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• W2007184804 abstract "The gene sequence of Manduca sextajuvenile hormone diol kinase (JHDK) codes for an enzyme that has 59% sequence identity to Drosophila melanogaster sarcoplasmic calcium-binding protein-2 (dSCP2). JHDK and dSCP2 are similar to G-proteins with three conserved sequence elements involved in purine nucleotide binding. Both proteins contain two pairs of EF-hand motifs. Characterization and partial purification of the D. melanogaster homolog of M. sexta JHDK from adultD. melanogaster gave material with JHDK activity. This activity has an experimental pI and molecular mass that are nearly identical to those of dSCP2. Moreover, D. melanogasterphosphotransferase activity has very similar chromatographic retention in three systems compared with M. sexta JHDK. Substrate docking to three-dimensional models of JHDK has shown that the three conserved nucleotide-binding elements surround the putative substrate-binding site and align with conserved sequence elements of p21Ras and adenylate kinase. D. melanogaster dSCP2 is a homolog of M. sexta JHDK, and these proteins constitute a novel kinase family that binds nucleotides using the scaffold of an SCP (Protein Data Bank code2SCP). The gene sequence of Manduca sextajuvenile hormone diol kinase (JHDK) codes for an enzyme that has 59% sequence identity to Drosophila melanogaster sarcoplasmic calcium-binding protein-2 (dSCP2). JHDK and dSCP2 are similar to G-proteins with three conserved sequence elements involved in purine nucleotide binding. Both proteins contain two pairs of EF-hand motifs. Characterization and partial purification of the D. melanogaster homolog of M. sexta JHDK from adultD. melanogaster gave material with JHDK activity. This activity has an experimental pI and molecular mass that are nearly identical to those of dSCP2. Moreover, D. melanogasterphosphotransferase activity has very similar chromatographic retention in three systems compared with M. sexta JHDK. Substrate docking to three-dimensional models of JHDK has shown that the three conserved nucleotide-binding elements surround the putative substrate-binding site and align with conserved sequence elements of p21Ras and adenylate kinase. D. melanogaster dSCP2 is a homolog of M. sexta JHDK, and these proteins constitute a novel kinase family that binds nucleotides using the scaffold of an SCP (Protein Data Bank code2SCP). juvenile hormone juvenile hormone diol kinase D. melanogaster sarcoplasmic calcium-binding protein-2 isoelectric focusing strong anion-exchange hydrophobic interaction chromatography weak anion-exchange reversed-phase liquid chromatography structurally conserved region root mean square 1,2-bis(2-aminophenoxy)ethane-N, N, N′, N′-tetraacetic acid. Note that “JH” or “JH diol” with no homolog designation is used in discussions relevant to all juvenile hormone homologs Endocrine control of embryonic and post-embryonic development in insects is primarily accomplished by juvenile hormone (JH)1 and ecdysteroids (1Truman J.W. Riddiford L.M. Nature. 1999; 401: 447-452Crossref PubMed Scopus (366) Google Scholar). Development proceeds through a series of molts and metamorphosis, where the function of JH is to regulate the character of the molt; metamorphosis is characterized by a sharp decrease in JH, and adult molting by the absence of JH (1Truman J.W. Riddiford L.M. Nature. 1999; 401: 447-452Crossref PubMed Scopus (366) Google Scholar). JH levels are regulated by de novo biosynthesis (2Feyereisen R. Kerkut G.A. Gilbert L.I. Comprehensive Insect Physiology, Biochemistry and Pharmacology. 7. Pergamon Press, Oxford1985: 391-429Google Scholar) and catabolism (3De Kort C.A.D. Granger N.A. Arch. Insect Biochem. Physiol. 