Interaction of Creatine Kinase and Nucleoside Diphosphate Kinase with Mitochondrial Cardiolipin Membranes: Differences in Mechanism and in the Effect on Enzyme Catalysis

Abstract

Mitochondrial isoforms of creatine kinase (MtCK) and nucleoside diphosphate kinase (NDPK-D) have critical functions in bioenergetics, membrane topology and organelle morphology with roles in human health and disease. X-ray structural analysis, electron microscopy, surface plasmon resonance (SPR) and scanning calorimetry revealed that both kinases form large oligomers that bind to and cross-link mitochondrial membranes via anionic phospholipids, mainly cardiolipin; at least MtCK can also induce cardiolipin-rich membrane domains. First we used surface plasmon resonance combined with thermodynamic analysis to study kinase/cardiolipin interaction. The two kinases differed in their membrane binding mechanism: (i) NDPK-D showed monophasic binding due to electrostatic interactions of a triad of basic amino acids, while binding of MtCK was biphasic, with only the main component depending on electrostatic interactions of C-terminal basic amino acids. (ii) Rising temperature increased cardiolipin affinity of MtCK, in particular in the second binding component, but not of NDPK-D, indicating hydrophobic interactions in case of MtCK. (iv) Kinase/membrane interaction occurred to be an entropy-driven binding process, in particular for MtCK, possibly due to charge neutralization, release of bound water, and effects on membrane order. Second, we studied the effect of membrane-association on kinase enzyme activity. While basic cardiolipin had no effect on MtCK, NDPK-D was strongly inhibited. This inhibition was relieved by doxorubicin that strongly competes for cardiolipin binding. We propose a model for MtCK and NDPK-D interaction with cardiolipin-containing lipid membranes. For NDPK-D, a single phase, purely electrostatic binding would lead to a partial shielding of the enzymes’ active sites and thus catalytic inhibition. For MtCK, a two-phase binding model of rapid electrostatic docking and slower anchoring via hydrophobic stretches is proposed, which does not affect the active sites.