Dynamic features of cAMP-dependent protein kinase revealed by apoenzyme crystal structure

To better understand the mechanism of ligand binding and ligand-induced conformational change, the crystal structure of apoenzyme catalytic (C) subunit of adenosine-3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) was solved. The apoenzyme structure (Apo) provides a snapshot of the enzyme in the first step of the catalytic cycle, and in this unliganded form the PKA C subunit adopts an open conformation. A hydrophobic junction is formed by residues from the small and large lobes that come into close contact. This "greasy" patch may lubricate the shearing motion associated with domain rotation, and the opening and closing of the active-site cleft. Although Apo appears to be quite dynamic, many important residues for MgATP binding and phosphoryl transfer in the active site are preformed. Residues around the adenine ring of ATP and residues involved in phosphoryl transfer from the large lobe are mostly preformed, whereas residues involved in ribose binding and in the Gly-rich loop are not. Prior to ligand binding, Lys72 and the C-terminal tail, two important ATP-binding elements are also disordered. The surface created in the active site is contoured to bind ATP, but not GTP, and appears to be held in place by a stable hydrophobic core, which includes helices C, E, and F, and beta strand 6. This core seems to provide a network for communicating from the active site, where nucleotide binds, to the peripheral peptide-binding F-to-G helix loop, exemplified by Phe239. Two potential lines of communication are the D helix and the F helix. The conserved Trp222-Phe238 network, which lies adjacent to the F-to-G helix loop, suggests that this network would exist in other protein kinases and may be a conserved means of communicating ATP binding from the active site to the distal peptide-binding ledge.     

Comparative surface geometry of the protein kinase family

Identifying conserved pockets on the surfaces of a family of proteins can provide insight into conserved geometric features and sites of protein–protein interaction. Here we describe mapping and comparison of the surfaces of aligned crystallographic structures, using the protein kinase family as a model. Pockets are rapidly computed using two computer programs, FADE and Crevasse. FADE uses gradients of atomic density to locate grooves and pockets on the molecular surface. Crevasse, a new piece of software, splits the FADE output into distinct pockets. The computation was run on 10 kinase catalytic cores aligned on the αF‐helix, and the resulting pockets spatially clustered. The active site cleft appears as a large, contiguous site that can be subdivided into nucleotide and substrate docking sites. Substrate specificity determinants in the active site cleft between serine/threonine and tyrosine kinases are visible and distinct. The active site clefts cluster tightly, showing a conserved spatial relationship between the active site and αF‐helix in the C‐lobe. When the αC‐helix is examined, there are multiple mechanisms for anchoring the helix using spatially conserved docking sites. A novel site at the top of the N‐lobe is present in all the kinases, and there is a large conserved pocket over the hinge and the αC‐β4 loop. Other pockets on the kinase core are strongly conserved but have not yet been mapped to a protein–protein interaction. Sites identified by this algorithm have revealed structural and spatially conserved features of the kinase family and potential conserved intermolecular and intramolecular binding sites.     

Crystal structure of glycinamide ribonucleotide transformylase from Escherichia coli at 3.0 A resolution. A target enzyme for chemotherapy.

The atomic structure of glycinamide ribonucleotide transformylase, an essential enzyme in purine biosynthesis, has been determined at 3.0 A resolution. The last three C-terminal residues and a sequence stretch of 18 residues (residues 113 to 130) are not visible in the electron density map. The enzyme forms a dimer in the crystal structure. Each monomer is divided into two domains, which are connected by a central mainly parallel seven-stranded beta-sheet. The N-terminal domain contains a Rossmann type mononucleotide fold with a phosphate ion bound to the C-terminal end of the first beta-strand. A long narrow cleft stretches from the phosphate to a conserved aspartic acid, Asp144, which has been suggested as an active-site residue. The cleft is lined by a cluster of residues, which are conserved between bacterial, yeast, avian and human enzymes, and likely represents the binding pocket and active site of the enzyme. GAR Tfase binds a reduced folate cofactor and glycinamide ribonucleotide for the catalysis of one of the initial steps in purine biosynthesis. Folate analogs and multi-substrate inhibitors of the enzyme have antineoplastic effects and the structure determination of the unliganded enzyme and enzyme-inhibitor complexes will aid the development of anti-cancer drugs.     

