into a transforming agent (21). 21. Seeburg, P. H., Colby, W. W., Capon, D. J., Goeddel, D. V. & Interestingly, the a-subunit of F1-ATPase, which has a Levinson, A. D. (1984) Nature (London) 312, 71-75. relatively large residue (arginine) at this site, is believed to 22. Hansen, E. B., Hansen, F. G. & von Meyenburg, K. (1982) Nucleic the Acids Res. 10, 7373-7385. bind ATP tightly and exhibit no ATPase activity, while 23. Bankier, A. T., Deininger, P. L., Farrell, P. J. & Barrell, B. G. (1983) 13-subunit with the smaller alanine at this position is believed Mol. Biol. Med. 1, 21-45. to be the site ofATP hydrolysis. In the other proteins in Table 24. Titani, K., Koide, A., Hermann, J., Ericsson, L. H., Kumar, S., Wade, 1 the amino acid identity at this position varies, and many of R. D., Walsh, K. A., Neurath, H. & Fischer, E. H. (1977) Proc. Natl. must Acad. Sci. USA 74, 4762-4766. them exhibit high levels of ATPase activity. Either p21 25. Joubert, F. J. & Haylett, T. (1981) Hoppe-Seyler's Z. Physiol. Chem. make a more specific conformational change at its loop, 362, 997-1006. which is subject to greater steric interference, or a nearby 26. Hashimoto, E., Takio, K. & Krebs, E. G. (1982) J. Biol. Chem. 257, residue present only in p21 or in the a-subunit of F1 might 727-733. 27. Kamps, M. P., Taylor, S. S. & Sefton, B. M. (1984) Nature (London) interact unfavorably with the position in question and prevent 310, 589-592. the appropriate conformational change. 28. Robson, R. L. (1984) FEBS Lett. 173, 394-398. The preceding model also provides a unifying explanation 29. Maloy, W. L., Bowien, B. U., Zwolinski, G. K., Kumar, K. G., Wood, for the transforming ability of ras proteins with mutations in H. G., Ericsson, L. W. & Walsh, K. A. (1979) J. Biol. Chem. 254, 11615-11622. diverse positions other than the glycine of segment 1, such as 30. Watson, H. C., Walker, N. P. C., Shaw, P. J., Bryant, T. N., Wendell, the substitution of alanine-59 by a threonine residue (48) and P. L., Fothergill, L. A., Perkins, R. E., Conroy, S. C., Dobson, M. J., the replacement of glutamine-61 by a leucine in the ras Tuite, M. F., Kingsman, A. J. & Kingsman, S. M. (1982) EMBO J. 1, product from human lung carcinoma cells (17). In the x-ray 1635-1640. studies kinase the 1 31. Banks, R. D., Blake, C. C. F., Evans, P. R., Haser, R., Rice, D. W., of adenylate (34), movement of segment Hardy, G. W., Merrett, M. & Phillips, A. W. (1979) Nature (London) loop brings it into close proximity with another section ofthe 279, 773-777. polypeptide chain near residues 120-123 (see Fig. 3). If the 32. Putney, S., Herlihy, W., Royal, N., Pang, H., Aposhian, H. V., homology between p21 and adenylate kinase at segment 3 is Pickering, L., Belagage, R., Biemann, K., Page, D., Kuby, S. A. & residues of kinase Schimmel, P. (1984) J. Biol. Chem. 259, 14317-14320. extended, 120-123 adenylate (-Ala-Gly- 33. Pai, E. F., Sachsenheimer, W., Schirmer, R. H. & Schulz, G. E. (1977) Pro-Glu-) correspond to residues 59-62 of p21 (Ala-Gly-Gln- J. Mol. Biol. 114, 37-45. Glu-) that include the mutagenic sites. The presence of 34. Sachsenheimer, W. & Schulz, G. E. (1977) J. Mol. Biol. 114, 23-36. certain side chains in this portion of p21 may result in 35. Rossman, M. G., Liljas, A., Branden, C. I. & Banaszak, L. J. (1975) hindered movement of the loop, inhibiting the hydrolysis of The Enzymes 11, 61-102. 36. Wierenga, R. K., DeMaeyer, M. C. H. & Hol, W. G. J. (1985) Bio- GTP, thus providing a unifying mechanistic explanation for chemistry 24, 1346-1357. all of the transforming variants of the ras protein. 37. Gibbs, J. B., Ellis, R. W. & Scolnick, E. M. (1984) Proc. Natl. Acad. Sci. USA 81, 2674-2678. We thank Eric Suchanek for his help with the computer graphics 38. Shih, T. Y., Stokes, P. E., Smythers, G. W., Dhar, R. & Oroszlan, S. system. This work was supported by National Institutes of Health (1982) J. Biol. Chem. 257, 11767-11773. Grants AM28616 39. Richardson, J. S. (1981) Adv. Protein Chem. 34, 167-339. and AM07824, and National Science Foundation 40. McLaughlin, P. J., Stuart, D. I., Klein, H. W., Oikonomakos, N. 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