title: Zn(II) coordination domain mutants of T4 gene 32 protein.
abstract: 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 113Cd NMR spectroscopy of 113Cd(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 1H NMR spectra recorded for wild-type and H81Q g32P-(A+B) as a function of pH allow the assignment of His81 ring proteins to delta = 6.81 and 6.57 ppm, respectively, at pH 7.8, corresponding to the C and D histidyl protons of 1H-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.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station 77843-2128.
authors.forename: D P
authors.initials: DP
authors.lastname: Giedroc
authors.forename: H W
authors.initials: HW
authors.lastname: Giu
authors.forename: R
authors.initials: R
authors.lastname: Khan
authors.forename: G C
authors.initials: GC
authors.lastname: King
authors.forename: K
authors.initials: K
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002104:Cadmium / Q000378:metabolism; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D013604:T-Phages / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; C060035:gp32 protein, Enterobacteria phage T4; D002104:Cadmium; D015032:Zinc