Crystal structures of the myristylated catalytic subunit of cAMP-dependent protein kinase reveal open and closed conformations.

Three crystal structures, representing two distinct conformational states, of the mammalian catalytic subunit of cAMP-dependent protein kinase were solved using molecular replacement methods starting from the refined structure of the recombinant catalytic subunit ternary complex (Zheng, J., et al., 1993a, Biochemistry 32, 2154-2161). These structures correspond to the free apoenzyme, a binary complex with an iodinated inhibitor peptide, and a ternary complex with both ATP and the unmodified inhibitor peptide. The apoenzyme and the binary complex crystallized in an open conformation, whereas the ternary complex crystallized in a closed conformation similar to the ternary complex of the recombinant enzyme. The model of the binary complex, refined at 2.9 A resolution, shows the conformational changes associated with the open conformation. These can be described by a rotation of the small lobe and a displacement of the C-terminal 30 residues. This rotation of the small lobe alters the cleft interface in the active-site region surrounding the glycine-rich loop and Thr 197, a critical phosphorylation site. In addition to the conformational changes, the myristylation site, absent in the recombinant enzyme, was clearly defined in the binary complex. The myristic acid binds in a deep hydrophobic pocket formed by four segments of the protein that are widely dispersed in the linear sequence. The N-terminal 40 residues that lie outside the conserved catalytic core are anchored by the N-terminal myristylate plus an amphipathic helix that spans both lobes and is capped by Trp 30. Both posttranslational modifications, phosphorylation and myristylation, contribute directly to the stable structure of this enzyme.     

Crystal structure of Onconase at 1.1 Å resolution--insights into substrate binding and collective motion

Onconase(?) (ONC) is an amphibian member of the pancreatic ribonuclease superfamily that is selectively toxic to tumor cells. It is a much less efficient enzyme than the archetypal ribonuclease A and, in an attempt to gain further insight, we report the first atomic resolution crystal structure of ONC, determined in complex with sulfate ions at 100 K. The electron density map is of a quality sufficient to reveal significant nonplanarity in several peptide bonds. The majority of active site residues are very well defined, with the exceptions being Lys31 from the catalytic triad and Lys33 from the B(1) subsite, which are relatively mobile but rigidify upon nucleotide binding. Cryocooling causes a compaction of the unit cell and the protein contained within. This is principally the result of an inward movement of one of the lobes of the enzyme (lobe 2), which also narrows the active site cleft. Binding a nucleotide in place of sulfate is associated with an approximately perpendicular movement of lobe 2 and has little further effect on the cleft width. Aspects of this deformation are present in the principal axes of anisotropy extracted from C(α) atomic displacement parameters, indicating its intrinsic nature. The three lowest-frequency modes of ONC motion predicted by an anisotropic network model are compaction/expansion variations in which lobe 2 is the prime mover. Two of these have high similarity to the cryocooling response and imply that the essential 'breathing' motion of ribonuclease A is conserved in ONC. Instead, shifts in conformational equilibria may contribute to the reduced ribonucleolytic activity of ONC.     

Interaction of N1-, N6- and C8-substituted derivatives of adenosine-5'-triphosphate with the catalytic subunit of cAMP-dependent protein kinase from rabbit skeletal muscles

In order to investigate the structure of the active site of the cAMP-dependent protein kinase catalytic subunit a synthesis of several previously unknown adenosine-5'-triphosphate (ATP) derivatives containing substituents of various nature at N(1), N(C6) and C(8) positions of the purine base was carried out. The interaction of these derivatives with a homogeneous preparation of the catalytic subunit of rabbit skeletal muscle cAMP-dependent protein kinase was investigated. All the nucleotide analogs were found to inhibit the enzyme activity; the inhibition was competitive with respect to ATP. It was assumed that the adenine moiety of the ATP molecule is bound to the active site of protein kinase by the hydrophobic interaction with the aromatic amino acid residues and by formation of the hydrogen bond between the exo-NH2-group of the substrate and a corresponding group of the enzyme. The "correct" binding of ATP to the enzyme active center is defined by the anti-conformation of the nucleotide.     

