Yasuo Kagawa

Small-angle X-ray scattering study of adenosine triphosphatase from thermophilic bacterium PS3.

Adenosine triphosphatase from the thermophilic bacterium PS3(TF1) has been studied by solution X-ray scattering. A structural change in TF1 caused by the binding of ADP was observed by examining the difference between the radii of gyration of the unligated and ligated forms. The radius of gyration of the unligated TF1 was found to be 49.5 +/- 0.3 A, and it decreased by approximately 3% after ligation with ADP. The positions and the amplitudes of a subsidiary maximum and a shoulder in the scattering profile showed subtle change on nucleotide binding. The lower limit of the maximum length of TF1 was determined to be 165 A for the unligated form and 150 A for the ligated form. The shape analysis of TF1 was performed by model calculations for simple triaxial bodies or their complexes. Among the various models tested, the one that gave the best fit with the experimental data consisted of seven ellipsoids of revolution; six identical ellipsoids with semi-axes: a = b = 18.5 A and c = 74 A. arranged hexagonally, and the other with a = b = 28 A and c = 45 A, located below the other six on the 6-fold axis. On the basis of this model it was suggested that there is a structural change on ligation with nucleotides, consisting of a shrinkage of the six long ellipsoids by 6% along their major axes.

The Structure and Expression of a Human Gene for a Nuclear-Coded Mitochondrial Adenosine Triphosphate Synthase Beta Subunit

The beta subunit of mitochondrial ATP synthase is coded on a nuclear genome, synthesized in the cytosol, and then assembled with the other subunits which are coded on the mitochondrial genome. To determine the molecular mechanism by which the expressions on these two genetic systems are coordinated, the gene structure of the human ATP synthase beta subunit was determined, and the structure involved in this expression was analyzed. The gene for the beta subunit was found to be composed of 10 exons. The first exon corresponded to the prepiece peptide for targeting mitochondria. The 5’ upstream region contained three CAT boxes (CCAAT), three GC boxes (CCGCCC) and four repeating sequences, but no typical TATA box. To determine the regulatory structure of the upstream region, fragments of various length were fused with a chloramphenicol acetyltransferase (CAT) gene and then transfected into a cultured cell. A 300 base pairs fragment was sufficient for expressing the CAT activity, and furthermore, the longer fragment (1300 base-pairs) enhanced the expression markedly. This result suggests that the gene for the human ATP synthase beta subunit has a regulatory structure. In addition, a restriction length fragment polymorphism in the gene and an interesting pseudo-gene are reported.

Formation and Function of Energy-transducing Biomembranes:The roles played by chaperonin and gene.

The energy transduction in biomembranes requires a special supramolecular structure. This structure is needed for converting proton motive energy into ATP synthesis. These biomembranes contain 4 enzyme complexes that are composed of many polypeptides. Here we describe the following 4 phenomena : 1) oligomer formation by self-assembly of the subunits, 2) incorporation of the oligomers into a lipid bilayer, 3) chaperonin-mediated formation of the oligomers and membranes, 4) coordination of the subunit biosyntheses by enhancers and the role of mitochondrial DNA.Polypeptides translated are assembled into the membrane complexes with or without the help of chaperonins. In the case of thermophilic proteins, the reconstitutability of polypeptides is excellent ; for example, the catalytic portion of ATP synthase, α1 β1 subunit complex, was reconstituted. On the other hand, mesophilic ATP synthase requires chaperonins for its formation. The transcription of mRNAs for these polypeptides is coordinated by special enhancer and silencer. The role of polypeptides encoded by mitochondrial DNA in the molecular assembly was studied with the wild-type and mutant cells.

Gene Structure of Human ATP Synthase Beta Subunit

The β subunit of ATP synthase is a catalytic subunit that is highly homologous among different species. Human nuclear genes for the mitochondrial enzymes including the β subunits and 8 related proteins were cloned to elucidate the coordinated transcriptional control of energy transduction.

Gene Structure and Function of Thermophilic ATP Synthase

An operon for thermophilic ATP synthase (TF0F1)was sequenced and mutated. The advantage of using TF0F1 for mechanistic studies is its reconstitutability without MgATP. All genes for TF0F1 were arranged in the order of promotors, structural genes coding for the I, TF0 subunits (a, c, b) and TF1 subunits (δ, α, γ, β and e), and a terminator. The cause of the stability of these subunits was deduced from their sequence. The site-directed mutagenesis of the α and β subunits revealed that the 4 ionizable residues corresponding to Lys 21 and Asp 119 in the MgATP binding segments of adenylate kinase, are essential for the normal catalytic activity of this enzyme. The resulting βI164 and βN252 mutant subunits were both noncatalytic after reassembly into the αβγ subunit complex, even though both subunits bound significant amounts of ADP. The resulting αI175 reassembled weakly into an oligomer, while the αN261 was reconstituted into an αβγ subunit complex that showed no intersubunit cooperativity.