ZN(II) COORDINATION DOMAIN MUTANTS OF T4 GENE-32 PROTEIN

Gene 32 protein (g32P), the replication accessory single-stranded nucleic acid binding protein from bacteriophage T4, contains 1 mol of Zn(II)/mol of protein. Zinc coordination provides structural stability to the DNA-binding core domain of the molecule, termed g32P-(A + B) (residues 22-253). Optical absorption studies with the Co(II)-substituted protein and Cd-113 NMR spectroscopy of Cd-113(II)-substituted g32P-(A + B) show that the metal coordination sphere in g32P is characterized by approximately tetrahedral ligand symmetry and ligation by the Cys-S- atoms of Cys77, Cys87, and Cys90. These studies predicted the involvement of a fourth protein-derived non-thiol ligand to complete the tetrahedral complex, postulated to be His81 on the basis of primary structure prediction and modeling [Giedroc, D. P., Johnson, B. A., Armitage, I. M., & Coleman, J. E. (1989) Biochemistry 28, 2410-2418]. To test this model, we have employed site-directed mutagenesis to substitute each of the two histidine residues in g32P (His64 and His81), accompanied by purification and structural characterization of these single-site mutant proteins. We show that g32P's containing any of three substitutions at residue 64 (H64Q, H64N, and H64L) are isolated from Escherichia coli in a Zn(II)-free form [less-than-or-equal-to 0.03 g.atom Zn(II)]. All derivatives show extremely weak affinity for the ssDNA homopolymer poly(dT). All are characterized by a far-UV-CD spectrum reduced in negative intensity relative to the wild-type protein. These structural features parallel those found for the known metal ligand mutant Cys87 --> Ser87 (C87S) g32P. In contrast, g32P-(A + B) containing a substitution of His81 with glutamine (H81Q), alanine (H81A) or cysteine (H81C), contains stoichiometric Zn(II) as isolated and binds to polynucleotides with an affinity comparable to the wild-type g32P-(A + B). Spin-echo H-1 NMR spectra recorded for wild-type and H81Q g32P-(A + B) as a function of pH allow the assignment of His81 ring protons to delta = 6.81 and 6.57 ppm, respectively, at pH 7.8, corresponding to the C and D histidyl protons of H-1-His-g32P-(A + B) [Pan, T., Giedroc, D. P., & Coleman, J. E. (1989) Biochemistry 28, 8828-8832]. These resonances shift downfield as the pH is reduced from 7.8 to 6.6 without metal dissociation, a result incompatible with His81 donating a ligand to the Zn(II) in wild-type g32P. Likewise, Cys81 in Zn(II) H81C g32P is readily reactive with 5,5'-dithiobis(2-nitrobenzoic acid), unlike metal ligands Cys77, Cys87, and Cys90. Upon substitution of H81Q, H81A, and H81C g32P-(A + B) with Co(II), the d-d ligand field transition envelope is not detectably different from that in the wild-type protein. The natural CD spectra of all three mutant Co(II) complexes show the same minor alterations in the S- --> Co(II) ligand-to-metal charge transfer region. Cd-113 substitution of the Gln, Ala, and Cys mutants gives a Cd-113 NMR signal at delta = 633 +/- 1 ppm, compared to delta = 639 ppm for the wild-type protein. We conclude that it is unlikely that His81 forms a coordination bond to Zn(II) in g32P, as the original model predicted. Metal coordination by His64 in g32P remains a possibility in light of the inability of His64 mutant proteins to coordinate Zn(II).