Two structures of the catalytic domain of phosphorylase kinase: an active protein kinase complexed with substrate analogue and product.

BACKGROUND Control of intracellular events by protein phosphorylation is promoted by specific protein kinases. All the known protein kinase possess a common structure that defines a catalytically competent entity termed the 'kinase catalytic core'. Within this common structural framework each kinase displays its own unique substrate specificity, and a regulatory mechanism that may be modulated by association with other proteins. Structural studies of phosphorylase kinase (Phk), the major substrate of which is glycogen phosphorylase, may be expected to shed light on its regulation. RESULTS We report two crystal structures of the catalytic core (residues 1-298; Phk gamma trnc) of the gamma-subunit of rabbit muscle phosphorylase kinase: the binary complex with Mn2+/beta-gamma-imidoadenosine 5'-triphosphate (AMPPNP) to a resolution of 2.6 A and the binary complex with Mg2+/ADP to a resolution of 3.0 A. The structures were solved by molecular replacement using the cAMP-dependent protein kinase (cAPK) as a model. CONCLUSIONS The overall structure of Phk gamma trnc is similar to that of the catalytic core of other protein kinases. It consists of two domians joined on one edge by a 'hinge', with the catalytic site located in the cleft between the domains. Phk gamma trnc is constitutively active, and lacks the need for an activatory phosphorylation event that is essential for many kinases. The structure exhibits an essentially 'closed' conformation of the domains which is similar to that of cAPK complexed with substrates. The phosphorylated residue that is located at the domain interface in many protein kinases and that is believed to stabilize an active conformation is substituted by a glutamate in Phk gamma trnc. The glutamate, in a similar manner to the phosphorylated residue in other protein kinases, interacts with an arginine adjacent to the catalytic aspartate but does not participate in interdomain contacts. The interactions between the enzyme and the nucleotide product of its activity, Mg2+/ADP, explain the inhibitory properties of the nucleotides that are observed in kinetic studies.

[1]  D. Graves,et al.  Properties of the gamma subunit of phosphorylase kinase. , 1987, The Journal of biological chemistry.

[2]  J. Zheng,et al.  Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. , 1991, Science.

[3]  S. Taylor,et al.  Phosphorylation modulates catalytic function and regulation in the cAMP-dependent protein kinase. , 1995, Biochemistry.

[4]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[5]  H. Paudel,et al.  Inhibition of the catalytic subunit of phosphorylase kinase by its alpha/beta subunits. , 1987, The Journal of biological chemistry.

[6]  J. Trewhella,et al.  Solution structure of the cAMP-dependent protein kinase catalytic subunit and its contraction upon binding the protein kinase inhibitor peptide. , 1993, Biochemistry.

[7]  D. Knighton,et al.  Structural basis of the intrasteric regulation of myosin light chain kinases. , 1992, Science.

[8]  L. Johnson,et al.  Expression of the phosphorylase kinase γ subunit catalytic domain in Escherichia coli , 1992 .

[9]  D. Knighton,et al.  2.0 A refined crystal structure of the catalytic subunit of cAMP-dependent protein kinase complexed with a peptide inhibitor and detergent. , 1993, Acta crystallographica. Section D, Biological crystallography.

[10]  J. Navaza,et al.  AMoRe: an automated package for molecular replacement , 1994 .

[11]  S. Hanks,et al.  Protein kinase catalytic domain sequence database: identification of conserved features of primary structure and classification of family members. , 1991, Methods in enzymology.

[12]  Susan S. Taylor,et al.  2.2 A refined crystal structure of the catalytic subunit of cAMP-dependent protein kinase complexed with MnATP and a peptide inhibitor. , 1993, Acta crystallographica. Section D, Biological crystallography.

[13]  P. Cohen,et al.  The role of protein phosphorylation in neural and hormonal control of cellular activity , 1982, Nature.

[14]  R. Read Improved Fourier Coefficients for Maps Using Phases from Partial Structures with Errors , 1986 .

[15]  D. Walsh,et al.  Analysis by mutagenesis of the ATP binding site of the gamma subunit of skeletal muscle phosphorylase kinase expressed using a baculovirus system. , 1992, Biochemistry.

[16]  Susan S. Taylor,et al.  Crystal structures of the myristylated catalytic subunit of cAMP‐dependent protein kinase reveal open and closed conformations , 1993, Protein science : a publication of the Protein Society.

[17]  L. Heilmeyer,et al.  The alpha and beta subunits of phosphorylase kinase are homologous: cDNA cloning and primary structure of the beta subunit. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[18]  R. Pearson,et al.  Intrasteric regulation of protein kinases and phosphatases. , 1991, Biochimica et biophysica acta.

[19]  D. Graves Use of peptide substrates to study the specificity of phosphorylase kinase phosphorylation. , 1983, Methods in enzymology.

[20]  D. Koshland Application of a Theory of Enzyme Specificity to Protein Synthesis. , 1958, Proceedings of the National Academy of Sciences of the United States of America.

[21]  D. Walsh,et al.  10 Phosphorylase Kinase , 1986 .

[22]  A. Mildvan,et al.  Magnetic resonance and kinetic studies of the manganese(II) ion and substrate complexes of the catalytic subunit of adenosine 3',5'-monophosphate dependent protein kinase from bovine heart. , 1979, Biochemistry.

[23]  Susan S. Taylor,et al.  Three protein kinase structures define a common motif. , 1994, Structure.

[24]  P. Cohen,et al.  Identification of the Ca2+‐dependent modulator protein as the fourth subunit of rabbit skeletal muscle phosphorylase kinase , 1978, FEBS letters.

