Giedroc D P, Giu H W, Khan R, King G C, Chen K
Department of Biochemistry and Biophysics, Texas A&M University, College Station 77843-2128.
Biochemistry. 1992 Jan 28;31(3):765-74. doi: 10.1021/bi00118a018.
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)
基因32蛋白（g32P）是来自噬菌体T4的复制辅助单链核酸结合蛋白，每摩尔蛋白含有1摩尔锌（II）。锌配位为该分子的DNA结合核心结构域（称为g32P-(A+B)，残基22 - 253）提供结构稳定性。用钴（II）取代的蛋白进行的光吸收研究以及对113镉（II）取代的g32P-(A+B)进行的113镉核磁共振光谱表明，g32P中的金属配位球的特征是具有近似四面体的配体对称性，并且由半胱氨酸77、半胱氨酸87和半胱氨酸90的半胱氨酸-S-原子进行配位。这些研究预测，需要第四个源自蛋白质的非硫醇配体来完成四面体络合物，根据一级结构预测和建模推测该配体为组氨酸81 [吉德罗克，D.P.，约翰逊，B.A.，阿米蒂奇，I.M.，& 科尔曼，J.E.（1989年）《生物化学》28卷，2410 - 2418页]。为了验证该模型，我们采用定点诱变技术替换g32P中的两个组氨酸残基（组氨酸64和组氨酸81），并对这些单点突变蛋白进行纯化和结构表征。我们发现，在残基64处含有三种取代之一（H64Q、H64N和H64L）的g32P以无锌（II）形式（小于或等于0.03克原子锌（II））从大肠杆菌中分离出来。所有衍生物对单链DNA均聚物聚（dT）表现出极弱的亲和力。所有衍生物的远紫外圆二色光谱相对于野生型蛋白在负强度上都有所降低。这些结构特征与已知的金属配体突变体半胱氨酸87→丝氨酸87（C87S）g32P的特征相似。相反，用谷氨酰胺（H81Q）；丙氨酸（H81A）或半胱氨酸（H81C）取代组氨酸81的g32P-(A+B)，分离时含有化学计量的锌（II），并且与多核苷酸结合的亲和力与野生型g32P-(A+B)相当。在pH值变化时记录的野生型和H81Q g32P-(A+B)的自旋回波1H核磁共振光谱表明，在pH 7.8时，组氨酸81的环质子分别位于δ = 6.81和6.57 ppm，对应于1H-组氨酸-g32P-(A+B)的C和D组氨酸质子[潘，T.，吉德罗克，D.P.，& 科尔曼，J.E.（1989年）《生物化学》28卷，8828 - 8832页]。当pH值从7.8降至6.6时，这些共振峰向低场移动，且没有金属解离，这一结果与野生型g32P中组氨酸81向锌（II）提供配体的情况不相符。同样，锌（II）H81C g32P中的半胱氨酸81很容易与5,5'-二硫代双（2-硝基苯甲酸）反应，这与金属配体半胱氨酸77、半胱氨酸87和半胱氨酸90不同。（摘要截于400字）
