title: C-terminal lysines determine phospholipid interaction of sarcomeric mitochondrial creatine kinase.
abstract: High affinity interaction between octameric mitochondrial creatine kinase (MtCK) and the phospholipid cardiolipin in the inner mitochondrial membrane plays an important role in metabolite channeling between MtCK and inner membrane adenylate translocator, which itself is tightly bound to cardiolipin. Three C-terminal basic residues revealed as putative cardiolipin anchors in the x-ray structures of MtCK and corresponding to lysines in human sarcomeric MtCK (sMtCK) were exchanged by in vitro mutagenesis (K369A/E, K379Q/A/E, K380Q/A/E) to yield double and triple mutants. sMtCK proteins were bacterially expressed, purified to homogeneity, and verified for structural integrity by enzymatic activity, gel filtration chromatography, and CD spectroscopy. Interaction with cardiolipin and other acidic phospholipids was quantitatively analyzed by light scattering, surface plasmon resonance, and fluorescence spectroscopy. All mutant sMtCKs showed a strong decrease in vesicle cross-linking, membrane affinity, binding capacity, membrane ordering capability, and binding-induced changes in protein structure as compared with wild type. These effects did not depend on the nature of the replacing amino acid but on the number of exchanged lysines. They were moderate for Lys-379/Lys-380 double mutants but pronounced for triple mutants, with a 30-fold lower membrane affinity and an entire lack of alterations in protein structure compared with wild-type sMtCK. However, even triple mutants partially maintained an increased order of cardiolipin-containing membranes. Thus, the three C-terminal lysines determine high affinity sMtCK/cardiolipin interaction and its effects on MtCK structure, whereas low level binding and some effect on membrane fluidity depend on other structural components. These results are discussed in regard to MtCK microcompartments and evolution.
authors.affiliation: Institute of Cell Biology, Swiss Federal Institute of Technology (ETH) Zürich, Hönggerberg, CH-8093 Zürich, Switzerland. uwe.schlattner@cell.biol.ethz.ch
authors.forename: Uwe
authors.initials: U
authors.lastname: Schlattner
authors.forename: Florian
authors.initials: F
authors.lastname: Gehring
authors.forename: Nathalie
authors.initials: N
authors.lastname: Vernoux
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Tokarska-Schlattner
authors.forename: Dietbert
authors.initials: D
authors.lastname: Neumann
authors.forename: Olivier
authors.initials: O
authors.lastname: Marcillat
authors.forename: Christian
authors.initials: C
authors.lastname: Vial
authors.forename: Theo
authors.initials: T
authors.lastname: Wallimann
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002308:Cardiolipins / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003402:Creatine Kinase / Q000737:chemistry*; D052281:Creatine Kinase, Mitochondrial Form; D003432:Cross-Linking Reagents / Q000494:pharmacology; D018360:Crystallography, X-Ray; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry*; D007700:Kinetics; D008027:Light; D008081:Liposomes / Q000737:chemistry; D008239:Lysine / Q000737:chemistry*; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010743:Phospholipids / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012518:Sarcomeres / Q000378:metabolism*; D012542:Scattering, Radiation; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D020349:Surface Plasmon Resonance; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry
chemical_list: D002308:Cardiolipins; D003432:Cross-Linking Reagents; D007527:Isoenzymes; D008081:Liposomes; D010743:Phospholipids; D014364:Tryptophan; D003402:Creatine Kinase; D052281:Creatine Kinase, Mitochondrial Form; D008239:Lysine