Characterization of Salmonella typhimurium YegS, a putative lipid kinase homologous to eukaryotic sphingosine and diacylglycerol kinases

Salmonella typhimurium YegS is a protein conserved in many prokaryotes. Although the function of YegS is not definitively known, it has been annotated as a potential diacylglycerol or sphingosine kinase based on sequence similarity with eukaryotic enzymes of known function. To further characterize YegS, we report its purification, biochemical analysis, crystallization, and structure determination. The crystal structure of YegS reveals a two-domain fold related to bacterial polyphosphate/ATP NAD kinases, comprising a central cleft between an N-terminal alpha/beta domain and a C-terminal two-layer beta-sandwich domain; conserved structural features are consistent with nucleotide binding within the cleft. The N-terminal and C-terminal domains of YegS are however counter-rotated, relative to the polyphosphate/ATP NAD kinase archetype, such that the potential nucleotide binding site is blocked. There are also two Ca2+ binding sites and two hydrophobic clefts, one in each domain of YegS. Analysis of mutagenesis data from eukaryotic homologues of YegS suggest that the N-terminal cleft may bind activating lipids while the C-terminal cleft may bind the lipid substrate. Microcalorimetry experiments showed interaction between recombinant YegS and Mg2+, Ca2+, and Mn2+ ions, with a weaker interaction also observed with polyphosphates and ATP. However, biochemical assays showed that recombinant YegS is endogenously neither an active diacylglycerol nor sphingosine kinase. Thus although the bioinformatics analysis and structure of YegS indicate that many of the ligand recognition determinants for lipid kinase activity are present, the absence of such activity may be due to specificity for a different lipid substrate or the requirement for activation by an, as yet, undetermined mechanism. In this regard the specific interaction of YegS with the periplasmic chaperone OmpH, which we demonstrate from pulldown experiments, may be of significance. Such an interaction suggests that YegS can be translocated to the periplasm and directed to the outer-membrane, an environment that may be required for enzyme activity.     

Catalytic independent functions of a protein kinase as revealed by a kinase-dead mutant: study of the Lys72His mutant of cAMP-dependent kinase

A highly conserved lysine in subdomain II is required for high catalytic activity among the protein kinases. This lysine interacts directly with ATP and mutation of this residue leads to a classical "kinase-dead" mutant. This study describes the biophysical and functional properties of a kinase-dead mutant of cAMP-dependent kinase where Lys72 was replaced with His. Although the mutant protein is less stable than the wild-type catalytic subunit, it is fully capable of binding ATP. The results highlight the effect of the mutation on stability and overall organization of the protein, especially the small lobe. Phosphorylation of the activation loop by a heterologous kinase, 3-phosphoinositide-dependent protein kinase-1 (PDK-1) also contributes dramatically to the global organization of the entire active site region. Deuterium-exchange mass spectrometry (DXMS) indicates a concerted stabilization of the entire active site following the addition of this single phosphate to the activation loop. Furthermore the mutant C-subunit is capable of binding both the type I and II regulatory subunits, but only after phosphorylation of the activation loop. This highlights the role of the large lobe as a scaffold for the regulatory subunits independent of catalytic competency and suggests that kinase dead members of the protein kinase superfamily may still have other important biological roles although they lack catalytic activity.     

Hydrogen exchange solvent protection by an ATP analogue reveals conformational changes in ERK2 upon activation

Structural and kinetic studies have provided extensive information about the molecular mechanisms of kinase activation by phosphorylation. However, it is still unclear how changes in protein dynamics and flexibility contribute to catalytic function. Mass spectrometry was used to probe changes in hydrogen/deuterium exchange in the MAP kinase, ERK2, in the presence and absence of the ATP analogue, AMP-PNP. In both active and inactive forms of ERK2, protection from hydrogen exchange by AMP-PNP binding was observed within conserved ATP binding motifs in the N-terminal lobe, which are known to directly interact with nucleotide in various protein kinases. In contrast, higher protection from exchange by AMP-PNP was observed in active ERK2 compared to inactive ERK2, in a region corresponding to the conserved DFG motif, which is located in the C-terminal lobe and coordinates Mg2+ at the catalytic site. Thus, AMP-PNP binding simultaneously protects residues within the N and C terminus in the active form of ERK2, but not the inactive form. This demonstrates that ERK2 binds nucleotide in two modes, in which active ERK2 adopts a closed conformation following nucleotide binding in solution, while inactive ERK2 adopts an open conformation. The finding provides novel evidence that phosphorylation of ERK2 facilitates interdomain closure, allowing proper orientation between ATP and substrate to facilitate phosphoryl transfer.     

