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Expression of active octameric chicken cardiac mitochondrial creatine kinase in Escherichia coli.

ABSTRACT: Sarcomeric mitochondrial creatine kinase (Mib-CK) of chicken was expressed in Escherichia coli as a soluble enzyme by using an inducible phage-T7 promoter. Up to one third of the protein in E. coli extracts consisted of soluble recombinant Mib-CK in an enzymically active form. Approx. 20 mg of nearly-homogenous Mib-CK was isolated in a two-step isolation procedure starting with 1 litre of isopropyl beta-D-thiogalactopyranoside-induced E. coli culture, whereas previous attempts to express other CK genes in E. coli have resulted in 20-fold lower yields and inclusion-body formation. Selection of the Mib-CK expression plasmid on media containing kanamycin rather than ampicillin extended the time period of maximal Mib-CK expression. Recombinant Mib-CK displayed an identical N-terminal amino acid sequence, identical Km for phosphocreatine and Vmax. values, the same electrophoretic behaviour and the same immunological cross-reactivity as the native enzyme isolated from chicken heart mitochondria. The recombinant Mib-CK had the same molecular mass as native chicken Mib-CK in m.s. analysis, indicating that post-translational modification of the enzyme in chicken tissue does not occur. As judged by gel-permeation chromatography and electron microscopy, recombinant enzyme formed predominantly octameric oligomers with the same overall structure as the chicken heart enzyme. Furthermore, the enzymes isolated from both sources formed protein crystals of space group P42(1)2, when grown in the absence of ATP, with one Mi-CK octamer per asymmetric unit. The indistinguishable X-ray-diffraction patterns indicate identical structures for the native and recombinant proteins.

SUBMITTER: Furter R

PROVIDER: S-EPMC1131953 | biostudies-other | 1992 Dec

REPOSITORIES: biostudies-other

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Project description:BackgroundAbnormalities in cardiac energy metabolism occur in heart failure (HF) and contribute to contractile dysfunction, but their role, if any, in HF-related pathologic remodeling is much less established. CK (creatine kinase), the primary muscle energy reserve reaction which rapidly provides ATP at the myofibrils and regenerates mitochondrial ADP, is down-regulated in experimental and human HF. We tested the hypotheses that pathologic remodeling in human HF is related to impaired cardiac CK energy metabolism and that rescuing CK attenuates maladaptive hypertrophy in experimental HF.MethodsFirst, in 27 HF patients and 14 healthy subjects, we measured cardiac energetics and left ventricular remodeling using noninvasive magnetic resonance 31P spectroscopy and magnetic resonance imaging, respectively. Second, we tested the impact of metabolic rescue with cardiac-specific overexpression of either Ckmyofib (myofibrillar CK) or Ckmito (mitochondrial CK) on HF-related maladaptive hypertrophy in mice.ResultsIn people, pathologic left ventricular hypertrophy and dilatation correlate closely with reduced myocardial ATP levels and rates of ATP synthesis through CK. In mice, transverse aortic constriction-induced left ventricular hypertrophy and dilatation are attenuated by overexpression of CKmito, but not by overexpression of CKmyofib. CKmito overexpression also attenuates hypertrophy after chronic isoproterenol stimulation. CKmito lowers mitochondrial reactive oxygen species, tissue reactive oxygen species levels, and upregulates antioxidants and their promoters. When the CK capacity of CKmito-overexpressing mice is limited by creatine substrate depletion, the protection against pathologic remodeling is lost, suggesting the ADP regenerating capacity of the CKmito reaction rather than CK protein per se is critical in limiting adverse HF remodeling.ConclusionsIn the failing human heart, pathologic hypertrophy and adverse remodeling are closely related to deficits in ATP levels and in the CK energy reserve reaction. CKmito, sitting at the intersection of cardiac energetics and redox balance, plays a crucial role in attenuating pathologic remodeling in HF. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT00181259.

Project description:Escherichia coli chemotaxis relies on control of the autophosphorylation activity of the histidine kinase CheA by transmembrane chemoreceptors. Core signaling units contain two receptor trimers of dimers, one CheA homodimer, and two monomeric CheW proteins that couple CheA activity to receptor control. Core signaling units appear to operate as two-state devices, with distinct kinase-on and kinase-off CheA output states whose structural nature is poorly understood. A recent all-atom molecular dynamic simulation of a receptor core unit revealed two alternative conformations, "dipped" and "undipped," for the ATP-binding CheA.P4 domain that could be related to kinase activity states. To explore possible signaling roles for the dipped CheA.P4 conformation, we created CheA mutants with amino acid replacements at residues (R265, E368, and D372) implicated in promoting the dipped conformation and examined their signaling consequences with in vivo Förster resonance energy transfer (FRET)-based kinase assays. We used cysteine-directed in vivo cross-linking reporters for the dipped and undipped conformations to assess mutant proteins for these distinct CheA.P4 domain configurations. Phenotypic suppression analyses revealed functional interactions among the conformation-controlling residues. We found that structural interactions between R265, located at the N terminus of the CheA.P3 dimerization domain, and E368/D372 in the CheA.P4 domain played a critical role in stabilizing the dipped conformation and in producing kinase-on output. Charge reversal replacements at any of these residues abrogated the dipped cross-linking signal, CheA kinase activity, and chemotactic ability. We conclude that the dipped conformation of the CheA.P4 domain is critical to the kinase-active state in core signaling units.IMPORTANCE Regulation of CheA kinase in chemoreceptor arrays is critical for Escherichia coli chemotaxis. However, to date, little is known about the CheA conformations that lead to the kinase-on or kinase-off states. Here, we explore the signaling roles of a distinct conformation of the ATP-binding CheA.P4 domain identified by all-atom molecular dynamics simulation. Amino acid replacements at residues predicted to stabilize the so-called "dipped" CheA.P4 conformation abolished the kinase activity of CheA and its ability to support chemotaxis. Our findings indicate that the dipped conformation of the CheA.P4 domain is critical for reaching the kinase-active state in chemoreceptor signaling arrays.

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Expression of active octameric chicken cardiac mitochondrial creatine kinase in Escherichia coli.

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