RESUMO
The crystal structure of a novel Cre-Lox synapse was solved using phases from multiple isomorphous replacement and anomalous scattering, and refined to 2.05 A resolution. In this complex, a symmetric protein trimer is bound to a Y-shaped three-way DNA junction, a marked departure from the pseudo-4-fold symmetrical tetramer associated with Cre-mediated LoxP recombination. The three-way DNA junction was accommodated by a simple kink without significant distortion of the adjoining DNA duplexes. Although the mean angle between DNA arms in the Y and X structures was similar, adjacent Cre trimer subunits rotated 29 degrees relative to those in the tetramers. This rotation was accommodated at the protein-protein and DNA-DNA interfaces by interactions that are "quasi-equivalent" to those in the tetramer, analogous to packing differences of chemically identical viral subunits at non-equivalent positions in icosahedral capsids. This structural quasi-equivalence extends to function as Cre can bind to, cleave and perform strand transfer with a three-way Lox substrate. The structure explains the dual recognition of three and four-way junctions by site-specific recombinases as being due to shared structural features between the differently branched substrates and plasticity of the protein-protein interfaces. To our knowledge, this is the first direct demonstration of quasi-equivalence in both the assembly and function of an oligomeric enzyme.
Assuntos
Sítios de Ligação Microbiológicos/genética , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Integrases/química , Integrases/metabolismo , Conformação de Ácido Nucleico , Recombinação Genética , Proteínas Virais/química , Proteínas Virais/metabolismo , Sequência de Bases , Sítios de Ligação , Catálise , Cristalografia por Raios X , DNA Bacteriano/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Modelos Moleculares , Ligação Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas , Rotação , Relação Estrutura-AtividadeRESUMO
The methylmercury-induced dealkylation of the corrinoid coenzyme methylcobalamin, yielding aquocobalamin and dimethylmercury as products, was studied spectrophotometrically at 350 nm using water as a solvent. Rate data were determined for the pH 7-9 region and also at pH 3.37. Evidence is provided which shows that CH3Hg+ serves as the species which accepts the methyl group and also that Hg2+ is methylated more rapidly than CH3Hg+ is.