Crystal structure of a transition state mimic of the catalytic subunit of cAMP-dependent protein kinase

Madhusudan, M. D.; Akamine, Pearl; Xuong, Nguyen‐Huu; Taylor, Susan S.

doi:10.1038/nsb780

Cited by 202 publications

(205 citation statements)

References 32 publications

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“…In particular, each analogue can make a key hydrogen bond with Phe54:N, thus stabilizing the tip of the Gly-rich loop. Comparable stability is only seen in closed conformation structures (2,27,28). The Gly-rich loop stabilization may be the structural basis for the enhanced thermostability conferred by the analogues (described above).…”

Section: Resultsmentioning

confidence: 97%

Balanol Analogues Probe Specificity Determinants and the Conformational Malleability of the Cyclic 3‘,5‘-Adenosine Monophosphate-Dependent Protein Kinase Catalytic Subunit^,

et al. 2003

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The protein kinase family is a prime target for therapeutic agents, since unregulated protein kinase activities are linked to myriad diseases. Balanol, a fungal metabolite consisting of four rings, potently inhibits Ser/Thr protein kinases and can be modified to yield potent inhibitors that are selectives characteristics of a desirable pharmaceutical compound. Here, we characterize three balanol analogues that inhibit cyclic 3′,5′-adenosine monophosphate-dependent protein kinase (PKA) more specifically and potently than calcium-and phospholipid-dependent protein kinase (PKC). Correlation of thermostability and inhibition potency suggests that better inhibitors confer enhanced protection against thermal denaturation. Crystal structures of the PKA catalytic (C) subunit complexed to each analogue show the Gly-rich loop stabilized in an "intermediate" conformation, disengaged from important phosphoryl transfer residues. An analogue that perturbs the PKA C-terminal tail has slightly weaker inhibition potency. The malleability of the PKA C subunit is illustrated by active site residues that adopt alternate rotamers depending on the ligand bound. On the basis of sequence homology to PKA, a preliminary model of the PKC active site is described. The balanol analogues serve to test the model and to highlight differences in the active site local environment of PKA and PKC. The PKA C subunit appears to tolerate balanol analogues with D-ring modifications; PKC does not. We attribute this difference in preference to the variable B helix and C-terminal tail. By understanding the details of ligand binding, more specific and potent inhibitors may be designed that differentiate among closely related AGC protein kinase family members.As cellular processes are better understood at the molecular level, especially in the context of recent genomic information, there has been an increased effort to target specific proteins that are linked to disease. Protein kinases are a diverse family of enzymes that have various regulatory roles yet function similarly by catalyzing the phosphoryl transfer of the γ-phosphate of ATP 1 to an enzyme-specific protein substrate. Since many diseases, including cancer, autoimmune disorders, cardiac disease, and diabetes, are associated with defects in protein phosphorylation, and there are an estimated ∼500 protein kinases in the human genome (1), this family is a major target in the design of pharmaceutical agents and inhibitors. A major challenge for the development of therapeutics is the identification of compounds that have high selectivity. To better understand binding diversity and how this correlates with specificity for protein kinases, three analogues of balanol were studied that specifically inhibit † Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory, a national user facility operated by Stanford University on behalf of the Office of Basic Energy Sciences, U.S.

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Section: Resultsmentioning

confidence: 97%

Balanol Analogues Probe Specificity Determinants and the Conformational Malleability of the Cyclic 3‘,5‘-Adenosine Monophosphate-Dependent Protein Kinase Catalytic Subunit^,

et al. 2003

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“…[58] 19 F NMR established the major presence of MgF3 -in the complex along with some octahedral AlF4 -, showing that charge balance predominates over geometry in selection of the TS analog (Section 4.2, Fig. 16B).…”

Section: Mgf3 -Misidentified As a Pentaoxyphosphoranementioning

confidence: 92%

Metal Fluorides as Analogues for Studies on Phosphoryl Transfer Enzymes

et al. 2017

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Abstract:The 1994 structure of a transition state analog with AlF4 -and GDP complexed to G1α, a small G protein, heralded a new field of research into structure and mechanism of enzymes that manipulate transfer of the phosphoryl (PO3 -) group. The list of enzyme structures that embrace metal fluorides, MFx, as ligands that imitate either the phosphoryl group or a phosphate, is now growing at over 80 per triennium. They fall into three distinct geometrical classes: (i) Tetrahedral complexes, based on BeF3 -, mimic ground state phosphates; (ii) Octahedral complexes, primarily based on AlF4 -, mimic "in-line" anionic transition state for phosphoryl transfer; and (iii) Trigonal bipyramidal complexes, represented by MgF3 -and putative AlF3 0 moieties, additionally mimic the tbp geometry of the transition state. The interpretation of these structures provides a deeper mechanistic understanding of the behavior and manipulation of phosphate monoesters in molecular biology. This review provides a comprehensive overview of these structures, their uses, and their computational development. It questions the identification of AlF3 0 and MgF4 = as tbp species in protein complexes and discusses the relevance of physical organic chemistry and water-based model studies for understanding phosphoryl group transfer in enzymes. It describes two roles for amino acid side-chains that mediate proton transfers during phosphoryl transfer, based on the analysis of protein/MFx structures. First, they deploy hydrogen bonding to neutral oxygen nucleophiles so as to orientate them for correct orbital overlap with the electrophilic phosphorus center. Secondly, they behave as classical general acid/base catalysts.

