Insights into the Phosphoryl Transfer Mechanism of Human Ubiquitous Mitochondrial Creatine Kinase.

Human ubiquitous mitochondrial creatine kinase (uMtCK) is responsible for the regulation of cellular energy metabolism. To investigate the phosphoryl-transfer mechanism catalyzed by human uMtCK, in this work, molecular dynamic simulations of uMtCK∙ATP-Mg2+∙creatine complex and quantum mechanism calculations were performed to make clear the puzzle. The theoretical studies hereof revealed that human uMtCK utilizes a two-step dissociative mechanism, in which the E227 residue of uMtCK acts as the catalytic base to accept the creatine guanidinium proton. This catalytic role of E227 was further confirmed by our assay on the phosphatase activity. Moreover, the roles of active site residues in phosphoryl transfer reaction were also identified by site directed mutagenesis. This study reveals the structural basis of biochemical activity of uMtCK and gets insights into its phosphoryl transfer mechanism.

Symmetry-adapted digital modeling I. Axial symmetric proteins.

Considered are axial symmetric proteins exemplified by the octameric mitochondrial creatine kinase, the Pyr RNA-binding attenuation protein, the D-aminopeptidase and the cyclophilin A-cyclosporin complex, with tetragonal (422), trigonal (32), pentagonal (52) and pentagonal (52) point-group symmetry, respectively. One starts from the protein enclosing form, which is characterized by vertices at points of a lattice (the form lattice) whose dimension depends on the point group. This allows the indexing of Cα's at extreme radial positions. The indexing is extended to additional residues on the basis of a finer lattice, the digital modeling lattice Λ, which includes the form lattice as a sublattice. This leads to a coarse-grained description of the protein. In the crystallographic point-group case, the planar indices are obtained from a projection of atomic positions along the rotation axis, taken as the z axis. The planar indices of a Cα are then those of the nearest projected lattice point. In the non-crystallographic case, low indices are an additional requirement. The coarse-grained bead follows from the condition imposed on the residues selected to have a z coordinate within a band of value δ above and below the height of lattice points. The choice of δ permits a variation of the coarse-grained bead model. For example, the value δ = 0.5 leads to a fine-grained indexing of the full set of residues, whereas with δ = 0.25 one gets a coarse-grained model which includes only about half of these residues. Within this procedure, the indexing of the Cα only depends on the choice of the digital modeling lattice and not on the value of δ. The characteristics which distinguish the present approach from other coarse-grained models of proteins on lattices are summarized at the end.

Protein "amyloid-like" networks at the phospholipid membrane formed by 4-hydroxy-2-nonenal-modified mitochondrial creatine kinase.

4-Hydroxy-2-nonenal (4-HNE) is a reactive aldehyde and a lipid peroxidation product formed in biological tissues under physiological and pathological conditions. Its concentration increases with oxidative stress and induces deleterious modifications of proteins and membranes. Mitochondrial and cytosolic isoforms of creatine kinase were previously shown to be affected by 4-HNE. In the present study, we analyzed the effect of 4-HNE on mitochondrial creatine kinase, an abundant protein from the mitochondrial intermembrane space with a key role in mitochondrial physiology. We show that this effect is double: 4-HNE induces a step-wise loss of creatine kinase activity together with a fast protein aggregation. Protein-membrane interaction is affected and amyloid-like networks formed on the biomimetic membrane. These fibrils may disturb mitochondrial organisation both at the membrane and in the inter membrane space.

Purification and stability of octameric mitochondrial creatine kinase isoform from herring (Clupea harengus) organ of vision.

Creatine kinases (CKs) constitute a large family of isoenzymes that are involved in intracellular energy homeostasis. In cells with high and fluctuating energy requirements ATP level is maintained via phosphocreatine hydrolysis catalyzed by creatine kinase. In contrast to invertebrates and higher vertebrates, in poikilothermic vertebrates the adaptations for the regulation of energy metabolism by changes in the oligomeric state of CK isoforms are not well known. The present study aimed at identification of herring eye CK isoforms and focuses on factors affecting the CK-octamer stability. In addition to the CK octamer, three different dimeric isoforms of CK were detected by cellulose acetate native electrophoresis. Destabilization of octamer was studied in the presence of TSAC substrates and about 50% of octamers dissociated into dimers within 24h. Moreover, we found that the increase of temperature from 4 °C to 30 °C caused rapid inactivation of dimers in TSAC-treated samples but did not affect octameric structures. In a thermostability assay we demonstrated that octamers retain their activity even at 50 °C. Our results indicate that destabilization of the octameric structure can lead to loss of enzyme activity at higher temperatures (above 30 °C). Furthermore, our results based on N-terminal sequence analysis suggest that probably the mitochondrial s-type CK, rather than u-type, is predominantly expressed in herring eye. In conclusion the existence of four various CK isoforms in one organ may reflect complex regulation of energy metabolism in the phototransduction process in teleost fishes.