1996; 33: 1-26Crossref Scopus (101) Google Scholar). Cellular JH metabolism involves a microsomal JH epoxide hydrolase that metabolizes JH to JH diol and a soluble JH diol kinase (JHDK) that catalyzes the formation of JH diol phosphate. JHDK is a homodimer with a subunit molecular mass of 20 kDa that uses MgATP or MgGTP as a phosphate donor to effectively catabolize JH I, II, or III diol by placing a phosphate group on the C-10 hydroxyl, thereby creating JH diol phosphate, the water-soluble end metabolite of JH catabolism (4Maxwell R.A. Welch W.H. Schooley D.A. J. Biol. Chem. 2002; 277: 21874-21881Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar).In this study, we report the sequencing and cloning of Manduca sexta JHDK, the molecular modeling of JHDK and Drosophila melanogaster sarcoplasmic calcium-binding protein-2 (dSCP2), and the partial purification and characterization of the D. melanogaster homolog of M. sexta JHDK. We show with high probability that the product of the D. melanogasterdSCP2 gene is the D. melanogaster homolog of M. sexta JHDK. Each is a member of a novel kinase family that is structurally similar to SCPs.DISCUSSIONPurine recognition for G-proteins is highly specific for GTP; the first and last residues of the NKXD sequence (∑3) specifically hydrogen-bond to the guanine ring of the bound nucleotide. The charged residue corresponding to Lys in ∑3 is highly conserved among all P-loop-containing nucleotide-triphosphate hydrolases; this residue is known to provide a hydrophobic surface for the purine ring and often forms a hydrogen bond with the endocyclic oxygen of the ribose ring (19Via A. Ferre F. Brannetti B. Valencia A. Helmer-Citterich M. J. Mol. Biol. 2000; 303: 455-465Crossref PubMed Scopus (69) Google Scholar, 28Sprang S.R. Annu. Rev. Biochem. 1997; 66: 639-678Crossref PubMed Scopus (879) Google Scholar). G-proteins have a low affinity for ATP due to the loss of a hydrogen bond donor at position 1 of the purine and the unfavorable positioning of two hydrogen bond donors: exocyclic NH2 at purine position 6 and the amido group of Asn (28Sprang S.R. Annu. Rev. Biochem. 1997; 66: 639-678Crossref PubMed Scopus (879) Google Scholar). Considering that M. sexta JHDK has a high affinity for ATP, but will also use GTP as a phosphate donor, it was not surprising that the conserved Lys of NKXD (∑3) is spatially quite similar to p21Ras (Fig. 6). However, the Asn of ∑3 is not always conserved (described below). As expected, ∑3 of M. sextaJHDK is positioned in such a way as to accommodate either purine, although in slightly different conformations (data not shown).Two forms of adenylate kinase each have Glu substituted for Asp in the ∑3 motif (NKXD), the same substitution as found for JHDK and dSCP2. One such adenylate kinase is known to use GTP (code 2AK3), and the other is known to use ATP (archaeal adenylate kinase (code 1NKS)). Interestingly, in the unoccupied protein crystal structure of each adenylate kinase, the Glu residue was in the (−)-conformation (pointing away from a possible contact with a nucleotide). Upon GTP binding, the Glu residue changed to a (+)-conformation to form a hydrogen bond with the exocyclic amino group at position 2 of the nucleotide (19Via A. Ferre F. Brannetti B. Valencia A. Helmer-Citterich M. J. Mol. Biol. 2000; 303: 455-465Crossref PubMed Scopus (69) Google Scholar). Furthermore, ATP did not confer this conformational change upon binding, indicating that a hydrogen bond is not formed between Glu and the exocyclic amino group at position 6 of the adenine nucleotide (19Via A. Ferre F. Brannetti B. Valencia A. Helmer-Citterich M. J. Mol. Biol. 2000; 303: 455-465Crossref PubMed Scopus (69) Google Scholar). We found that JHDK and dSCP2 also have a Glu substitution in this position and that it was in the (−)-conformation. Interestingly, by using only weak constraints, the Glu of JHDK could form the (+)-conformation (data not shown).A select group of ATP/GTP-binding proteins contain a conserved Asn residue in the ∑3 motif (NKXD): archaeal adenylate kinase (code 1NKS), deoxynucleotide kinase (code 1DEK), guanylate kinase (code 1GKY), and the nitrogenase ion protein (code 1NIP) (19Via A. Ferre F. Brannetti B. Valencia A. Helmer-Citterich M. J. Mol. Biol. 2000; 303: 455-465Crossref PubMed Scopus (69) Google Scholar). Although the exact role of the Asn residue is unknown, it is thought to be indirectly involved in the stabilization of NTP binding. However, the majority of ATP/GTP-binding proteins do not contain a conserved Asn. Via et al. (19Via A. Ferre F. Brannetti B. Valencia A. Helmer-Citterich M. J. Mol. Biol. 2000; 303: 455-465Crossref PubMed Scopus (69) Google Scholar) have proposed that the Asn residue is an evolutionary relic of an ancestral protein able to bind