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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 Cterminal 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 Cterminal 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.

Section: Resultssupporting

confidence: 57%

mentioning

confidence: 99%

mentioning

confidence: 99%

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C-terminal Lysines Determine Phospholipid Interaction of Sarcomeric Mitochondrial Creatine Kinase

Schlattner

Gehring

Vernoux

et al. 2004

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“…by protein sequencing and thorough studies of the reconstituted CRT protein(s), work which is currently in progress. Open questions are also whether Cr transported into mitochondria is immediately recharged via mitochondrial creatine kinase to PCr (36,38), e.g. for energetic purposes, or whether Cr, a highly abundant zwitterionic guanidino compound in the cytosol, could fulfill some protective role as an osmolyte to guarantee mitochondrial integrity under conditions of cellular stress.…”

Section: Table I Inhibition Of Cr Uptake By Structurally Related Compmentioning

confidence: 99%

Novel Mitochondrial Creatine Transport Activity

Walzel¹,

Speer²,

Zanolla³

et al. 2002

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Immunoblotting of isolated mitochondria from rat heart, liver, kidney, and brain with antibodies made against N-and C-terminal peptide sequences of the creatine transporter, together with in situ immunofluorescence staining and immunogold electron microscopy of adult rat myocardium, revealed two highly related polypeptides with molecular masses of ϳ70 and ϳ55 kDa in mitochondria. These polypeptides were localized by immunoblotting of inner and outer mitochondrial membrane fractions, as well as by immunogold labeling in the mitochondrial inner membrane. In addition, a novel creatine uptake via a mitochondrial creatine transport activity was demonstrated by [ 14 C]creatine uptake studies with isolated mitochondria from rat liver, heart, and kidney showing a saturable low affinity creatine transporter, which was largely inhibited in a concentrationdependent manner by the sulfhydryl-modifying reagent NEM, as well as by the addition of the above anti-creatine transporter antibodies to partially permeabilized mitochondria. Mitochondrial creatine transport was to a significant part dependent on the energetic state of mitochondria and was inhibited by arginine, and to some extent also by lysine, but not by other creatine analogues and related compounds. The existence of an active creatine uptake mechanism in mitochondria indicates that not only creatine kinase isoenzymes, but also creatine transporters and thus a certain proportion of the creatine kinase substrates, might be subcellularly compartmentalized. Our data suggest that mitochondria, shown here to possess creatine transport activity, may harbor such a creatine/phosphocreatine pool.

“…The reaction is central to short-term temporal energy buffering (1,2) as well as in spatial shuttling of energy from production to consumption sites (3)(4)(5). A wide array of endergonic processes is driven by nucleotide hydrolysis, from motion in molecular motors, active transport, and synthetic metabolism to signal transduction.…”

mentioning

confidence: 99%

The Structure of Lombricine Kinase

Bush

Kirillova

Clark

et al. 2011

Lombricine kinase is a member of the phosphagen kinase family and a homolog of creatine and arginine kinases, enzymes responsible for buffering cellular ATP levels. Structures of lombricine kinase from the marine worm Urechis caupo were determined by x-ray crystallography. One form was crystallized as a nucleotide complex, and the other was substrate-free. The two structures are similar to each other and more similar to the substrate-free forms of homologs than to the substrate-bound forms of the other phosphagen kinases. Active site specificity loop 309 -317, which is disordered in substrate-free structures of homologs and is known from the NMR of arginine kinase to be inherently dynamic, is resolved in both lombricine kinase structures, providing an improved basis for understanding the loop dynamics. Phosphagen kinases undergo a segmented closing on substrate binding, but the lombricine kinase ADP complex is in the open form more typical of substrate-free homologs. Through a comparison with prior complexes of intermediate structure, a correlation was revealed between the overall enzyme conformation and the substrate interactions of His 178 . Comparative modeling provides a rationale for the more relaxed specificity of these kinases, of which the natural substrates are among the largest of the phosphagen substrates.Lombricine, arginine, and creatine kinases (EC 2.7.3) are homologous phosphagen kinases that catalyze the buffering of cellular ATP levels through phosphoryl transfer to/from their respective guanidino-containing substrates. The reaction is central to short-term temporal energy buffering (1, 2) as well as in spatial shuttling of energy from production to consumption sites (3-5). A wide array of endergonic processes is driven by nucleotide hydrolysis, from motion in molecular motors, active transport, and synthetic metabolism to signal transduction. Thus, the maintenance of a constant ATP/ADP ratio, displaced far from thermodynamic equilibrium in the face of high and variable rates of ATP turnover, is crucial for cellular homeostasis (2).Different organisms use different phosphagen substrates, usually only one and each with its own specific phosphagen kinase ( Fig. 1) (2). Lombricine kinase, as well as taurocyamine kinase and glycocyamine kinase, is found exclusively in annelids and allied groups (2). Phylogenetic analyses and studies of the intron/exon organization of the genes of these phosphagen kinases unique to annelids have shown that they are more closely related to creatine kinases (with which they share 50 -60% sequence identity) than typical monomeric arginine kinases such as that from the horseshoe crab (6) (40% sequence identity). Annelids are more diverse in their choice of phosphagen, and the substrate specificities of the corresponding kinases are often lower (6).Structural work has concentrated on the presumptive ancestral arginine kinase and the vertebrate creatine kinase (7, 8), but sequence alignment suggests that subunit fold, if not quaternary structure, is conserved across the fa...