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).