Crystal structures of biotin protein ligase from Pyrococcus horikoshii OT3 and its complexes: structural basis of biotin activation

Biotin protein ligase (EC 6.3.4.15) catalyses the synthesis of an activated form of biotin, biotinyl-5'-AMP, from substrates biotin and ATP followed by biotinylation of the biotin carboxyl carrier protein subunit of acetyl-CoA carboxylase. The three-dimensional structure of biotin protein ligase from Pyrococcus horikoshii OT3 has been determined by X-ray diffraction at 1.6A resolution. The structure reveals a homodimer as the functional unit. Each subunit contains two domains, a larger N-terminal catalytic domain and a smaller C-terminal domain. The structural feature of the active site has been studied by determination of the crystal structures of complexes of the enzyme with biotin, ADP and the reaction intermediate biotinyl-5'-AMP at atomic resolution. This is the first report of the liganded structures of biotin protein ligase with nucleotide and biotinyl-5'-AMP. The structures of the unliganded and the liganded forms are isomorphous except for an ordering of the active site loop upon ligand binding. Catalytic binding sites are suitably arranged to minimize the conformational changes required during the reaction, as the pockets for biotin and nucleotide are located spatially adjacent to each other in a cleft of the catalytic domain and the pocket for biotinyl-5'-AMP binding mimics the combination of those of the substrates. The exact locations of the ligands and the active site residues allow us to propose a general scheme for the first step of the reaction carried out by biotin protein ligase in which the positively charged epsilon-amino group of Lys111 facilitates the nucleophilic attack on the ATP alpha-phosphate group by the biotin carboxyl oxygen atom and stabilizes the negatively charged intermediates.     

The crystal structure of E. coli rRNA pseudouridine synthase RluE

Pseudouridine synthase RluE modifies U2457 in a stem of 23 S RNA in Escherichia coli. This modification is located in the peptidyl transferase center of the ribosome. We determined the crystal structures of the C-terminal, catalytic domain of E. coli RluE at 1.2 A resolution and of full-length RluE at 1.6 A resolution. The crystals of the full-length enzyme contain two molecules in the asymmetric unit and in both molecules the N-terminal domain is disordered. The protein has an active site cleft, conserved in all other pseudouridine synthases, that contains invariant Asp and Tyr residues implicated in catalysis. An electropositive surface patch that covers the active site cleft is just wide enough to accommodate an RNA stem. The RNA substrate stem can be docked to this surface such that the catalytic Asp is adjacent to the target base, and a conserved Arg is positioned to help flip the target base out of the stem into the enzyme active site. A flexible RluE specific loop lies close to the conserved region of the stem in the model, and may contribute to substrate specificity. The stem alone is not a good RluE substrate, suggesting RluE makes additional interactions with other regions in the ribosome.     

Importance of the A-helix of the catalytic subunit of cAMP-dependent protein kinase for stability and for orienting subdomains at the cleft interface.

