FETT STRUCTURE AND FUNCTION OF CREATINE KINASE
Karin Fritz-Wolf1, Ilme Schlichting2, Thomas Schnyder3, Michael Forstner3, Michael Eder3, Theo Wallimann3, Wolfgang Kabsch1
1 Max-Planck-Institut für
medizinische Forschung, Abteilung Biophysik, Jahnstrasse 29,
69028 Heidelberg, Germany
e-mail: kabsch@mpimf-heidelberg.mpg.de
2 Max-Planck-Institut für
molekulare Physiologie, Rheinlanddamm 201, 44139 Dortmund,
Germany
3 Institute for Cell Biology,
ETH-Hönggerberg, CH-8093 Zürich, Switzerland
Creatine kinase (CK, EC 2.7.3.2) catalyzes the reversible phosphoryl transfer from phosphocreatine to ADP. The enzyme is present in tissues with high and fluctuating energy requirements and exists in tissue-specific (M, muscle type; B, brain type) as well as in compartment-specific (cytosolic and mitochondrial) isoforms. The cytosolic forms are homo- or heterodimeric (MM-, BB-, or MB-CK), whereas the mitochondrial creatine kinases (Mi-CK) form octamers. Mi-CKs are found exclusively in the mitochondrial intermembrane compartment being attached to the inner membrane, but only the octameric form can interact with both the inner and outer mitochondrial membranes. It is functionally and perhaps also physically coupled to the adenine nucleotide translocator (ANT) of the inner membrane and with porin of the outer membrane. This enables the enzyme to utilize the intramitochondrially produced ATP for synthesis of phosphocreatine, which is exported into the cytosol for regeneration of ATP by the cytosolic isoforms. This interplay between the cytosolic and mitochondrial CKs, with the easily diffusable (phospho)creatine serving as an energy shuttle between the cellular compartments, is referred to as the 'phosphocreatine circuit' (for review see [1]).
The crystal structure of sarcomeric chicken cardiac Mib-CK both with and without NaATP was solved at 3 A resolution [2]. The octamer has 422 point group symmetry and appears as a cube of side length 93 A with a 20 A wide channel extending along the fourfold axis. A cluster of positively charged amino acids at the fourfold faces of the octamer possibly interacts with negatively charged mitochondrial membranes. Each monomer consists of a small a-helical domain and a large domain containing an eight-stranded antiparallel b-sheet flanked by seven a-helices. The conserved residues of the CK-family form a compact cluster that covers the active site between the domains. As shown by small-angle scattering, Mib-CK undergoes large conformational changes upon binding of MgATP. So far, crystallographic data of the ATP binding site were obtained only in the absence of Mg2+ and the protein cocrystallized with NaATP hardly changes its structure. Although none of the nucleotides is found at its correct place required for catalysis, they cannot be far off. ATP is located in the cleft between the domains in the highly conserved region of all guanidino kinases and many of the active site residues implicated by various other methods, namely Cys 278, Val 232--Lys 237, Val 275--Arg 287, Asp 335, Trp 223, and Ala 323 are found in the vicinity of the nucleotides. Recently, we have solved the structure of chicken BB-CK, a homodimeric brain type cytosolic isoform, by molecular replacement and present details at the meeting.