RESUMO
Given the broad range of substrates hydrolyzed by Nudix (nucleoside diphosphate linked to X) enzymes, identification of sequence and structural elements that correctly predict a Nudix substrate or characterize a family is key to correctly annotate the myriad of Nudix enzymes. Here, we present the structure determination and characterization of Bd3179 -- a Nudix hydrolase from Bdellovibrio bacteriovorus-that we show localized in the periplasmic space of this obligate Gram-negative predator. We demonstrate that the enzyme is a nucleoside diphosphate sugar hydrolase (NDPSase) and has a high degree of sequence and structural similarity to a canonical ADP-ribose hydrolase and to a nucleoside diphosphate sugar hydrolase (1.4 and 1.3 Å Cα RMSD respectively). Examination of the structural elements conserved in both types of enzymes confirms that an aspartate-X-lysine motif on the C-terminal helix of the α-ß-α NDPSase fold differentiates NDPSases from ADPRases.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Bdellovibrio/enzimologia , Pirofosfatases/química , Pirofosfatases/metabolismo , Proteínas de Bactérias/genética , Bdellovibrio/genética , Domínio Catalítico , Clonagem Molecular , Modelos Moleculares , Açúcares de Nucleosídeo Difosfato/metabolismo , Periplasma/metabolismo , Estrutura Terciária de Proteína , Pirofosfatases/genética , Homologia de Sequência de Aminoácidos , Nudix HidrolasesRESUMO
We provide direct evidence that Bin/Amphiphysin/Rvs (BAR) family members bend the steady state membrane architecture of organelles in intact cells. In response to inducible BAR molecular actuators, organelles exhibit distinct changes to the orientation and degree of their membrane curvature. This rapidly inducible system may offer a mechanism by which to better understand the structure-function relationship of intracellular organelles.
Assuntos
Membrana Celular/metabolismo , Membrana Celular/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Relação Estrutura-AtividadeRESUMO
Using a newly synthesized gibberellin analog containing an acetoxymethyl group (GA(3)-AM) and its binding proteins, we developed an efficient chemically inducible dimerization (CID) system that is completely orthogonal to existing rapamycin-mediated protein dimerization. Combining the two systems should allow applications that have been difficult or impossible with only one CID system. By using both chemical inputs (rapamycin and GA(3)-AM), we designed and synthesized Boolean logic gates in living mammalian cells. These gates produced output signals such as fluorescence and membrane ruffling on a timescale of seconds, substantially faster than earlier intracellular logic gates. The use of two orthogonal dimerization systems in the same cell also allows for finer modulation of protein perturbations than is possible with a single dimerizer.
