2.0 Å refined crystal structure of the catalytic subunit of cAMP-dependent protein kinase complexed with a peptide inhibitor and detergent

Knighton, Daniel R.; Bell, Sean; Zheng, Jimin; Eyck, Lynn F. Ten; Xuong, Nguyen‐Huu; Taylor, Susan S.; Sowadski, Janusz M.

doi:10.1107/s0907444993000502

Cited by 73 publications

(80 citation statements)

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“…The indole ring of Trp30 is stabilized, in particular, by stacking between Arg93 from the small lobe and Arg190 from the large lobe. The close proximity of Thr'97, an essential phosphorylation site in m-C (22), is also shown in Figs. 2 and 3A.…”

Section: Methodsmentioning

confidence: 54%

A conserved helix motif complements the protein kinase core.

Véron

Radzio‐Andzelm

Tsigelny

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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Residues 40-300 of the mammalian catalytic (C) subunit of cAMP-dependent protein kinase define a conserved biobal catalytic core shared by all eukaryotic protein kinases. Contiguous to the core is an extended amphipathic a-helix (A helix). Trp3O, a prominent feature of this helix, fills a deep hydrophobic pocket between the two lobes on the surface opposite to the active site. The C subunit in Dictyostelium discoideum shows sequence conservation of residues 40-350 with the mouse enzyme but contains an N-terminal extension of 332 residues. A sequence corresponding to the A helix contiguous to the core is absent. However, we have now identified a remote A-helix motif (residues 77-98). When the core of the Dictyostelium C subunit was modeled, based on the mouse C subunit, complementarity between this putative A helix and the surface of the core was found to be conserved. Analysis of other protein kinases reveals that the A-helix motif is not restricted to cAMP-dependent protein kinase. In the Src-related family of protein kinases, for example, an A helix is very likely contiguous to the core, thus serving as a linker between the conserved catalytic core and the Src homology 2 domain. We predict that an A-helix motif complementary to the core will be a conserved feature of most eukaryotic protein kinases.

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Section: Methodsmentioning

confidence: 54%

A conserved helix motif complements the protein kinase core.

Véron

Radzio‐Andzelm

Tsigelny

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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“…Third, each of the three mutated systems was energy minimized for 1000 iterations with (i) relaxed side chains, a fixed backbone, and a fixed MgATP level or (ii) relaxed side chains, a relaxed NPL backbone, and a fixed MgATP level. Crystallographic temperature factors (B-factors) were compared for C-subunit crystal structures solved for complexes with ATP (36) and without (38) ATP to determine the overall flexibility of the C-subunit. Temperature factors for amino acid backbones were calculated as an average of B-factors for four atoms, C R , C , C, and N.…”

Section: Methodsmentioning

confidence: 99%

Kinetic Analyses of Mutations in the Glycine-Rich Loop of cAMP-Dependent Protein Kinase

et al. 1998

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The conserved glycines in the glycine-rich loop (Leu-Gly 50 -Thr-Gly 52 -Ser-Phe-Gly 55 -ArgVal) of the catalytic (C) subunit of cAMP-dependent protein kinase were each mutated to Ser (G50S, G52S, and G55S). The effects of these mutations were assessed here using both steady-state and presteady-state kinetic methods. While G50S and G52S reduced the apparent affinity for ATP by approximately 10-fold, substitution at Gly55 had no effect on nucleotide binding. In contrast to ATP, only mutation at position 50 interfered with ADP binding. These three mutations lowered the rate of phosphoryl transfer by 7-300-fold. The combined data indicate that G50 and G52 are the most critical residues in the loop for catalysis, with replacement at position 52 being the most extreme owing to a larger decrease in the rate of phosphoryl transfer (29 vs 1.6 s -1 in contrast to 500 s -1 for wild-type C). Surprisingly, all three mutations lowered the affinity for Kemptide by approximately 10-fold, although none of the loop glycines makes direct contact with the substrate. The inability to correlate the rate constant for net product release with the dissociation constant for ADP implies that other steps may limit the decomposition of the ternary product complex. The observations that G52S (a) selectively affects ATP binding and (b) significantly lowers the rate of phosphoryl transfer without making direct contact with either the nucleotide or the peptide imply that this residue serves a structural role in the loop, most likely by positioning the backbone amide of Ser53 for contacting the γ-phosphate of ATP. Energyminimized models of the mutant proteins are consistent with the observed kinetic consequences of each mutation. The models predict that only mutation of Gly52 will interfere with the observed hydrogen bonding between the backbone and ATP.Many nucleotide binding enzymes utilize glycine-rich loops that help position nucleotides for catalysis (1-3). Crystal structures of dinucleotide and mononucleotide binding enzymes define several glycine-rich loops that are distinct both in their amino acid sequence and in their orientation relative to the nucleotide. The glycine-rich loops found in dinucleotide binding enzymes [e.g., pyruvate dehydrogenase (4) and dihydrofolate reductase (5)] are typically Gly-X-Gly-X-X-Gly and provide space for the dinucleotide pyrophosphate moiety. The glycine-rich loops, or P-loops, found in mononucleotide binding enzymes [e.g., p21ras (6), adenylate kinase (7), Rec A (8), and elongation factor Tu (9)] have the consensus sequence Gly-X-X-X-X-Gly-Lys-Ser/Thr and interact with the γ-phosphate of ATP (10). In contrast, the protein kinase family of enzymes contain a glycine-rich loop with a consensus sequence Gly-X-Gly-X-X-Gly-X-Val. On the basis of numerous crystal structures, this loop is part of a -hairpin that connects -strands 1 and 2 and serves as a nucleotide-positioning loop (NPL) 1 that interacts with all three phosphates of ATP (11)(12)(13)(14)(15). The glycines in the protein kinase NPL are ...