All eukaryotic protein kinases share a conserved catalytic core. In the catalytic (C) subunit of cAMP-dependent protein kinase (cAPK) this core is preceded by a myristylation motif followed by a long helix with Trp 30 at the end of this A-helix filling a hydrophobic cavity between the two lobes of the core. To understand the importance of the A-helix, the myristylation motif (delta 1-14) as well as the entire N-terminal segment (delta 1 -39) were deleted. In addition, Trp 30 was replaced with both Tyr and Ala. All proteins were overexpressed in E. coli and purified to homogeneity. rC(delta 1-14), rC(W30Y), and rC(W30A) all had reduced thermostability, but were catalytically indistinguishable from wild-type C. Based on Surface Plasmon Resonance, all three also formed stable holoenzyme complexes with the RI-subunit, although the appKds were reduced by more than 10-fold due to decrease in the association rate. Surprisingly, however, the holoenzymes were even more thermostable than wild-type holoenzyme. To obtain active enzyme, it was necessary to purify rC(delta 1-39) as a fusion protein with glutathione-S-transferase (GST-rC(delta 1-39), although its thermostability (Tm) was decreased by 12.5 degrees C, was catalytically similar to wild-type C and was inhibited by both the type I and II R-subunits and the heat-stable protein kinase inhibitor (PKI). The Tm for holoenzyme II formed with GST-rC(delta 1-39) was 16.5 degrees C greater than the Tm for free GST-rC(delta 1-39), and the Ka(cAMP) was increased nearly 10-fold. These mutants point out striking and unanticipated differences in how the RI and RII subunits associate with the C-subunit to form a stable holoenzyme and indicate, furthermore, that this N-terminal segment, far from the active site cleft, influences those interactions. The importance of the A-helix and Trp 30 for stability correlates with its location at the cleft interface where it orients the C-helix in the small lobe and the activation loop in the large so that these subdomains are aligned in a way that allows for correct configuration of residues at the active site. This extensive network of contacts that links the A-helix directly to the active site in cAPK is compared to other kinases whose crystal structures have been solved.     

Mechanism of Aurora B activation by INCENP and inhibition by hesperadin

Aurora family serine/threonine kinases control mitotic progression, and their deregulation is implicated in tumorigenesis. Aurora A and Aurora B, the best-characterized members of mammalian Aurora kinases, are approximately 60% identical but bind to unrelated activating subunits. The structure of the complex of Aurora A with the TPX2 activator has been reported previously. Here, we report the crystal structure of Aurora B in complex with the IN-box segment of the inner centromere protein (INCENP) activator and with the small molecule inhibitor Hesperadin. The Aurora B:INCENP complex is remarkably different from the Aurora A:TPX2 complex. INCENP forms a crown around the small lobe of Aurora B and induces the active conformation of the T loop allosterically. The structure represents an intermediate state of activation of Aurora B in which the Aurora B C-terminal segment stabilizes an open conformation of the catalytic cleft, and a critical ion pair in the kinase active site is impaired. Phosphorylation of two serines in the carboxyl terminus of INCENP generates the fully active kinase.     

A three-dimensional model of the Cdc2 protein kinase: localization of cyclin- and Suc1-binding regions and phosphorylation sites.

The Cdc2 protein kinase requires cyclin binding for activity and also binds to a small protein, Suc1. Charged-to-alanine scanning mutagenesis of Cdc2 was used previously to localize cyclin A- and B- and Suc1-binding sites (B. Ducommun, P. Brambilla, and G. Draetta, Mol. Cell. Biol. 11:6177-6184, 1991). Those sites were mapped by building a Cdc2 model based on the crystallographic coordinates of the catalytic subunit of cyclic AMP-dependent protein kinase (cAPK) (D. R. Knighton, J. Zheng, L. F. Ten Eyck, V. A. Ashford, N.-H. Xuong, S. S. Taylor, and J. M. Sowadski, Science 253:407-414, 1991). On the basis of this model, additional mutations were made and tested for cyclin A and Suc1 binding and for kinase activity. Mutations that interfere with cyclin A binding are localized primarily on the small lobe near its interface with the cleft and include an acidic patch on the B helix and R-50 in the highly conserved PSTAIRE sequence. Two residues in the large lobe, R-151 and T-161, influence cyclin binding, and both are at the surface of the cleft near its interface with the PSTAIRE motif. Cyclin-dependent phosphorylation of T-161 in Cdc2 is essential for activation, and the model provides insights into the importance of this site. T-161 is equivalent to T-197, a stable phosphorylation site in cAPK. On the basis of the model, cyclin binding very likely alters the surface surrounding T-161 to allow for T-161 phosphorylation. The two major ligands to T-197 in cAPK are conserved as R-127 and R-151 in Cdc2. The equivalent of the third ligand, H-87, is T-47 in the PSTAIRE sequence motif. Once phosphorylated, T-161 is predicted to play a major structural role in Cdc2, comparable to that of T-197 in cAPK, by assembling the active conformation required for peptide recognition. The inhibitory phosphorylation at Y-15 also comes close to the cleft interface and on the basis of this model would disrupt the cleft interface and the adjacent peptide recognition site rather than prevent ATP binding. In contrast to cyclin A, both lobes influence Suc1 binding; however, the Suc1-binding sites are far from the active site. Several mutants map to the surface in cAPK, which is masked in part by the N-terminal 40 residues that lie outside the conserved catalytic core. The other Suc1-binding site maps to the large lobe near a 25-residue insert and includes R-215.     

