Structural and functional analysis of a truncated form of Saccharomyces cerevisiae ATP sulfurylase: C-terminal domain essential for oligomer formation but not for activity.

ATP sulfurylase catalyzes the first step in the activation of sulfate by transferring the adenylyl-moiety (AMP approximately ) of ATP to sulfate to form adenosine 5'-phosphosulfate (APS) and pyrophosphate (PP(i)). Subsequently, APS kinase mediates transfer of the gamma-phosphoryl group of ATP to APS to form 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and ADP. The recently determined crystal structure of yeast ATP sulfurylase suggests that its C-terminal domain is structurally quite independent from the other domains, and not essential for catalytic activity. It seems, however, to dictate the oligomerization state of the protein. Here we show that truncation of this domain results in a monomeric enzyme with slightly enhanced catalytic efficiency. Structural alignment of the C-terminal domain indicated that it is extremely similar in its fold to APS kinase although not catalytically competent. While carrying out these structural and functional studies a surface groove was noted. Careful inspection and modeling revealed that the groove is sufficiently deep and wide, as well as properly positioned, to act as a substrate channel between the ATP sulfurylase and APS kinase-like domains of the enzyme.

Structure of mitochondrial creatine kinase.

Creatine kinase (CK, EC 2.7.3.2), an enzyme important for energy metabolism in cells of high and fluctuating energy requirements, catalyses the reversible transfer of a phosphoryl goup from phosphocreatine to ADP. We have solved the structure of the octameric mitochondrial isoform, Mib-CK, which is located in the intermembrane compartment and along the cristae membranes. Mib-CK consumes ATP produced in the mitochondria for the production of phosphocreatine, which is then exported into the cytosol for fast regeneration of ATP by the cytosolic CK isoforms. The octamer has 422 point-group symmetry, and appears as a cube of side length 93 angstrom with a channel 20 angstrom wide extending along the four-fold axis. Positively charged amino acids at the four-fold faces of the octamer possibly interact with negatively charged mitochondrial membranes. Each monomer consists of a small alpha-helical domain and a large domain containing an eight-stranded antiparallel beta-sheet flanked by seven alpha-helices. The conserved residues of the CK family form a compact cluster that covers the active site between the domains.

Localization of reactive cysteine residues by maleidoyl undecagold in the mitochondrial creatine kinase octamer.

Octamers of mitochondrial creatine kinase (Mi-CK) were modified with the thiol-specific reagents N-ethylmaleimide or the gold-coupled derivative, maleidoyl undecagold. The kinetics of inhibition of the Mi-CK catalysis was shown to be comparable for both reagents, suggesting that the large gold cluster complex is accessible to the reactive cysteines. SDS-PAGE analysis revealed that two of eight cysteines per Mi-CK monomer were labeled with maleidoyl undecagold with a similar affinity for the functional maleimide group. Gel exclusion chromatography of labeled molecules showed that the octameric structure of Mi-CK was preserved after thiol modification. Freeze-dried gold-labeled octamers visualized by electron microscopy under cryo-conditions were enhanced in contrast and showed a well-preserved fourfold symmetry of the end-on view. Image analysis of gold-labeled Mi-CK exhibited an averaged end-on view with four strong contrast signals located at the periphery of the octamer, whereas the center of the molecule remained electron translucent. We conclude that the two cysteine residues per monomer labeled with maleidoyl undecagold are located at the octamer's perimeter and we discuss the possible role of these reactive cysteines in enzyme catalysis.

The structure of mitochondrial creatine kinase and its membrane binding properties.

The biochemical and biophysical characterization of the mitochondrial creatine kinase (Mi-CK) from chicken cardiac muscle is reviewed with emphasis on the structure of the octameric oligomer by electron microscopy and on its membrane binding properties. Information about shape, molecular symmetry and dimensions of the Mi-CK octamer, as obtained by different sample preparation techniques in combination with image processing methods, are compared. The organization of the four dimeric subunits into the Mi-CK complex as apparent as apparent in the end-on projections is discussed and the consistently observed high binding affinity of the four-fold symmetric end-on faces towards many support films and towards each other is outlined. A study on the oligomeric state of the enzyme in solution and in intact mitochondria, using chemical crosslinking reagents, is presented together with the results of a search for a possible linkage of Mi-CK with the adenine nucleotide translocator (ANT). The nature of Mi-CK binding to model membranes, demonstrating that rather the octameric than the dimeric subspecies is involved in lipid interaction and membrane contact formation, is resumed and put into relation to our structural observations. The findings are discussed in light of a possible in vivo function of the Mi-CK octamer bridging the gap between outer and inner mitochondrial membranes at the contact sites.

Crystallization of mitochondrial creatine kinase on negatively charged lipid layers.