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“…To further understand the detailed structural rearrangements accompanying kinase catalytic events, crystal structures of complexes involving the C subunit of cAPK subsequently were solved (38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50), as were numerous crystal structures of diverse kinases (51)(52)(53)(54)(55)(56)(57)(58)(59)(60)(61)(62)(63)(64)(65) and inhibitor-kinase complexes (66)(67)(68)(69)(70)(71)(72)(73)(74)(75). Not only do these structures show how kinase core catalytic activity is affected either by endogenous inhibitors (e.g., R subunits, PKI and self-associating protein segments) or by exogenous ones (e.g., divalent metal ions, the natural product balanol and a host of synthetic analogues), they also provide greater insight into the specific interactions underlying inhibition.…”

mentioning

confidence: 99%

Crystal Structure of the Potent Natural Product Inhibitor Balanol in Complex with the Catalytic Subunit of cAMP-Dependent Protein Kinase

et al. 1999

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Endogenous protein kinase inhibitors are essential for a wide range of physiological functions. These endogenous inhibitors may mimic peptide substrates as in the case of the heat-stable protein kinase inhibitor (PKI), or they may mimic nucleotide triphosphates. Natural product inhibitors, endogenous to the unique organisms producing them, can be potent exogenous inhibitors against foreign protein kinases. Balanol is a natural product inhibitor exhibiting low nanomolar K i values against serine and threonine specific kinases, while being ineffective against protein tyrosine kinases. To elucidate balanol's specific inhibitory effects and provide a basis for understanding inhibition-regulated biological processes, a 2.1 Å resolution crystal structure of balanol in complex with cAMP-dependent protein kinase (cAPK) was determined. The structure reveals conserved binding regions and displays extensive complementary interactions between balanol and conserved cAPK residues. This report describes the structure of a protein kinase crystallized with a natural ATP mimetic in the absence of metal ions and peptide inhibitor.Protein kinases, of which the human genome is predicted to encode several thousand, reversibly phosphorylate diverse molecular targets and are critical enzymes in the initiation, regulation, and abrogation of both normal and abnormal cellular functions (reviewed in refs 1-6). These often exquisitely specific kinases play essential roles in apoptosis, cell proliferation, gene expression, glycogen metabolism, immune response, neurotransmission, oncogenesis, and secondary messenger signal transduction (7-18). This biological pervasiveness underscores the importance of more explicitly characterizing kinase activation and inhibition. Additionally, there is significant therapeutic value in achieving selective pharmacological control of members of this important class of enzymes, since unregulated or otherwise defective protein kinase activities to date have been implicated in asthma, cancer, cardiovascular disorders, central nervous system (CNS) 1 diseases, diabetes, human immunodeficiency virus (HIV) infections, inflammation, psoriasis, and rheumatoid arthritis (19)(20)(21)(22)(23)(24).To more explicitly characterize protein kinase activation and inhibition, cAMP-dependent protein kinase, the most completely characterized and mechanistically simplest protein kinase (25, reviewed in refs 26-31), is a logical singular choice for biochemical and structural research focusing on cellular phosphorylation events at the molecular level. The inactive cAPK holoenzyme consists of two regulatory (R) and two catalytic (C) subunits that dissociate in response to elevated levels of intracellular cAMP. The triply phosphorylated, active C subunit then phosphorylates serine or threonine residues of peptide substrates containing the consensus sequence Arg-Arg-X-Ser[Thr]-Hyd, where X is variable and Hyd is any hydrophobic residue (3,14,32,33). The C subunit of cAPK shares with other kinase family members a conserved cat...

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2.0 Å refined crystal structure of the catalytic subunit of cAMP-dependent protein kinase complexed with a peptide inhibitor and detergent

Cited by 73 publications

References 0 publications

A conserved helix motif complements the protein kinase core.

A conserved helix motif complements the protein kinase core.

Kinetic Analyses of Mutations in the Glycine-Rich Loop of cAMP-Dependent Protein Kinase

Crystal Structure of the Potent Natural Product Inhibitor Balanol in Complex with the Catalytic Subunit of cAMP-Dependent Protein Kinase

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