Dynamics of signaling by PKA

The catalytic and regulatory subunits of cAMP-dependent protein kinase (PKA) are highly dynamic signaling proteins. In its dissociated state the catalytic subunit opens and closes as it moves through its catalytic cycle. In this subunit, the core that is shared by all members of the protein kinase family is flanked by N- and C-terminal segments. Each are anchored firmly to the core by well-defined motifs and serve to stabilize the core. Protein kinases are not only catalysts, they are also scaffolds. One of their major functions is to bind to other proteins. In addition to its interactions with the N- and C- termini, the catalytic subunit interacts with its inhibitor proteins, PKI and the regulatory subunits. Both bind with subnanomolar affinity. To achieve this tight binding requires docking of a substrate mimetic to the active site cleft as well as a peripheral docking site. The peripheral site used by PKI is distinct from that used by RIalpha as revealed by a recent structure of a C:RIalpha complex. Upon binding to the catalytic subunit, the linker region of RIalpha becomes ordered. In addition, cAMP-binding domain A undergoes major conformational changes. RIalpha is a highly malleable protein. Using small angle X-ray scattering, the overall shape of the regulatory subunits and corresponding holoenzymes have been elucidated. These studies reveal striking and surprising isoform differences.     

Structural basis for the low affinities of yeast cAMP-dependent and mammalian cGMP-dependent protein kinases for protein kinase inhibitor peptides.

Affinities of the catalytic subunit (C1) of Saccharomyces cerevisiae cAMP-dependent protein kinase and of mammalian cGMP-dependent protein kinase were determined for the protein kinase inhibitor (PKI) peptide PKI(6-22)amide and seven analogues. These analogues contained structural alterations in the N-terminal alpha-helix, the C-terminal pseudosubstrate portion, or the central connecting region of the PKI peptide. In all cases, the PKI peptides were appreciably less active as inhibitors of yeast C1 than of mammalian C alpha subunit. Ki values ranged from 5- to 290-fold higher for the yeast enzyme than for its mammalian counterpart. Consistent with these results, yeast C1 exhibited a higher Km for the peptide substrate Kemptide. All of the PKI peptides were even less active against the mammalian cGMP-dependent protein kinase than toward yeast cAMP-dependent protein kinase, and Kemptide was a poorer substrate for the former enzyme. Alignment of amino acid sequences of these homologous protein kinases around residues in the active site of mammalian C alpha subunit known to interact with determinants in the PKI peptide [Knighton, D. R., Zheng, J., Ten Eyck, L. F., Xuong, N-h, Taylor, S. S., & Sowadski, J. M. (1991) Science 253, 414-420] provides a structural basis for the inherently lower affinities of yeast C1 and cGMP-dependent protein kinase for binding peptide inhibitors and substrates. Both yeast cAMP-dependent and mammalian cGMP-dependent protein kinases are missing two of the three acidic residues that interact with arginine-18 in the pseudosubstrate portion of PKI. Further, the cGMP-dependent protein kinase appears to completely lack the hydrophobic/aromatic pocket that recognizes the important phenylalanine-10 residue in the N-terminus of the PKI peptide, and binding of the inhibitor by the yeast protein kinase at this site appears to be partially compromised.     

Crystal structure of Escherichia coli PurE, an unusual mutase in the purine biosynthetic pathway