[25]  R. Pearson,et al.  Protein kinase phosphorylation site sequences and consensus specificity motifs: tabulations. , 1991, Methods in enzymology.

[26]  Elizabeth J. Goldsmith,et al.  Atomic structure of the MAP kinase ERK2 at 2.3 Å resolution , 1994, Nature.

[27]  R. Huber,et al.  Phosphotransferase and substrate binding mechanism of the cAMP‐dependent protein kinase catalytic subunit from porcine heart as deduced from the 2.0 A structure of the complex with Mn2+ adenylyl imidodiphosphate and inhibitor peptide PKI(5‐24). , 1993, The EMBO journal.

[28]  M. Way,et al.  Identification of a region in segment 1 of gelsolin critical for actin binding. , 1990, The EMBO journal.

[29]  J. Zheng,et al.  Structure of a peptide inhibitor bound to the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. , 1991, Science.

[30]  W. Kabsch,et al.  Dictionary of protein secondary structure: Pattern recognition of hydrogen‐bonded and geometrical features , 1983, Biopolymers.

[31]  E F Garman,et al.  Expression, purification and crystallisation of phosphorylase kinase catalytic domain. , 1995, Journal of molecular biology.

[32]  Sung-Hou Kim,et al.  Crystal structure of cyclin-dependent kinase 2 , 1993, Nature.

[33]  Nguyen-Huu Xuong,et al.  Crystal structure of the catalytic subunit of cAMP-dependent protein kinase complexed with magnesium-ATP and peptide inhibitor , 1993 .

[34]  P. Evans,et al.  Site-directed mutagenesis identifies catalytic residues in the active site of Escherichia coli phosphofructokinase. , 1992, Biochemistry.

[35]  A. Lesk,et al.  The relation between the divergence of sequence and structure in proteins. , 1986, The EMBO journal.

[36]  T. Teng,et al.  Mounting of crystals for macromolecular crystallography in a free-standing thin film , 1990 .

[37]  D. Knighton,et al.  Systematic mutational analysis of cAMP-dependent protein kinase identifies unregulated catalytic subunits and defines regions important for the recognition of the regulatory subunit. , 1992, The Journal of biological chemistry.

[38]  M. Wigler,et al.  A mutation in the catalytic subunit of cAMP-dependent protein kinase that disrupts regulation. , 1988, Science.

[39]  S. Hubbard,et al.  Crystal structure of the tyrosine kinase domain of the human insulin receptor , 1994, Nature.

[40]  E. Krebs,et al.  An adenosine 3',5'-monophosphate-dependant protein kinase from rabbit skeletal muscle. , 1968, The Journal of biological chemistry.

[41]  K. Gould,et al.  Phosphorylation at Thr167 is required for Schizosaccharomyces pombe p34cdc2 function. , 1991, The EMBO journal.

[42]  G J Barton,et al.  ALSCRIPT: a tool to format multiple sequence alignments. , 1993, Protein engineering.

[43]  H. Paudel,et al.  The ATPase activity of phosphorylase kinase is regulated in parallel with its protein kinase activity. , 1991, The Journal of biological chemistry.

[44]  R. Sousa,et al.  The use of glycerol in crystallization of T7 RNA polymerase: Implications for the use of cosolvents in crystallizing flexible proteins , 1990 .

[45]  W. Hackmann Tesla's sparks of imagination , 1993, Nature.

[46]  D. Blumenthal,et al.  The gamma-subunit of skeletal muscle phosphorylase kinase contains two noncontiguous domains that act in concert to bind calmodulin. , 1989, The Journal of biological chemistry.

[47]  R. D. Wade,et al.  Homology of the gamma subunit of phosphorylase b kinase with cAMP-dependent protein kinase. , 1984, Biochemistry.

[48]  S. Taylor,et al.  Energetic limits of phosphotransfer in the catalytic subunit of cAMP-dependent protein kinase as measured by viscosity experiments. , 1992, Biochemistry.

[49]  E. Krebs,et al.  Structure of the site phosphorylated in the phosphorylase b to a reaction. , 1959, The Journal of biological chemistry.

[50]  D. Graves,et al.  Kinetic mechanism and specificity of the phosphorylase kinase reaction. , 1978, The Journal of biological chemistry.

[51]  L. Johnson,et al.  The effects of phosphorylation on the structure and function of proteins. , 1993, Annual review of biophysics and biomolecular structure.

[52]  J. Knowles Enzyme-catalyzed phosphoryl transfer reactions. , 1980, Annual review of biochemistry.

[53]  H. Shuntoh,et al.  Autoactivation of catalytic (C alpha) subunit of cyclic AMP-dependent protein kinase by phosphorylation of threonine 197 , 1993, Molecular and cellular biology.

[54]  E. Krebs,et al.  Conversion of phosphorylase b to phosphorylase a in muscle extracts. , 1955, The Journal of biological chemistry.

[55]  B. Kemp,et al.  Insights into autoregulation from the crystal structure of twitchin kinase , 1994, Nature.

[56]  Wolfram Saenger,et al.  Principles of Nucleic Acid Structure , 1983 .

[57]  B. Honig,et al.  A rapid finite difference algorithm, utilizing successive over‐relaxation to solve the Poisson–Boltzmann equation , 1991 .

[58]  L. Johnson,et al.  Electrostatic effects in the control of glycogen phosphorylase by phosphorylation , 1994, Protein science : a publication of the Protein Society.

[59]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[60]  N. Xuong,et al.  Structure of the mammalian catalytic subunit of cAMP-dependent protein kinase and an inhibitor peptide displays an open conformation. , 1993, Acta crystallographica. Section D, Biological crystallography.