Structure of mitochondrial creatine kinase

Abstract

Creatine kinase (CK, EC 2.7.3.2), an enzyme important for energy metabolism in cells of high and fluctuating energy requirements, catalyses the reversible transfer of a phosphoryl goup from phosphocreatine to ADP. We have solved the structure of the octameric mitochondrial isoform, Mib-CK, which is located in the intermembrane compartment and along the cristae membranes. Mib-CK consumes ATP produced in the mitochondria for the production of phosphocreatine, which is then exported into the cytosol for fast regeneration of ATP by the cytosolic CK isoforms. The octamer has 422 point-group symmetry, and appears as a cube of side length 93 angstrom with a channel 20 angstrom wide extending along the four-fold axis. Positively charged amino acids at the four-fold faces of the octamer possibly interact with negatively charged mitochondrial membranes. Each monomer consists of a small alpha-helical domain and a large domain containing an eight-stranded antiparallel beta-sheet flanked by seven alpha-helices. The conserved residues of the CK family form a compact cluster that covers the active site between the domains.

Expression, purification from inclusion bodies, and crystal characterization of a transition state analog complex of arginine kinase: A model for studying phosphagen kinases

“…Arginine kinase is therefore likely to join a select handful of enzymes (Lolis & Petsko, 1990) for which high-resolution structures are available for a transition state analog complex. Attempts are underway to determine phases by isomorphous replacement, but it is also hoped that it will be possible to determine the structure by the molecular replacement method (Rossmann, 1972) using the recent Mi-CK structure (Fritz-Wolf et al, 1996), when the coordinates become available.…”

“…After many reports, over many years, describing crystals diffracting to -3 8, resolution, the first guanidino kinase structure was reported recently (Fritz-Wolf et at., 1996). The 3 8, structures of apo-Mi-CK with and without bound ATP revealed subunits with a 1 12-residue a-helical N-terminal domain followed by a 277-residue domain with an eight-stranded antiparallel P-sheet flanked by seven helices.…”

“…The active site is in the cleft between domains. but its details remain obscure, because of disorder and flexibility in the active site, and because the enzyme has been visualized before the substrate-induced conformational changes (Fritz-Wolf et at., 1996). The work described here is designed to complement the CK results with the development of an experimental system through which it will be possible to study the high resolution structure in its "active" state and perform mutational analysis.…”

The active site histidines of creatine kinase. A critical role of His 61 situated on a flexible loop

“…Chicken muscle Mih-CK has been crystallized in our laboratory (Schnyder et al, 1990) and its X-ray structure in the presence of bound ATP has been solved (Fritz-Wolf et al, 1996). Analysis of the ATP-binding region showed histidine residues that were close to the active site and might be involved in catalysis.…”

“…The flexible parts of the structure, as indicated by temperature factors above 40 A', i.e., the loop bearing His 61 (residues 60-65) and another flexible loop covering the ATP binding site (residues 314-331), are colored red and depicted by arrows. The figure was prepared from the X-ray coordinates of Mib-CK (Fritz-Wolf et al, 1996) using the program Molscript (Kraulis, 1991) and rendered with the use of the Raster3D software (Bacon & Anderson, 1988;Memtt & Murphy, 1994). in the reverse reaction was observed for the same His 61 Asp data were analyzed using software by W.W. Cleland (see Materials mutant that exhibited only 1.6% of the wt activity. and methods).…”