Regulation of sodium-calcium exchanger activity by creatine kinase.

It has been shown that in rat heart NCX1 exists in a macromolecular -complex including PKA, PKA-anchoring protein, PKC, and phosphatases PP1 and PP2A. In addition, several lines of evidence suggest that the interactions of the exchanger with other molecules are closely associated with its function in regulation of [Ca(2+)](i). NCX contains a large intracellular loop (NCXIL) that is responsible for regulating NCX activity. We used the yeast two-hybrid method to screen a human heart cDNA library and found that the C-terminal region of sarcomeric mitochondrial creatine kinase (sMiCK) interacted with NCX1IL. Among the four creatine kinase (CK) isozymes, both sMiCK and the muscle-type cytosolic creatine kinase (CKM) co-immunoprecipitated with NCX1. Both sMiCK and CKM were able to produce a recovery in the decreased NCX1 activity that was lost under energy-compromised conditions. This regulation is mediated through a putative PKC phosphorylation site of sMiCK and CKM. The catalytic activity of sMiCK and CKM is not required for their regulation of NCX1 activity. Our results suggest a novel mechanism for the regulation of NCX1 activity and a novel role for CK.

Macro CK2 accumulation in tenofovir-treated HIV patients is facilitated by CK oligomer stabilization but is not predictive for pathology.

BACKGROUND: Ubiquitous mitochondrial creatine kinase (uMtCK) accumulates as macroenzyme creatine kinase type 2 (macro CK2) in the serum of HIV-infected patients under a tenofovir disoproxil fumarate (TDF)-containing antiretroviral regimen. The genesis and clinical significance of this finding is unclear. METHODS: A prospective observational 5-year follow-up study was performed on those patients in which macro CK2 appearance was initially described ('TDF switch study' cohort). In addition, tenofovir (TFV), its prodrug TDF and its active, intracellular derivative TFV diphosphate (TDP) were tested in vitro for their effects on different key properties of uMtCK to clarify possible interactions of uMtCK with TFV compounds. RESULTS: In just under 5 years of continuous TDF treatment, only 4/12 (33%) patients remained macro CK2-positive, whereas 8/12 (66%) originally positive patients were macro CK2-negative at the end of follow-up. Prospective clinical follow-up data indicate that macro CK2 appearance under TDF is not associated with significant cell damage or occurrence of malignancies. A trend towards grade 1 hypophosphataemia suggests subclinical proximal tubular dysfunction in macro-CK2-positive patients, although it was not associated with a significant decrease in estimated glomerular filtration rate. In vitro, TFV, TDF and TDP did not interfere with uMtCK enzyme activity as competitive inhibitors or pseudo-substrates, but TFV and TDF stabilized the native uMtCK octameric structure in dilute solutions. CONCLUSIONS: Appearance of octameric uMtCK as macro CK2 in the serum of TDF-treated patients is suggested to result from a combination of low-level mitochondrial damage caused by subclinical renal tubular dysfunction together with possible compensatory uMtCK overexpression and a putative concomitant stabilization of uMtCK octamers by higher levels of TFV in proximal tubules.

Regulation of sodium-calcium exchanger activity by creatine kinase under energy-compromised conditions.

Na(+)/Ca(2+) exchanger (NCX) is one of the major mechanisms for removing Ca(2+) from the cytosol especially in cardiac myocytes and neurons, where their physiological activities are triggered by an influx of Ca(2+). NCX contains a large intracellular loop (NCXIL) that is responsible for regulating NCX activity. Recent evidence has shown that proteins, including kinases and phosphatases, associate with NCX1IL to form a NCX1 macromolecular complex. To search for the molecules that interact with NCX1IL and regulate NCX1 activity, we used the yeast two-hybrid method to screen a human heart cDNA library and found that the C-terminal region of sarcomeric mitochondrial creatine kinase (sMiCK) interacted with NCX1IL. Moreover, both sMiCK and the muscle-type creatine kinase (CKM) coimmunoprecipitated with NCX1 using lysates of cardiacmyocytes and HEK293T cells that transiently expressed NCX1 and various creatine kinases. Both sMiCK and CKM were able to produce a recovery in the decreased NCX1 activity that was lost under energy-compromised conditions. This regulation is mediated through a putative PKC phosphorylation site of sMiCK and CKM. The autophosphorylation and the catalytic activity of sMiCK and CKM are not required for their regulation of NCX1 activity. Our results suggest a novel mechanism for the regulation of NCX1 activity.

