Zinc coordination in bacteriophage T4 gene 32 protein

Abstract

Bacteriophage T4 gene 32 protein (gp32) is the prototype single-stranded (ss) nucleic acid binding protein. It contains an intrinsic Zn(II) ion within the postulated ssDNA binding core domain. Physical and biochemical characterization of the wild-type and a number of Zn(II) coordination domain mutant gp32s show that His$sp{81}$ does not coordinate to the metal ion, in contrast to a previous model proposed on the basis of amino acid sequence comparisons. A variety of substitutions at this position give rise to a metalloprotein which appears to have the same set of metal-ligand coordination bonds and nearly wild-type functional ssDNA binding and helix-destabilizing activity. We have proposed a new model which utilizes the side chains of His$sp{64}$, together with Cys$sp{77}$, Cys$sp{87}$ and Cys$sp{90}$, to form the metal coordination structure. Optical absorption studies on a metal-reconstituted His$sp{64} to$ Cys (H64C) mutant gp32 provide support for this novel coordination scheme, one which has not been observed in other zinc-containing nucleic acid binding proteins. The functional significance of zinc coordination in gp32 has been addressed using an in vitro biochemical assay of the relative activity of gp32 derivatives to function as accessory factors in recombination. A metal-free derivative of gene 32 protein, prepared from Zn(II) metalloprotein upon treatment with methyl methanethiolsulfonate, binds ssDNA very weakly and fails to stimulate a homologous pairing reaction catalyzed by the T4 recombinase uvsX protein. These data are the first of any kind which directly couple the formation of the Zn(II) complex in gp32 to functionally significant ssDNA binding by gp32.