both ATP and GTP nucleotides. This is consistent with the fact that M. sexta JHDK can bind both nucleotides and has the conserved Asn. Interestingly, D. melanogaster phosphotransferase activity could not be detected using GTP as a phosphate donor (data not shown). Moreover, dSCP2 does not contain the conserved Asn of the ∑3 motif (NKXD).Purification and characterization of M. sexta JHDK revealed a highly specific kinase well suited for the catabolism of low nanomolar concentrations of JH diol (4Maxwell R.A. Welch W.H. Schooley D.A. J. Biol. Chem. 2002; 277: 21874-21881Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Sequence analysis and comparison of M. sexta JHDK revealed a high sequence identity to D. melanogaster dSCP2 (also classified as calexcitin) (10Kelly L.E. Phillips A.M. Delbridge M. Stewart R. Insect Biochem. Mol. Biol. 1997; 27: 783-792Crossref PubMed Scopus (24) Google Scholar). M. sexta JHDK and D. melanogaster dSCP2 are similar to G-proteins in that they each have three conserved consensus sequence elements involved in nucleotide binding (16Kjeldgaard M. Nyborg J. Clark B.F. FASEB J. 1996; 10: 1347-1368Crossref PubMed Scopus (230) Google Scholar). In addition, JHDK and dSCP2 contain two pairs of EF-hand motifs (29Lewit-Bentley A. Rety S. Curr. Opin. Struct. Biol. 2000; 10: 637-643Crossref PubMed Scopus (414) Google Scholar). All currently known kinases use a nucleotide-binding fold structure that can be simplified to a residual core for NTP binding composed of four α-helices and four β-strands in a fixed architecture (15Vetter I.R. Wittinghofer A. Q. Rev. Biophys. 1999; 32: 1-56Crossref PubMed Scopus (142) Google Scholar, 30Milner-White E.J. Coggins J.R. Anton I.A. J. Mol. Biol. 1991; 221: 751-754Crossref PubMed Scopus (40) Google Scholar). However, molecular models of JHDK and dSCP2 indicate a high probability of an all-α-helical structure similar to Ca2+-binding proteins.The charged residue corresponding to ∑2 (DXXG) is highly conserved among all P-loop-containing nucleotide-triphosphate hydrolases and is known to be directly involved in phosphate binding through a coordination of Mg2+ (31Saraste M. Sibbald P.R. Wittinghofer A. Trends Biochem. Sci. 1990; 15: 430-434Abstract Full Text PDF PubMed Scopus (1734) Google Scholar, 32Bourne H.R. Sanders D.A. McCormick F. Nature. 1991; 349: 117-127Crossref PubMed Scopus (2660) Google Scholar, 33Valencia A. Chardin P. Wittinghofer A. Sander C. Biochemistry. 1991; 30: 4637-4648Crossref PubMed Scopus (56) Google Scholar). In most cases, the coordination is via a water molecule. M. sexta JHDK and dSCP2 each contain the conserved Asp in ∑2, which has been shown to be superimposable with the equivalent Asp in adenylate kinase and p21Ras (Fig. 6). Fig. 7 illustrates the position of the C-10 hydroxyl (phosphate acceptor) in relation to the aforementioned Asp residue and the γ-phosphate of ATP. The position of the substrates in relation to one another and the conserved NTP fingerprint leads us to speculate on a possible catalytic mechanism.Thompson et al. (34Thompson P.R. Hughes D.W. Cianciotto N.P. Wright G.D. J. Biol. Chem. 1998; 273: 14788-14795Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar) and Izard and Ellis (35Izard T. Ellis J. EMBO J. 2000; 19: 2690-2700Crossref PubMed Scopus (49) Google Scholar) proposed that phosphoryl transfer proceeds though an in-line mechanism to a primary or secondary alcohol of a small molecule. According to their hypothetical mechanism, JH diol could undergo anSn2-type phosphate transfer initiated by the abstraction of a hydroxyl proton by the catalytic base Asp18. Our models of JHDK and dSCP2 have similar residues in position to catalyze this type of reaction. Our models predict that Asp18 (M. sexta JHDK) and Asp20(dSCP2) are within hydrogen-bonding distance of the phosphate acceptor (JH diol, C-10) and phosphate donor (γ-phosphate of ATP). Moreover, this is the only enzyme functional group within 9 Å capable of acting as a catalytic base. Thompson et al. (34Thompson P.R. Hughes D.W. Cianciotto N.P. Wright G.D. J. Biol. Chem. 1998; 273: 14788-14795Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar) and Izard and Ellis (35Izard T. Ellis J. EMBO J. 2000; 19: 2690-2700Crossref PubMed Scopus (49) Google