Mitochondrial creatine kinase interaction with cardiolipin-containing biomimetic membranes is a two-step process involving adsorption and insertion.

Mitochondrial creatine kinase (mtCK) binding to the mitochondrial inner membrane largely determines its biological functions in cellular energy homeostasis, mitochondrial physiology, and dynamics. The membrane binding mechanism is, however, not completely understood. Recent data suggest that a hydrophobic component is involved in mtCK binding to cardiolipin at the outer face of the inner mitochondrial membrane, in addition to the well known electrostatically driven process. In this manuscript, using an electrochemical method derived from alternating current polarography for differential capacity measurements, we distinctly reveal that protein-cardiolipin interaction has a two-step mechanism. For short incubation time, protein adsorption to the phospholipid charged headgroup was the only process detected, whereas on a longer time scale evidence of protein insertion was observed.

Elevación crónica de creatincinasa / Chronic creatinine elevation

Las miopatías son entidades en las que se produce afectación musculo esquelética con elevación de creatincinasa en suero. La Enfermedad de Addison y la Hipertermia Maligna constituyen causas poco comunes de miopatía. La Enfermedad de Addison corresponde a una insuficiencia suprarrenal primaria producida por un déficit de glucocorticoides, mineral corticoides y andrógenos por destrucción de la corteza suprarrenal, cuyos síntomas son inespecíficos y el tratamiento es con hidrocortisona. La Hipertermia Maligna se produce por una alteración genética en la que los anestésicos inhalados y los relajantes musculares desencadenan una hipertermia progresiva, con rigidez muscular, taquicardia, hipotensión, rabdomiolisis, y coagulación intravascular diseminada (AU)

Myopathy is a disease that affects the musculoskeletal system with elevated serum creatinine. Addison’s Disease and Malignant Hyperthermia, are rare causes of myopathy. Addison’s disease, also known as primary adrenal insufficiency, occurs when the adrenal gland does not produce enough glucocorticoids, mineralocorticoids and androgens because of the destruction of the adrenal cortex. Symptoms are non-specific and it is treated with hydrocortisone. Malignant Hyperthermia is a genetic disorder, in which inhaled anaesthetics and muscle relaxants, trigger progressive hyperthermia with muscle rigidity, tachycardia, hypotension, rhabdomyolysis, and disseminated intravascular coagulation (AU)

Mitochondrial creatine kinase binding to phospholipid monolayers induces cardiolipin segregation.

It is well established that the octameric mitochondrial form of creatine kinase (mtCK) binds to the outer face of the inner mitochondrial membrane mainly via electrostatic interactions with cardiolipin (CL). However, little is known about the consequences of these interactions on membrane and protein levels. Brewster angle microscopy investigations provide, for the first time to our knowledge, images indicating that mtCK binding induced cluster formation on CL monolayers. The thickness of the clusters (10-12 nm) corresponds to the theoretical height of the mtCK-CL complex. Protein insertion into a condensed CL film, together with monolayer stabilization after protein addition, was observed by means of differential capacity measurements. Polarization modulation infrared reflection-absorption spectroscopy showed that the mean orientation of alpha-helices within the protein shifted upon CL binding from 30 degrees to 45 degrees with respect to the interface plane, demonstrating protein domain movements. A comparison of data obtained with CL and phosphatidylcholine/phosphatidylethanolamine/CL (2:1:1) monolayers indicates that mtCK is able to selectively recruit CL molecules within the mixed monolayer, consolidating and changing the morphology of the interfacial film. Therefore, CL-rich domains induced by mtCK binding could modulate mitochondrial inner membrane morphology into a raft-like organization and influence essential steps of mitochondria-mediated apoptosis.

Morphology modifications in negatively charged lipid monolayers upon mitochondrial creatine kinase binding.