Polymorphic forms of crystals of mitochondrial creatine kinase (Mi-CK) octamers were generated by the lipid-layer technique, using cardiolipin as interphase adhesion matrix. Depending on the protein and lipid concentration, different types of monolayers and 3-D stacks thereof assembled in a low ionic strength crystallization buffer. Sodium tungstate was found to promote and stabilize the crystal formation, though in-plane crystallization was also possible in the absence of tungstate. All crystal forms exhibited a p4 symmetry with lattice parameters (a = b) ranging from 10.6 to 24.0 nm and with one or four octamers per unit cell in end-on orientation. In ice-embedded crystals, which showed a molecular packing different from that of negatively stained preparations, structural features of the Mi-CK octamer were observed at a resolution of 1 nm. The crystallization process took advantage of the electrostatic interaction between negatively charged lipid head groups of cardiolipin and positive charges located at the top/bottom faces of the Mi-CK octamer. In the absence of a cardiolipin support, Mi-CK formed linear filaments from a solution of phosphotungstate by association of octamers via their top/bottom faces. When tungstate was used instead of phosphotungstate, the filaments aligned themselves into large crystalline assemblies.

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

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.

Circular harmonic averaging of rotary-shadowed and negatively stained creatine kinase macromolecules.

The structure of mitochondrial creatine kinase is investigated by high-resolution shadowing at very low temperature and conventional negative staining. The electron microscopic images are analyzed with circular harmonic averaging, a method suited for the processing of single molecules. The rotational alignment and averaging is performed with the circular harmonic components, which allows data compression and several steps of noise reduction to be carried out within the averaging procedure. In addition, the symmetry can be deduced. For the mitochondrial creatine kinase, a fourfold symmetry is found that is compatible with the biochemical and biophysical characterization of the molecule.

Crystallization of mitochondrial creatine kinase. Growing of large protein crystals and electron microscopic investigation of microcrystals consisting of octamers.

Mitochondrial creatine kinase isolated from chicken cardiac muscle was crystallized by vapor diffusion techniques. Depending on the growth conditions, fine needles and platelets as well as large single crystals appeared after a few days. Large crystals were shown to diffract to at least 3.2 A resolution (Schnyder, T., Winkler, H., Gross, H., Sargent, D., Eppenberger, H. M., and Wallimann, T. (1990) Biophys J. 57, 420 and thus are suited for a detailed X-ray analysis in the future. The relatively high density of single crystals measured by a linear organic solvent density gradient indicates a tight packing of mitochondrial creatine kinase molecules within the crystals. Microcrystals, however, were subjected to electron optical examination either after prefixation with glutaraldehyde followed by conventional negative staining or by freeze-fracturing crystals in mother liquor and heavy metal replication with platinum/carbon. In both cases the crystals exhibited a square lattice with parameters of a = b = 139 A and a = b = 132 A in negatively stained and replicated crystals, respectively. No other lattice parameters were found, suggesting that these microcrystals represent a quasi-cubic three-dimensional lattice, which is in accordance with the finding that the building blocks of the crystals are the cube-like octamers described (Schnyder, T., Engel, A., Lustig, A., and Wallimann, T. (1988) J. Biol. Chem. 263, 16954-16962). Digital image processing applied to electron micrographs of crystals clearly revealed the arrangement of mitochondrial creatine kinase octamers in the crystal lattice as well as the subdivision of the octamer into its subdomains at a resolution of 23 A.

Structure of the mitochondrial creatine kinase octamer: high-resolution shadowing and image averaging of single molecules and formation of linear filaments under specific staining conditions.

The combination of high-resolution tantalum/tungsten (Ta/W) shadowing at very low specimen temperature (-250 degrees C) under ultrahigh vacuum (less than 2 x 10(-9) mbar) with circular harmonic image averaging revealed details on the surface structure of mitochondrial creatine kinase (Mi-CK) molecules with a resolution less than 2.5 nm. Mi-CK octamers exhibit a cross-like surface depression dividing the square shaped projection of 10 x 10 nm into four equally sized subdomains, which correspond to the four dimers forming the octameric Mi-CK molecule. By a combination of positive staining (with uranyl acetate) and heavy metal shadowing, internal structures as well as the surface relief of Mi-CK were visualized at the same time at high resolution. Computational image analysis revealed only a single projection class of molecules, but the ability of Mi-CK to form linear filaments, as well as geometrical considerations concerning the formation of octamers by four equal, asymmetric dimers, suggest the existence of at least two distinct faces on the molecule. By image processing of Mi-CK filaments a side view of the octamer differing from the top-bottom projections of single molecules became evident showing a funnel-like access each form the top and bottom of the octamer connected by a central channel. The general structure of the Mi-CK octamer described here is relevant to the localization of the molecule at the inner-outer mitochondrial contact sites and to the function of Mi-CK as an "energy channeling" molecule.