Scholar) found that mutation of the essential Asp residue resulted in a nearly complete loss of activity. However, a mechanism similar to that of adenylate kinase (code 1AKY) involving the attack of a catalytic water molecule cannot be discounted. Mutation of the corresponding Asp residue of adenylate kinase results in loss of NTP binding (36Schlauderer G.J. Proba K. Schulz G.E. J. Mol. Biol. 1996; 256: 223-227Crossref PubMed Scopus (80) Google Scholar).Figs. 2 and 7 illustrate the nucleotide-binding site of M. sexta JHDK and D. melanogaster dSCP2. The binding site is composed of amino acids from ∑2 (loop region between helices 1 and 2), ∑3 (part of helix 10), and ∑1 (unstructured C-terminal end). Uncharacteristic of the fixed P-loop containing proteins, a high degree of flexibility associated with the C-terminal phosphate-binding site (∑1, P-loop) was observed during modeling of the target proteins. As a result, many nucleotide-binding conformations were encountered. Fig.7 illustrates one such conformation in which all three nucleotide-binding motifs (∑1, ∑2, and ∑3) are involved in nucleotide recognition, and the γ-phosphate of ATP is within 4 Å of the phosphate-accepting hydroxyl of JH diol.Several factors support the reliability and validity of our models forM. sexta JHDK and D. melanogaster dSCP2. 1) Both sequences were analyzed in two automated molecular modeling programs. In each case, the top five matches were the same as those we obtained by sequence threading. 2) The average positional energy of M. sexta JHDK (−0.26 kiloteslas) is quite similar to that of the template crystal structure (code 2SCP) (−0.30 kiloteslas). The backbone alignment of the target proteins with the template resulted in low (3 Å or less) r.m.s. deviation. 3) A single binding pocket is present in both target proteins. The character and dimensions of the binding pocket are well suited for JH diol and correlate with the observed phosphate selectivity of M. sexta JHDK. The position of the binding pocket is within 2 Å of the C-terminally located phosphate-binding motif. 4) Substrate docking has shown that three conserved sequence elements surround the putative substrate and align (within 3 Å or less) with conserved sequence elements of p21Ras and adenylate kinase. NTP binding is consistent with known NTP-binding proteins. It is extremely unlikely that chance can explain the simultaneous energetic profile, the three-dimensional alignment of signature residues, and the formation of a correctly sized pocket (lacking in the template). In support of this point, scrambled sequences or alternate folds failed to produce a model.All currently known kinases contain a nucleotide-binding fold structure that can be simplified to a residual core for NTP binding, composed of four α-helices and four β-strands in a fixed architecture (15Vetter I.R. Wittinghofer A. Q. Rev. Biophys. 1999; 32: 1-56Crossref PubMed Scopus (142) Google Scholar, 30Milner-White E.J. Coggins J.R. Anton I.A. J. Mol. Biol. 1991; 221: 751-754Crossref PubMed Scopus (40) Google Scholar). This α,β-structural core of several β-strands alternating with α-helices is also referred to as the “ancestral core structure” (30Milner-White E.J. Coggins J.R. Anton I.A. J. Mol. Biol. 1991; 221: 751-754Crossref PubMed Scopus (40) Google Scholar) and is present in most nucleotide-binding proteins (15Vetter I.R. Wittinghofer A. Q. Rev. Biophys. 1999; 32: 1-56Crossref PubMed Scopus (142) Google Scholar). However, examples exist such as phosphoenolpyruvate carboxykinase (37Matte A. Goldie H. Sweet R.M. Delbaere L.T. J. Mol. Biol. 1996; 256: 126-143Crossref PubMed Scopus (93) Google Scholar, 38Matte A. Tari L.W. Goldie H. Delbaere L.T. J. Biol. Chem. 1997; 272: 8105-8108Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) in which the α,β-arrangements are different.There are only two known examples of nucleotide-binding proteins that do not contain the α,β-structure in any arrangement: dUTPase and chaperonins. The dUTPase structure consists of a trimeric all-β-domain. Chaperonins such as GroEL/GroES are large multisubunit assemblies that are essential for the folding of many proteins. They require the energy derived from hydrolyzing ATP to initiate a conformational change. The GroEL equatorial