BACKGROUND: Conversion of 5-aminoimidazole ribonucleotide (AIR) to 4-carboxyaminoimidazole ribonucleotide (CAIR) in Escherichia coli requires two proteins - PurK and PurE. PurE has recently been shown to be a mutase that catalyzes the unusual rearrangement of N(5)-carboxyaminoimidazole ribonucleotide (N(5)-CAIR), the PurK reaction product, to CAIR. PurEs from higher eukaryotes are homologous to E. coli PurE, but use AIR and CO(2) as substrates to produce CAIR directly. RESULTS: The 1.50 A crystal structure of PurE reveals an octameric structure with 422 symmetry. A central three-layer (alphabetaalpha) sandwich domain and a kinked C-terminal helix form the folded structure of the monomeric unit. The structure reveals a cleft at the interface of two subunits and near the C-terminal helix of a third subunit. Co-crystallization experiments with CAIR confirm this to be the mononucleotide-binding site. The nucleotide is bound predominantly to one subunit, with conserved residues from a second subunit making up one wall of the cleft. CONCLUSIONS: The crystal structure of PurE reveals a unique quaternary structure that confirms the octameric nature of the enzyme. An analysis of the native crystal structure, in conjunction with sequence alignments and studies of co-crystals of PurE with CAIR, reveals the location of the active site. The environment of the active site and the analysis of conserved residues between the two classes of PurEs suggests a model for the differences in their substrate specificities and the relationship between their mechanisms.     

Distinct in vitro interaction pattern of dopamine receptor subtypes with adaptor proteins involved in post-endocytotic receptor targeting

The sequences contributing to the catalytic site of protein kinases are not all comprised within the highly conserved catalytic core. Thus, in mammalian cAMP-dependent protein kinase (PKA), the C-terminal sequence participates in substrate binding. Using synthetic peptides mimicking the FxxF motif present at most C-termini of AGC kinases, we have raised highly specific antibodies which are potent and specific inhibitors of the catalytic activity of the cognate protein kinase. Taking into account the structure of PKA, these results point to the potential of the C-terminal region of protein kinases as a target for designing specific protein kinase inhibitors.     

Analysis of micronuclear, macronuclear and cDNA sequences encoding the regulatory subunit of cAMP-dependent protein kinase of Euplotes octocarinatus: evidence for a ribosomal frameshift

We have isolated and characterized the micronuclear gene encoding the regulatory subunit of cAMP-dependent protein kinase of the ciliated protozoan Euplotes octocarinatus, as well as its macronuclear version and the corresponding cDNA. Analyses of the sequences revealed that the micronuclear gene contains one small 69-bp internal eliminated sequence (IES) that is removed during macronuclear development. The IES is located in the 5'-noncoding region of the micronuclear gene and is flanked by a pair of tetranucleotide 5'-TACA-3' direct repeats. The macronuclear DNA molecule carrying this gene is approximately 1400 bp long and is amplified to about 2000 copies per macronucleus. Sequence analysis suggests that the expression of this gene requires a +1 ribosomal frameshift. The deduced protein shares 31% identity with the cAMP-dependent protein kinase type I regulatory subunit of Homo sapiens, and 53% identity with the regulatory subunit R44 of one of the two cAMP-dependent protein kinases of Paramecium. In addition, it contains two highly conserved cAMP binding sites in the C-terminal domain. The putative autophosphorylation site ARTSV of the regulatory subunit of E. octocarinatus is similar to that of the regulatory subunit R44 of Paramecium but distinct from the consensus motif RRXSZ of other eukaryotic regulatory subunits of cAMP-dependent protein kinases.     

Structure of the Methanococcus jannaschii mevalonate kinase, a member of the GHMP kinase superfamily.

The mevalonate-dependent pathway is used by many organisms to synthesize isopentenyl pyrophosphate, the building block for the biosynthesis of many biologically important compounds, including farnesyl pyrophosphate, dolichol, and many sterols. Mevalonate kinase (MVK) catalyzes a critical phosphoryl transfer step, producing mevalonate 5'-phosphate. The crystal structure of thermostable MVK from Methanococcus jannaschii has been determined at 2.4 A, revealing an overall fold similar to the homoserine kinase from M. jannaschii. In addition, the enzyme shows structural similarity with mevalonate 5-diphosphate decarboxylase and domain IV of elongation factor G. The active site of MVK is in the cleft between its N- and C-terminal domains. Several structural motifs conserved among species, including a phosphate-binding loop, have been found in this cavity. Asp(155), an invariant residue among MVK sequences, is located close to the putative phosphate-binding site and has been assumed to play the catalytic role. Analysis of the MVK model in the context of the other members of the GHMP kinase family offers the opportunity to understand both the mechanism of these enzymes and the structural details that may lead to the design of novel drugs.