Mitochondrial creatine kinase (mtCK) may participate to membrane organization at the mitochondrial level by modulating lipid state and fluidity. The effect of the protein on lipid phase behaviour of different acyl chain length phosphatidylglycerol monolayers was analyzed from pressure-area isotherms and from the compressional modulus variation with respect to the surface pressure. Monolayer morphology was visualized by Brewster angle microscopy. No condensation effect was visible on dimyristoylphosphatidylglycerol (DMPG). For the other PG monolayers tested, dipalmitoylphosphatidylglycerol (DPPG) and distearoylphosphatidylglycerol (DSPG), mtCK facilitated the formation of a liquid condensed phase. The effect depended on the surface pressure at which transition phase occurred. The effect of mtCK was more pronounced for tetramyristoylcardiolipin (TMCL) monolayers, as liquid condensed regions appeared 10 mN/m below the transition phase of the pure TMCL monolayer. The observed domains were circular and rather uniform, indicating a stabilization of the condensed phase. The same effect, namely an overall condensation of the monolayer with formation of circular domains, was observed upon protein injection beneath TMCL monolayers in different condensation states at constant area. MtCK ability to induce and stabilize a LC phase on monolayers could have important consequences in membrane organization and emphasize its structural role at mitochondrial level.

Cytoplasmic and mitochondrial creatine kinases from the skeletal muscle of sperm whale (Physeter macrocephalus). Molecular cloning and enzyme characterization.

We have amplified two cDNAs, coding for creatine kinases (CKs), from the skeletal muscle of sperm whale Physeter macrocephalus by PCR, and cloned these cDNAs into pMAL plasmid. These are the first CK cDNA and deduced amino acid sequences from cetaceans to be reported. One of the two amino acid sequences is a cytoplasmic, muscle-type isoform (MCK), while the other was identified as a sarcomeric, mitochondrial isoform (sMiCK) that included a mitochondrial targeting peptide. The amino acid sequences of sperm whale MCK and sMiCK showed 94-96% sequence identity with corresponding isoforms of mammalian CKs, and all of the key residues necessary for CK function were conserved. The phylogenetic analyses of vertebrate CKs with three independent methods (neighbor-joining, maximum-likelihood and Bayes) supported the clustering of sperm whale MCK with Bos and Sus MCKs, in agreement with the contemporary view that these groups are closely related. Sperm whale MCK and sMiCK were expressed in Escherichia coli as a fusion protein with maltose-binding protein, and the kinetic constants (K (m), K (d) and k (cat)) were determined for the forward reaction. Comparison of kinetic constants with those of human and mouse CKs indicated that sperm whale MCK has a comparable affinity for creatine (K (m) (Cr) = 9.38 mM) to that of human MCK, and the sMiCK has two times higher affinity for creatine than the human enzyme. Both the MCK and sMiCK of sperm whale display a synergistic substrate binding (K (d) /K (m) = 3.1-7.8) like those of other mammalian CKs.

Mitochondrial creatine kinase adsorption to biomimetic membranes: a Langmuir monolayer study.

Interaction of mitochondrial creatine kinase (mtCK) with either synthetic or natural zwitterionic or acidic phospholipids was monitored by surface pressure measurements. Injection of mtCK beneath a monolayer at very low surface pressure results in a large increase in the apparent area per lipid molecule reflecting the intrinsic surface activity of the protein. This effect is particularly pronounced with anionic phospholipid-containing films. Upon compression to high lateral pressure, the protein is squeezed out of the lipid monolayer. On the contrary, mtCK injected beneath a monolayer compressed at 30 mN/m, does not insert into the monolayer but is concentrated below the surface by anionic phospholipids as evidenced by the immediate and strong increase in the apparent molecular area occurring upon decompression. Below 8 mN/m the protein adsorbs to the interface and remains intercalated until the lateral pressure increases again. The critical pressure of insertion is higher for anionic lipid-containing monolayers than for films containing only zwitterionic phospholipids. In the former case it is markedly diminished by NaCl. The adsorption of mtCK depends on the percentage of negative charges carried by the monolayer and is reduced by increasing NaCl concentrations. However, the residual interaction existing in the absence of a global negative charge on the membrane may indicate that this interaction also involves a hydrophobic component.

The role of an absolutely conserved tryptophan residue in octamer formation and stability in mitochondrial creatine kinases.

In most organisms, mitochondrial creatine kinase (MtCK) is present as dimers and octamers with the latter predominating under physiological conditions. An absolutely conserved tryptophan residue (Trp-264 in chicken sarcomeric MtCK) appears to play a key role in octamer stability. Recently, it has been shown that the sponge Tethya aurantia, a member of the most ancient group of living multi-cellular animals, expresses an obligate, dimeric MtCK that lacks this absolutely conserved tryptophan residue, instead possessing a tyrosine in this position. In the present study we confirm that the absolutely conserved tryptophan residue is lacking in other sponge MtCKs where it is instead substituted by histidine or asparagine. Site directed mutations of the Trp-264 in expression constructs of chicken sarcomeric MtCK and the octameric MtCK from the marine worm Chaetopterus destabilized the octameric quaternary structure producing only dimers. A Tyr-->Trp mutation in an expression construct of the Tethya MtCK construct failed to produce octamerization; Tyr-->His and Tyr-->Asn mutations also yielded dimers. These results, in conjunction with analysis of homology models of Chaetopterus and Tethya MtCKs, strongly support the view that while the absolutely conserved tryptophan residue is important in octamer stability, octamer formation involves a complex suite of interactions between a variety of residues.