domain binds ATP with a structure composed of four helical subdomains (39Llorca O. Marco S. Carroscosa J.L. Valpuesta J.M. J. Struct. Biol. 1997; 118: 31-42Crossref PubMed Scopus (33) Google Scholar). To our knowledge, GroEL is the only nucleotide-binding protein that uses an all-α-helical structure to bind nucleotides. Chaperonins have a unique sequence to bind ATP that is quite different from M. sexta JHDK, P-loop proteins, or nucleotide-binding proteins.Kelly et al. (10Kelly L.E. Phillips A.M. Delbridge M. Stewart R. Insect Biochem. Mol. Biol. 1997; 27: 783-792Crossref PubMed Scopus (24) Google Scholar) isolated a cDNA clone corresponding toD. melanogaster dSCP2 using antibodies raised against theDrosophila Ca2+-binding protein DCABP-23 to screen an expression library. In situ hybridization using adult Drosophila indicated that the expression pattern of the transcript is exclusive to neural tissue. Later, Quattrone et al. 5A. Quattrone, H. Xu, A. Pacini, and D. L. Alkon, unpublished data. reclassified dSCP2 as a Ca2+-sensing low molecular mass GTPase (GenBankTM/EBI accession number AAC62484). M. sexta JHDK shares 59% sequence identity and 80+% sequence similarity with dSCP2. Modeling of dSCP2 indicates that it has a tertiary structure very similar to SCPs. The conserved NTP-binding motifs in dSCP2 spatially align with the equivalent motifs in the GTP-binding protein p21Ras, adenylate kinase, and the theoretical model of M. sexta JHDK. Models predict that dSCP2 and M. sexta JHDK bind JH diol and MgATP in a similar fashion. We have shown that the phosphotransferase responsible for production of JH diol phosphate in adult Drosophila has a pI and active molecular mass that correspond to those predicted for dSCP2. The phosphotransferase activity has the same retention properties upon HIC and SAX chromatography as well as similar retention upon WAX chromatography as M. sexta JHDK. Given this information, we conclude that dSCP2 is the probableDrosophila homolog of M. sexta JHDK and that dSCP2 should be known as Drosophila JHDK.What is the relationship of JHDK to Ca2+-binding proteins? The exact function of SCPs is unknown. One speculation is that they are responsible for the maintenance of a buffered intracellular Ca2+ concentration (40Vijay-Kumar S. Cook W.J. J. Mol. Biol. 1992; 224: 413-426Crossref PubMed Scopus (59) Google Scholar). However, there are some protein families (S100 protein) that possess EF-hand motifs that are unable to bind any metal (29Lewit-Bentley A. Rety S. Curr. Opin. Struct. Biol. 2000; 10: 637-643Crossref PubMed Scopus (414) Google Scholar, 41Rety S. Sopkova J. Renouard M. Osterloh D. Gerke V. Tabaries S. Russo-Marie F. Lewit-Bentley A. Nat. Struct. Biol. 1999; 6: 89-95Crossref PubMed Scopus (258) Google Scholar). Interestingly, Nelson et al. (42Nelson T.J. Cavallaro S., Yi, C.L. McPhie D. Schreurs B.G. Gusev P.A. Favit A. Zohar O. Kim J. Beushausen S. Ascoli G. Olds J. Neve R. Alkon D.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13808-13813Crossref PubMed Scopus (51) Google Scholar) identified calexcitin as a signaling protein that binds calcium and GTP and shares sequence homology with AmphioxusSCP. Apparently, this is the first known instance of a GTP-binding protein that also binds Ca2+. Although a tertiary structure has not been proposed for calexcitin, it does have partial sequence similarity to some of the conserved GTP-binding motifs.The role of divalent cations in JHDK is unclear. Models of M. sexta JHDK indicate that at least two EF-hand motifs have a high probability for divalent cation binding. In fact, the ∑2 motif (DXXG) is part of the first N-terminal Ca2+-binding motif (Fig. 2). M. sexta JHDK was found to be inhibited by metals during purification and characterization (4Maxwell R.A. Welch W.H. Schooley D.A. J. Biol. Chem. 2002; 277: 21874-21881Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The use of chelators restored activity. In addition, M. sexta JHDK activity was inhibited by micromolar levels of free Mg2+ and Ca2+ (4Maxwell R.A. Welch W.H. Schooley D.A. J. Biol. Chem. 2002; 277: 21874-21881Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). However, Ca2+ inhibition was observed well above physiological levels. Several attempts were made to remove