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“…Threonine residues at this position play a conserved role in serine/threonine protein kinases, serving as an anchor point for the C-terminal end of the T-loop in the activated, substratebound kinase. Ternary complex structures of protein kinase A (51) show that this residue stabilizes the conformation of catalytically important residues during phosphoryl transfer, explaining why phosphorylation of Thr-179 is inhibitory (Fig. 5B).…”

Section: Bacterial Expression Of Nek2 Constructs and Phosphorylationmentioning

confidence: 99%

Structure and Regulation of the Human Nek2 Centrosomal Kinase

Rellos

Ivins

Baxter

et al. 2007

Journal of Biological Chemistry

125

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The dimeric Ser/Thr kinase Nek2 regulates centrosome cohesion and separation through phosphorylation of structural components of the centrosome, and aberrant regulation of Nek2 activity can lead to aneuploid defects characteristic of cancer cells. Mutational analysis of autophosphorylation sites within the kinase domain identified by mass spectrometry shows a complex pattern of positive and negative regulatory effects on kinase activity that are correlated with effects on centrosomal splitting efficiency in vivo. The 2.2-Å resolution x-ray structure of the Nek2 kinase domain in complex with a pyrrole-indolinone inhibitor reveals an inhibitory helical motif within the activation loop. This helix presents a steric barrier to formation of the active enzyme and generates a surface that may be exploitable in the design of specific inhibitors that selectively target the inactive state. Comparison of this "auto-inhibitory" conformation with similar arrangements in cyclin-dependent kinase 2 and epidermal growth factor receptor kinase suggests a role for dimerization-dependent allosteric regulation that combines with autophosphorylation and protein phosphatase 1c phosphatase activity to generate the precise spatial and temporal control required for Nek2 function in centrosomal maturation.Protein kinase activity is crucial for the precise regulation of the eukaryotic cell cycle. Although cyclin-dependent kinases remain the master regulators of cell cycle progression, it is clear that a variety of other proteins kinases also play important roles.Nek2 is a member of the NIMA 6 -related kinase (or Nek) family of serine/threonine protein kinases, so named because all are related to the NIMA kinase of Aspergillus nidulans (for review, see Ref. 1). In Aspergillus, NIMA is essential for mitotic entry with temperature-sensitive nimA mutations leading to cells that are "never in mitosis" (2). Like Aspergillus, other fungi also express a single NIMA-related kinase, e.g. Kin3 in Saccharomyces cerevisiae and Fin1 in Schizosaccharomyces pombe. Although the yeast Neks do not appear to be essential for cell cycle progression, elegant studies on Fin1 have revealed key functions in chromosome segregation and mitotic exit (3, 4). In contrast, higher eukaryotes express multiple Neks with 11 genes (Nek1 to Nek11) encoded within the human genome (1). With the exception of human Nek6 and Nek7 and Nek10, all Neks have an N-terminal kinase domain followed by a C-terminal non-catalytic regulatory domain. However, the length and composition of motifs present within these C-terminal extensions vary considerably, reflecting diverse roles in cell regulation.Of the human proteins, Nek2 is the most closely related to the fungal kinases, being 47% identical to NIMA within the amino acid sequence of their catalytic domains. Nek2 localizes to centrosomes. Here, it contributes to spindle pole formation during mitosis (5) through phosphorylation of proteins involved in centriolar cohesion, including C-Nap1 and rootletin, to allow spindle pole separation (6 ...

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Crystal structure of a transition state mimic of the catalytic subunit of cAMP-dependent protein kinase

Cited by 202 publications

References 32 publications

Balanol Analogues Probe Specificity Determinants and the Conformational Malleability of the Cyclic 3‘,5‘-Adenosine Monophosphate-Dependent Protein Kinase Catalytic Subunit^,

Balanol Analogues Probe Specificity Determinants and the Conformational Malleability of the Cyclic 3‘,5‘-Adenosine Monophosphate-Dependent Protein Kinase Catalytic Subunit^,

Metal Fluorides as Analogues for Studies on Phosphoryl Transfer Enzymes

Structure and Regulation of the Human Nek2 Centrosomal Kinase

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Crystal structure of a transition state mimic of the catalytic subunit of cAMP-dependent protein kinase

Cited by 202 publications

References 32 publications

Balanol Analogues Probe Specificity Determinants and the Conformational Malleability of the Cyclic 3‘,5‘-Adenosine Monophosphate-Dependent Protein Kinase Catalytic Subunit,

Balanol Analogues Probe Specificity Determinants and the Conformational Malleability of the Cyclic 3‘,5‘-Adenosine Monophosphate-Dependent Protein Kinase Catalytic Subunit,

Metal Fluorides as Analogues for Studies on Phosphoryl Transfer Enzymes

Structure and Regulation of the Human Nek2 Centrosomal Kinase

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Balanol Analogues Probe Specificity Determinants and the Conformational Malleability of the Cyclic 3‘,5‘-Adenosine Monophosphate-Dependent Protein Kinase Catalytic Subunit^,

Balanol Analogues Probe Specificity Determinants and the Conformational Malleability of the Cyclic 3‘,5‘-Adenosine Monophosphate-Dependent Protein Kinase Catalytic Subunit^,