Daniel R. Knighton

2.2 A refined crystal structure of the catalytic subunit of cAMP-dependent protein kinase complexed with MnATP and a peptide inhibitor.

. The crystal structure of a ternary complex containing the catalytic subunit of cAMP-dependent protein kinase, ATP and a 20-residue inhibitor peptide was refined at a resolution of 2.2 A to an R value of 0.177. In order to identify the metal binding sites, the crystals, originally grown in the presence of low concentrations of Mg(2+), were soaked in Mn(2+). Two Mn(2+) ions were identified using an anomalous Fourier map. One Mn(2+) ion bridges the gamma- and beta-phosphates and interacts with Asp184 and two water molecules. The second Mn(2+) ion interacts with the side chains of Asn171 and Asp l84 as well as with a water molecule. Modeling a serine into the P site of the inhibitor peptide suggests a mechanism for phosphotransfer.

A template for the protein kinase family

The crystal structure of the catalytic subunit of cAMP-dependent protein kinase, complexed with ATP and a 20-residue inhibitor peptide, is reviewed and correlated with chemical and genetic data. The striking convergence of the structure with the biochemistry and genetics provides for the first time a molecular basis for understanding how this enzyme functions, as well as an explanation for the highly conserved residues that are scattered throughout the molecule. Because these residues probably serve a common role in all eukaryotic protein kinases, this first protein kinase structure serves as a general template for the entire family of enzymes.

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

. A mutant (Serl39Ala) of the mouse recombinant catalytic (C) subunit of cAMP-dependent protein kinase was co-crystallized with a peptide inhibitor, PKI(5-24), and MEGA-8 (octanoyl-N-methylglucamide) detergent. This structure was refined using all observed data (30 248 reflections) between 30 and 1.95 A resolution to an R factor of 0.186. R.m.s. deviations of bond lengths and bond angles are 0.013 A and 2.3 degrees, respectively. The final model has 3075 atoms (207 solvent) with a mean B factor of 31.9 A(2). The placement of invariant protein-kinase residues and most C:PKI(5-24) interactions were confirmed, but register errors affecting residues 55-64 and 309-339 were corrected during refinement by shifting the affected sequences toward the C terminus along the previously determined backbone path. New details of C:PKI(5-24) interactions and the Ser338 autophosphorylation site are described, and the acyl group binding site near the catalytic subunit NH(2) terminus is identified.

High-affinity FKBP-12 ligands derived from (R)-(−)-carvone. Synthesis and evaluation of FK506 pyranose ring replacements

Abstract The preparation and evaluation of potent small molecule inhibitors of FKBP-12 rotamase activity is described. These ligands contain many of the structural features of the FK506 pyranose ring region, yet are synthetically more accessible. The versatility of these FKBP-12 ligands is demonstrated with respect to effector domain exploration.

crystallization of catalytic subunit of adenosine cyclic monophosphate-dependent protein kinase

Publisher Summary Extensive crystallographic work on the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase (cAPK) has established reproducible crystallization conditions that generate a set of different crystal forms. These different crystal forms almost certainly represent different conformational states of the enzyme and provides a comparative framework that significantly enlarges the scope of the crystallographic work. The crystallographic work described in the chapter is carried out on the catalytic subunit purified from porcine heart, as well as on the catalytic subunit expressed in and purified from E. coli. Through systematic adjustments of crystallization conditions, crystals representing three forms of the protein–the apoenzyme, a binary complex with a specific peptide inhibitor, and a ternary complex with a specific peptide inhibitor and MgATP– are obtained in four different space groups. Each of these crystals and the three factors influencing the reproducibility and control of crystallization conditions are discussed. Crystallographic results correlate with data from the SANS experiments, and indicate that the enzyme is flexible and that crystals in different space groups very likely represent different conformational states of the enzyme. The best crystals are formed by the binary and ternary complexes of the recombinant catalytic subunit and thus emphasize the importance of being able to take advantage of recombinant techniques.

cAMP-dependent protein kinase and the protein kinase family

The structure of the catalytic subunit of cAMP-dependent protein kinase, the first protein kinase structure to be solved, is reviewed. The general architecture of the enzyme is described as well as the active site regions associated with substrate binding and catalysis. In particular, the unique features of the protein kinase nucleotide fold are outlined. While the catalytic subunit is one of the simplest of the protein kinases, it nevertheless serves as a structural framework for the catalytic core of the entire protein kinase family which now includes over 200 important regulatory enzymes. The essential and conserved features of this core are summarized, and a preliminary model of myosin light-chain kinase, based on the structure of the catalytic subunit, is also discussed.