endogenous bound divalent cations from native M. sexta JHDK (4Maxwell R.A. Welch W.H. Schooley D.A. J. Biol. Chem. 2002; 277: 21874-21881Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). In all cases, Ca2+ was not required for activity. Due to the low quantities of native M. sexta JHDK obtained through purification, an expression system will be required to further characterize the role of divalent cations and EF-hand motifs in JHDK.45Ca binding assays and overlay blots will determine whether JHDK binds Ca2+ (42Nelson T.J. Cavallaro S., Yi, C.L. McPhie D. Schreurs B.G. Gusev P.A. Favit A. Zohar O. Kim J. Beushausen S. Ascoli G. Olds J. Neve R. Alkon D.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13808-13813Crossref PubMed Scopus (51) Google Scholar). Circular dichroism spectroscopy and fluorescence spectroscopy should determine whether divalent cation binding is initiating structural changes (43Gombos Z. Jeromin A. Mal T.K. Chakrabartty A. Ikura M. J. Biol. Chem. 2001; 276: 22529-22536Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar).Interestingly, the top 20 matches from a BLAST search of M. sexta JHDK were all calcium-binding proteins, hypothetical or otherwise. Of these, many of the various forms of calexcitin and SCP also showed conservation of an Asp at ∑2, a conserved Lys and Asp at ∑3 (in some cases, Lys was substituted with Cys, and Asp was substituted with Glu), and a partial phosphate-binding sequence at the C terminus for ∑1 (GXXXXG always conserved) when aligned with the primary sequence of M. sexta JHDK. One may hypothesize a function other than Ca2+ binding for these proteins, such as nucleotide binding and small molecule phosphotransferase function. Future molecular modeling may show whether these NTP-binding motifs are also superimposable with those of p21Ras and adenylate kinase.It is not surprising that knowledge-based modeling strongly indicated a Ca2+-binding protein tertiary fold given the conservation of three divalent cation-binding motifs (EF-hands) for JHDK and dSCP2. It is surprising, however, to find the presence and superimposability of all three highly conserved nucleotide-binding motifs (fingerprint of GTPases) associated with the Ca2+-binding protein tertiary fold. JHDK and dSCP2 are members of a novel class of nucleotide-binding proteins that use the all-α-helical Ca2+-binding protein scaffold to arrange the highly evolved fingerprint of GTPase motifs for nucleotide binding. In addition, they appear to be the first example of a hormone-inactivating phosphotransferase (4Maxwell R.A. Welch W.H. Schooley D.A. J. Biol. Chem. 2002; 277: 21874-21881Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). It would seem that JHDK and dSCP2 fill a structural gap linking kinase function to the tertiary fold of the Ca2+-binding protein. Endocrine control of embryonic and post-embryonic development in insects is primarily accomplished by juvenile hormone (JH)1 and ecdysteroids (1Truman J.W. Riddiford L.M. Nature. 1999; 401: 447-452Crossref PubMed Scopus (366) Google Scholar). Development proceeds through a series of molts and metamorphosis, where the function of JH is to regulate the character of the molt; metamorphosis is characterized by a sharp decrease in JH, and adult molting by the absence of JH (1Truman J.W. Riddiford L.M. Nature. 1999; 401: 447-452Crossref PubMed Scopus (366) Google Scholar). JH levels are regulated by de novo biosynthesis (2Feyereisen R. Kerkut G.A. Gilbert L.I. Comprehensive Insect Physiology, Biochemistry and Pharmacology. 7. Pergamon Press, Oxford1985: 391-429Google Scholar) and catabolism (3De Kort C.A.D. Granger N.A. Arch. Insect Biochem. Physiol. 