Effects of Substitution of Proposed Zn(II) Ligand His81 or His64 in Phage T4 Gene 32 Protein: Spectroscopic Evidence for a Novel Zinc Coordination Complex

Abstract

T4 gene 32 protein (gp32), the prototype helix-destabilizing or single-stranded (ss) DNA binding protein, contains one tightly coordinated Zn2+ ion bound tetrahedrally by three cysteines (residues 77, 87, and 90) and a fourth non-thiol donor. In previous work, it was shown that the proposed non-thiol ligand His81 could be readily substituted with nonliganding glutamine and alanine residues without deleterious effects on gp32 structure and simple assays of ssDNA binding. In this paper we show that exchange broadening of bulk 35Cl- anion by protein-bound Zn(II) is not observed in the His81-->Ala (H81A) mutant, unless the coordination site is disrupted with an organomercurial, p-mercuriphenylsulfonate. This suggests that, in the mutant protein, anions, and by implication solvent molecules, do not gain access to a newly formed inner shell Zn(II) coordination site as a result of mutagenesis. H81A gp32 is characterized by nearly wild-type helix-destabilizing activity on poly(d[A-T]) and highly cooperative binding to the polynucleotide poly(A) at pH 7.7 over the temperature range from 20 to 42 degrees C at 0.35 M NaCl, exhibiting only a approximately 2.5-4-fold decrease in poly(A) affinity. Limited proteolysis experiments show that an additional tryptic cleavage site maps to the Arg111-Lys112 bond within the protease-resistant core domain of the H81A gp32 following long incubation times and results in the accumulation of a 16-kDa subcore fragment. This new cleavage site is within the internal LAST motif, which has been proposed to be directly involved in cooperative ssDNA binding [Casas-Finet, J. R., & Karpel, R. L. (1993) Biochemistry 32, 9735-9744]. Thus substitution of His81 with Ala subtly alters the conformation or dynamics of the backbone around the LAST motif, which may be manifest as a moderately lower cooperative binding affinity of H81A gp32 for polynucleotides. H81A gp32, however, is fully functional in stimulating in vitro homologous pairing catalyzed by the T4 recombinase uvsX protein. Since substitution of His81 with a nonliganding Ala is nearly silent, we propose an alternative mode of Zn(II) coordination in T4 gene 32 protein, involving His64 rather than His81 as the fourth non-thiol ligand. That replacement of His64, and not His81, with Cys results in marked changes in the first coordination sphere of ligands as evidenced by the optical spectrum of Co(II)-substituted H64C gp32 is consistent with the noninvolvement of His81 and implicates a novel His64-X12-Cys77-X9-Cys87-X2-Cys90 coordination motif, unique among zinc-containing nucleic acid binding proteins.