The archaeal triphosphate tunnel metalloenzyme SaTTM defines structural determinants for the diverse activities in the CYTH protein family.

CYTH proteins make up a large superfamily that is conserved in all three domains of life. These enzymes have a triphosphate tunnel metalloenzyme (TTM) fold, which typically results in phosphatase functions, e.g., RNA triphosphatase, inorganic polyphosphatase, or thiamine triphosphatase. Some CYTH orthologs cyclize nucleotide triphosphates to 3',5'-cyclic nucleotides. So far, archaeal CYTH proteins have been annotated as adenylyl cyclases, although experimental evidence to support these annotations is lacking. To address this gap, we characterized a CYTH ortholog, SaTTM, from the crenarchaeote Sulfolobus acidocaldarius. Our in silico studies derived ten major subclasses within the CYTH family implying a close relationship between these archaeal CYTH enzymes and class IV adenylyl cyclases. However, initial biochemical characterization reveals inability of SaTTM to produce any cyclic nucleotides. Instead, our structural and functional analyses show a classical TTM behavior, i.e., triphosphatase activity, where pyrophosphate causes product inhibition. The Ca2+-inhibited Michaelis complex indicates a two-metal-ion reaction mechanism analogous to other TTMs. Cocrystal structures of SaTTM further reveal conformational dynamics in SaTTM that suggest feedback inhibition in TTMs due to tunnel closure in the product state. These structural insights combined with further sequence similarity network-based in silico analyses provide a firm molecular basis for distinguishing CYTH orthologs with phosphatase activities from class IV adenylyl cyclases.

Isoform-specific monoclonal antibodies against HSP90.

The purpose of this study was to develop monoclonal antibodies (mAbs) that distinguish between the two isoforms of human 90-kDa heat shock protein (HSP90), i.e., HSP90 alpha and beta. Human HSP90 alpha and beta isoforms expressed in Escherichia coli were separately used as antigens for developing the mAbs. Twenty-three and ten mAbs were obtained by immunization of mice with HSP90 alpha and beta, respectively. Among them, ten and three mAbs specifically recognized HSP90 alpha and beta isoforms, respectively, on the criteria of both enzyme-linked immunosorbent and immunoblotting analyses. Immunochemical analysis by use of the mAbs revealed that both of the HSP90 isoforms were present in human cells even under unstressed conditions and that the expression of HSP90 alpha was more strongly induced when the cells were exposed to arsenate. This is the first report of the development of the mAbs discriminating between the two isoforms of HSP90. The mAbs specific for HSP90 isoforms should be useful for the regulational and functional analyses of HSP90 isoforms.