1996; 33: 1-26Crossref Scopus (101) Google Scholar). Cellular JH metabolism involves a microsomal JH epoxide hydrolase that metabolizes JH to JH diol and a soluble JH diol kinase (JHDK) that catalyzes the formation of JH diol phosphate. JHDK is a homodimer with a subunit molecular mass of 20 kDa that uses MgATP or MgGTP as a phosphate donor to effectively catabolize JH I, II, or III diol by placing a phosphate group on the C-10 hydroxyl, thereby creating JH diol phosphate, the water-soluble end metabolite of JH catabolism (4Maxwell R.A. Welch W.H. Schooley D.A. J. Biol. Chem. 2002; 277: 21874-21881Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). In this study, we report the sequencing and cloning of Manduca sexta JHDK, the molecular modeling of JHDK and Drosophila melanogaster sarcoplasmic calcium-binding protein-2 (dSCP2), and the partial purification and characterization of the D. melanogaster homolog of M. sexta JHDK. We show with high probability that the product of the D. melanogasterdSCP2 gene is the D. melanogaster homolog of M. sexta JHDK. Each is a member of a novel kinase family that is structurally similar to SCPs. DISCUSSIONPurine recognition for G-proteins is highly specific for GTP; the first and last residues of the NKXD sequence (∑3) specifically hydrogen-bond to the guanine ring of the bound nucleotide. The charged residue corresponding to Lys in ∑3 is highly conserved among all P-loop-containing nucleotide-triphosphate hydrolases; this residue is known to provide a hydrophobic surface for the purine ring and often forms a hydrogen bond with the endocyclic oxygen of the ribose ring (19Via A. Ferre F. Brannetti B. Valencia A. Helmer-Citterich M. J. Mol. Biol. 2000; 303: 455-465Crossref PubMed Scopus (69) Google Scholar, 28Sprang S.R. Annu. Rev. Biochem. 1997; 66: 639-678Crossref PubMed Scopus (879) Google Scholar). G-proteins have a low affinity for ATP due to the loss of a hydrogen bond donor at position 1 of the purine and the unfavorable positioning of two hydrogen bond donors: exocyclic NH2 at purine position 6 and the amido group of Asn (28Sprang S.R. Annu. Rev. Biochem. 1997; 66: 639-678Crossref PubMed Scopus (879) Google Scholar). Considering that M. sexta JHDK has a high affinity for ATP, but will also use GTP as a phosphate donor, it was not surprising that the conserved Lys of NKXD (∑3) is spatially quite similar to p21Ras (Fig. 6). However, the Asn of ∑3 is not always conserved (described below). As expected, ∑3 of M. sextaJHDK is positioned in such a way as to accommodate either purine, although in slightly different conformations (data not shown).Two forms of adenylate kinase each have Glu substituted for Asp in the ∑3 motif (NKXD), the same substitution as found for JHDK and dSCP2. One such adenylate kinase is known to use GTP (code 2AK3), and the other is known to use ATP (archaeal adenylate kinase (code 1NKS)). Interestingly, in the unoccupied protein crystal structure of each adenylate kinase, the Glu residue was in the (−)-conformation (pointing away from a possible contact with a nucleotide). Upon GTP binding, the Glu residue changed to a (+)-conformation to form a hydrogen bond with the exocyclic amino group at position 2 of the nucleotide (19Via A. Ferre F. Brannetti B. Valencia A. Helmer-Citterich M. J. Mol. Biol. 2000; 303: 455-465Crossref PubMed Scopus (69) Google Scholar). Furthermore, ATP did not confer this conformational change upon binding, indicating that a hydrogen bond is not formed between Glu and the exocyclic amino group at position 6 of the adenine nucleotide (19Via A. Ferre F. Brannetti B. Valencia A. Helmer-Citterich M. J. Mol. Biol. 2000; 303: 455-465Crossref PubMed Scopus (69) Google Scholar). We found that JHDK and dSCP2 also have a Glu substitution in this position and that it was in the (−)-conformation. Interestingly, by using only weak constraints, the Glu of JHDK could form the (+)-conformation (data not shown).A select group of ATP/GTP-binding proteins contain a conserved Asn residue in the ∑3 motif (NKXD): archaeal adenylate kinase (code 1NKS), deoxynucleotide kinase (code 1DEK), guanylate kinase (code 1GKY), and the nitrogenase ion protein (code 1NIP) (19Via A. Ferre F. Brannetti B. Valencia A. Helmer-Citterich M. J. Mol. Biol. 2000; 303: 455-465Crossref PubMed Scopus (69) Google Scholar). Although the exact role of the Asn residue is unknown, it is thought to be indirectly involved in the stabilization of NTP binding. However, the majority of ATP/GTP-binding proteins do not contain a conserved Asn. Via et al. (19Via A. Ferre F. Brannetti B. Valencia A. Helmer-Citterich M. J. Mol. Biol. 2000; 303: 455-465Crossref PubMed Scopus (69) Google Scholar) have proposed that the Asn residue is an evolutionary relic of an ancestral protein able to bind both ATP and GTP nuc" @default.
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