Crystallization and preliminary X-ray analysis of two different forms of mitochondrial creatine kinase from chicken cardiac muscle.

Crystals of mitochondrial creatine kinase isolated from chicken heart were grown by precipitation with polyethylene glycol 1000. The enzyme has been crystallized in the absence and presence of ATP in two different space groups. Crystals are tetragonal, with space group P42(1)2, a = b = 171 A, c = 150 A in the absence of ATP; and P422, a = b = 101 A, c = 114.4 A in the presence of ATP. We suggest that there is one octamer (346 kDa) per asymmetric unit without ATP and one dimer (86 kDa) per asymmetric unit with ATP. Using synchrotron radiation, the octameric form diffracts to at least 3 A resolution.

Native mitochondrial creatine kinase forms octameric structures. II. Characterization of dimers and octamers by ultracentrifugation, direct mass measurements by scanning transmission electron microscopy, and image analysis of single mitochondrial creatine kinase octamers.

Electron micrographs of negatively stained and metal-shadowed mitochondrial creatine kinase (Mi-CK) molecules purified as described by Schlegel et al. (Schlegel, J., Zurbriggen, B., Wegmann, E., Wyss, M., Eppenberger, H. M., and Wallimann, T. (1988) J. Biol Chem. 263, 16942-16953) revealed a homogeneous population (greater than or equal to 95%) of distinctly sized square-shaped, octameric particles with a side length of 10 nm that frequently exhibited a pronounced 4-fold axis of symmetry. The cube-like molecules consist of four dimers that are arranged around a stain-accumulating central cavity of 2.5-3 nm in diameter. This interpretation is supported by single particle averaging including correlation analysis by computer. Upon prolonged storage or high dilution, the cube-like octamers tended to dissociate into "banana-shaped" dimers. Sedimentation velocity and sedimentation equilibrium experiments yielded an s value of 12.8-13.5 S and an Mr of 328,000 +/- 25,000 for the octameric cubes. An s value of 5.0 S and a Mr of 83,000 +/- 8,000 was found under conditions which revealed banana-shaped dimers. These dimers proved to be very stable, as their dissociation into monomers of 45 kDa (s value = 2.0 S) required 6 M guanidine HCl. Thus, the oligomeric structures observed in the electron microscope are identified as Mi-CK dimers (banana-shaped structures) and cubical Mi-CK octamers assembled from four Mi-CK dimers. The octameric nature of native Mi-CK and the formation of Mi-CK dimers were confirmed by direct mass measurements of individual molecules by scanning transmission electron microscopy yielding a molecular mass of 340 +/- 55 kDa for the octamer and 89 +/- 27 kDa for the dimer. A structural model of Mi-CK octamers and the possible interaction with ATP/ADP-translocator molecules as well as with the outer mitochondrial membrane is proposed. The implications with respect to the physiological function of Mi-CK as an energy-channeling molecule at the producing side of the phosphoryl creatine shuttle are discussed.

Mitochondrial creatine kinase from cardiac muscle and brain are two distinct isoenzymes but both form octameric molecules.

Mitochondrial creatine kinase (Mi-CK) from chicken cardiac muscle and brain, recently shown to differ in their N-terminal amino acid sequences and to be encoded by multiple mRNAs (Hossle, H.P., Schlegel, J., Wegmann, G., Wyss, M., Böhlen, P., Eppenberger, H. M., Wallimann, T., and Perriard, J.C. (1988) Biochim. Biophys. Res. Commun. 151, 408-416) were separated on two-dimensional nonequilibrium pH-gradient electrophoresis gels and visualized as two distinct protein spots by immunoblotting. Analysis of the two proteins purified by specific elution from Blue-Sepharose with ADP (Wallimann, T., Zurbriggen, B., and Eppenberger, H. M. (1985) Enzyme 33, 226-231) followed by fast protein liquid chromatography cation exchange chromatography showed obvious differences in peptide maps, in immunological cross-reactivity with monoclonal antibodies, and in kinetic parameters. However, even though the two proteins were different, tissue-specific mitochondrial isoforms, both formed regularly-sized, perforated cube-like octameric structures with Mr of 364,000 +/- 25,000 and 352,000 +/- 20,000 for the cardiac and brain isoform, respectively. Electron microscopy of cardiac and brain Mi-CK octamers revealed cube-like molecules with a central cavity or transverse channel filled by negative stain. The octameric molecular structure of Mi-CK isoforms differs from the generally accepted dimeric arrangement of "cytosolic" muscle MM- and brain BB-CK.