Prediction of protein orientation upon immobilization on biological and nonbiological surfaces.

We report on a rapid simulation method for predicting protein orientation on a surface based on electrostatic interactions. New methods for predicting protein immobilization are needed because of the increasing use of biosensors and protein microarrays, two technologies that use protein immobilization onto a solid support, and because the orientation of an immobilized protein is important for its function. The proposed simulation model is based on the premise that the protein interacts with the electric field generated by the surface, and this interaction defines the orientation of attachment. Results of this model are in agreement with experimental observations of immobilization of mitochondrial creatine kinase and type I hexokinase on biological membranes. The advantages of our method are that it can be applied to any protein with a known structure; it does not require modeling of the surface at atomic resolution and can be run relatively quickly on readily available computing resources. Finally, we also propose an orientation of membrane-bound cytochrome c, a protein for which the membrane orientation has not been unequivocally determined.

Interfacial behavior of cytoplasmic and mitochondrial creatine kinase oligomeric states.

Adsorption to the air/water interface of isoenzymes of creatine kinase was investigated using surface pressure-area isotherms and Brewster angle microscopy (BAM) observations. Octameric mitochondrial creatine kinase (mtCK) exhibits a significant affinity for the air/water interface. Whatever the mode of formation of the interfacial film, i.e., injection of the protein in the subphase or spreading onto the buffer surface, the final arrangement and conformation adopted by mtCK molecules lead to a similar result. In contrast, the dimeric isoenzymes mtCK and cytosolic MMCK do not induce any surface pressure variation. However, when the subphase contains 0.3M NaCl, both isoenzymes adsorb to the interface. When treated with 0.8 or 3M GdnHCl, muscle creatine kinase (MMCK) becomes surface active and occupies a greater surface than mtCK. This result contrasts with previous observations, often derived from monomeric proteins, that their surface activity is increased upon unfolding. It underlines the possible influence exerted by the protein oligomeric state on its interfacial activity. At a subphase pH of 8.8, which corresponds to the pI of octameric mtCK, the profiles of the isotherms obtained with dimeric and octameric states and the resistance to compression of the protein monolayers are significantly affected when compared to those recorded at pH 7.4. These data suggest that the octamer is more hydrophobic than the dimer and may contribute to explaining why octamers bind to the inner mitochondrial membrane while dimers do not.

Macroenzyme creatine kinase (CK) type 2 in HIV-infected patients is significantly associated with TDF and consists of ubiquitous mitochondrial CK.

OBJECTIVE: To evaluate the prevalence and origin of macroenzyme creatine kinase type 2 (Macro CK2) in HIV-1-infected patients on antiretroviral treatment. DESIGN: CK, CK-MB activity and protein weight, electrophoretic behaviour, glomerular filtration rate (GFR), aspartate aminotransferase (AST), alanine aminotransferase (ALT), bone alkaline phosphatase (AP), beta2-microglobulin serum levels and proteinuria were analysed in 468 HIV-infected outpatients. Sera with detectable Macro CK2 were further analysed using immunoblotting. RESULTS: CK-MB isoenzyme activity and mass concentration revealed the presence of Macro CK2 in 32/408 (7.8%) outpatients. Tenofovir DF (TDF) treatment was a prominent common feature in these patients. Prospective examination of sera from 41 patients collected prior to and during TDF exposure showed Macro CK2 in 20/41 (48%) TDF-treated patients and in 0/19 control sera from patients with TDF-free regimens. Macro CK2 was not present prior to TDF exposure. Patients with Macro CK2 showed a significant elevation of serum beta2-microglobulin levels. GFR, AST/ALT ratio, bone AP and proteinuria remained unchanged. Electrophoresis and immunoblotting demonstrated that the Macro CK2 in TDF-treated patients consisted of the ubiquitous (uMtCK) and not the sarcomeric type (sMtCK) of mitochondrial CK (MtCK). CONCLUSIONS: Macro CK2 consisting of uMtCK is associated with the use of TDF-containing regimens. Whether the appearance of uMtCK in these patients reflects mitochondrial damage remains to be clarified.