C-terminal lysines determine phospholipid interaction of sarcomeric mitochondrial creatine kinase.

Uwe Schlattner,Florian Gehring,Dietbert Neumann,Theo Wallimann,Olivier Marcillat,Nathalie Vernoux,Malgorzata Tokarska-Schlattner,Christian Vial

doi:10.1074/jbc.m314158200

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.

Highlights

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
This intimate exchange of substrates and products, the so-called functional coupling or metabolite channeling [10, 11], fulfills important functions that may vary among different tissues, species, and developmental states [12, 13]: (i) phosphocreatine becomes the high energy intermediate that is exported from mitochondria into the cytosol [3]; (ii) locally generated ADP stimulates oxidative phosphorylation [11]; and (iii) ADP channeled through the MtCK/adenylate translocator (ANT) interaction inhibits the Ca2ϩ-induced opening of the mitochondrial permeability transition pore [14, 15], a well known trigger of apoptosis [16, 17]
Mutation of these lysines drastically reduces the capacity of sarcomeric MtCK (sMtCK) to attach to and to cross-link mitochondrial model membranes containing CL by at least an order of magnitude

Summary

EXPERIMENTAL PROCEDURES

Plasmids and Chemicals—Plasmid pUS01 carrying the gene for human sMtCK between the NdeI and BamHI sites has been described before [40]. Phospholipids were obtained from Lipid Products for light scattering and Biacore experiments (South Nutfield, UK) or from Sigma for fluorescence experiments. Dry lipids were hydrated (5–20 mg/ml) in 10 mM TES, pH 6.5, 50 mM potassium acetate (light scattering and Biacore experiments) or 20 mM Tris, pH 7.4, 0.1 mM EDTA (fluorescence experiments) with subsequent heating to 70 °C when containing laurdan and dispersed by vortexing to produce multilamellar vesicles. Binding of sMtCK to a model lipid membrane was measured by surface plasmon resonance (SPR) with a Biacore 2000TM instrument (Biacore, Uppsala, Sweden) according to Schlattner and Wallimann [29, 43], using standard buffer with 2 mM ␤-mercaptoethanol. The maximum emission wavelength was measured by using excitation wavelengths ranging from 275 to 307 nm

RESULTS

Relative Vmax

DISCUSSION