X-ray structures of several ternary substrate and product complexes of the catalytic subunit of cAMP-dependent protein kinase (PKAc) have been determined with different bound metal ions. In the PKAc complexes, Mg2+, Ca2+, Sr2+, and Ba2+ metal ions could bind to the active site and facilitate the phosphoryl transfer reaction. ATP and a substrate peptide (SP20) were modified, and the reaction products ADP and the phosphorylated peptide were found trapped in the enzyme active site. Finally, we determined the structure of a pseudo-Michaelis complex containing Mg2+, nonhydrolyzable AMP-PCP (β,γ-methyleneadenosine 5′-triphosphate) and SP20. The product structures together with the pseudo-Michaelis complex provide snapshots of different stages of the phosphorylation reaction. Comparison of these structures reveals conformational, coordination, and hydrogen bonding changes that might occur during the reaction and shed new light on its mechanism, roles of metals, and active site residues.

HIV-1 protease is an important target for the development of antiviral inhibitors to treat AIDS. A room-temperature joint X-ray/neutron structure of the protease in complex with clinical drug amprenavir has been determined at 2.0 Å resolution. The structure provides direct determination of hydrogen atom positions in the enzyme active site. Analysis of the enzyme-drug interactions suggests that some hydrogen bonds may be weaker than deduced from the non-hydrogen interatomic distances. This information may be valuable for the design of improved protease inhibitors.

Post-translational protein phosphorylation by protein kinase A (PKA) is a ubiquitous signalling mechanism which regulates many cellular processes. A low-temperature X-ray structure of the ternary complex of the PKA catalytic subunit (PKAc) with ATP and a 20-residue peptidic inhibitor (IP20) at the physiological Mg(2+) concentration of ∼0.5 mM (LT PKA-MgATP-IP20) revealed a single metal ion in the active site. The lack of a second metal in LT PKA-MgATP-IP20 renders the β- and γ-phosphoryl groups of ATP very flexible, with high thermal B factors. Thus, the second metal is crucial for tight positioning of the terminal phosphoryl group for transfer to a substrate, as demonstrated by comparison of the former structure with that of the LT PKA-Mg(2)ATP-IP20 complex obtained at high Mg(2+) concentration. In addition to its kinase activity, PKAc is also able to slowly catalyze the hydrolysis of ATP using a water molecule as a substrate. It was found that ATP can be readily and completely hydrolyzed to ADP and a free phosphate ion in the crystals of the ternary complex PKA-Mg(2)ATP-IP20 by X-ray irradiation at room temperature. The cleavage of ATP may be aided by X-ray-generated free hydroxyl radicals, a very reactive chemical species, which move rapidly through the crystal at room temperature. The phosphate anion is clearly visible in the electron-density maps; it remains in the active site but slides about 2 Å from its position in ATP towards Ala21 of IP20, which mimics the phosphorylation site. The phosphate thus pushes the peptidic inhibitor away from the product ADP, while resulting in dramatic conformational changes of the terminal residues 24 and 25 of IP20. X-ray structures of PKAc in complex with the nonhydrolysable ATP analogue AMP-PNP at both room and low temperature demonstrated no temperature effects on the conformation and position of IP20.

The DNA-dependent protein kinase (DNA-PK) is a trimeric enzyme consisting of a 460-kDa catalytic subunit (DNA-PKcs) and a heterodimeric regulatory complex called Ku, which is comprised of 70 (Ku70) and 86 (Ku80) kDa subunits. Mutations that affect the expression of the catalytic or Ku80 subunits of DNA-PK disrupt both V(D)J recombination and DNA double-stranded break repair pathways. In this report, we show that two previously uncharacterized rodent cell lines that are defective in DNA double-stranded break repair express catalytically inactive DNA-PK. The DNA-PKcs from the DNA double-stranded break repair mutant cell lines IRS-20 and SX-9 assembles on double-stranded DNA but fails to function as a protein kinase. In addition to the kinase defect, the abundance of the DNA-PKcs from both of these cell lines is reduced relative to wild-type controls. These results suggest that the DNA-PKcs gene from each of these cell lines contains mutations that inactivate the enzymatic activity and the expression or stability of the gene product. These data further strengthen the hypothesis that DNA-PK-mediated protein phosphorylation is a necessary component of the DNA double-stranded break repair pathway.The rejoining of double-stranded DNA breaks induced by ionizing radiation or occurring as intermediates of V(D)J recombination is performed via a biochemical pathway that includes the DNA-dependent protein kinase holoenzyme. DNA-PK 1 is a trimeric complex consisting of a DNA-binding component made up of the 70 and 86 kd subunits of the Ku autoantigen (1, 2) and a catalytic subunit of approximately 460 kDa (3). Cells from the x-ray-sensitive complementation group (xrs)-7, which includes the severe combined immunodeficiency (scid) mouse and the Chinese hamster ovary (CHO) V3 cell line, exhibit reduced expression of the DNA-PKcs, lack measurable DNA-stimulated kinase activity, and are defective for DNA double-stranded break repair and V(D)J recombination (4 -6). Similarly, cells from the xrs-6 complementation group, which contain mutations that reduce the expression of the Ku80 subunit of DNA-PK, exhibit losses of Ku-specific DNA-ending binding activity (7-9) DNA-PK kinase activity (10) and are also defective for DNA double-stranded break repair and V(D)J recombination (11-13).Molecular analysis of these DNA-repair mutant cells indicates that DNA-PK is required for the rejoining of doublestranded DNA breaks, but the mechanism by which DNA-PK functions in this process has yet to be elucidated. DNA-PK is a serine and threonine protein kinase that is activated by doublestranded DNA containing single-stranded to double-stranded transitions, such as DNA-ends, nicks, gaps, and stem-loop structures (14). In vitro, the Ku and catalytic subunits of DNA-PK assemble in a DNA-dependent manner (15), and the DNA-bound holoenzyme preferentially phosphorylates substrates that are bound to the same DNA molecule (1, 16). DNA-PK has been shown to phosphorylate a broad range of proteins in vitro, most of which are DNA-binding proteins (17), includi...

The Protein Crystallography Station at Los Alamos Neutron Science Center is a high‐performance beamline that forms the core of a capability for neutron macromolecular structure and function determination. This capability also includes the Macromolecular Neutron Crystallography (MNC) consortium between Los Alamos (LANL) and Lawrence Berkeley National Laboratories for developing computational tools for neutron protein crystallography, a biological deuteration laboratory, the National Stable Isotope Production Facility, and an MNC drug design consortium between LANL and Case Western Reserve University.