Structural basis for the nucleic acid binding cooperativity of bacteriophage T4 gene 32 protein: the (Lys/Arg)3(Ser/Thr)2 (LAST) motif.

To identify the functional residues of the N-terminal B region of bacteriophage T4 gene 32 protein involved in its cooperative binding to single-stranded nucleic acids, a process dependent on homotypic protein-protein interaction, we have studied the interaction of the protein with synthetic peptides containing different portions of this domain. Gel-permeation chromatography showed that a 6-acryloyl-2-dimethylaminonaphthalene (acrylodan)-labeled fluorescent peptide corresponding to the first 17 residues of gene 32 protein formed a complex with whole protein. The fluorescence was blue-shifted 14 nm upon interaction with intact protein, and somewhat less so (7-11 nm) with cleavage products of the protein lacking B domains. The intrinsic tryptophan fluorescence of whole and truncated protein was quenched by this peptide and by the nonderivatized peptide. The peptide bound tightly to truncated protein at both 0.015 and 0.44 M Na+, with a stoichiometry of 1:1. Similar tryptophan quenching or acrylodan blue shifts were obtained with peptides corresponding to residues 1-9 and 3-8, but not residues 1-4, 5-9, or 5-17, indicating that the essential amino acids are contained within positions 3-8, Lys-Arg-Lys-Ser-Thr-Ala. Several other DNA binding proteins contain a LAST motif with documented involvement of these residues in nucleic acid interaction. The amino acid and coding sequence of residues 110-114, a region proposed to be involved in nucleic acid binding, is virtually identical to that of residues 3-7. Based on these observations, we have formulated a model for the cooperative interactions of gene 32 protein with single-stranded nucleic acids.

为鉴定T4噬菌体基因32蛋白（bacteriophage T4 gene 32 protein）N端B结构域中参与其与单链核酸（single-stranded nucleic acids）协同结合的功能性残基——这一结合过程依赖于同源蛋白质相互作用（homotypic protein-protein interaction）——我们研究了该蛋白与包含该结构域不同区段的合成肽段之间的相互作用。凝胶渗透色谱法（gel-permeation chromatography）结果显示，对应基因32蛋白前17个残基、经6-丙烯酰基-2-二甲基氨基萘（acrylodan）标记的荧光肽段，可与完整蛋白形成复合物。该肽段与完整蛋白相互作用时，荧光发生14 nm的蓝移；而与缺失B结构域的蛋白裂解产物相互作用时，蓝移幅度仅为7~11 nm，相对较小。完整蛋白与截短蛋白的内源色氨酸荧光（intrinsic tryptophan fluorescence）均可被该肽段以及未衍生化的肽段所猝灭。该肽段在0.015 M与0.44 M的钠离子浓度下，均可与截短蛋白紧密结合，化学计量比为1:1。针对对应残基1-9与3-8的肽段，可观测到类似的色氨酸猝灭或acrylodan蓝移效应，但对应残基1-4、5-9或5-17的肽段则无此现象，这表明关键氨基酸残基位于3-8位区段，即Lys-Arg-Lys-Ser-Thr-Ala（赖氨酸-精氨酸-赖氨酸-丝氨酸-苏氨酸-丙氨酸）。另有多种DNA结合蛋白包含LAST基序（LAST motif），且已有研究证实该基序内的此类残基参与核酸结合过程。被推测参与核酸结合的110-114位残基区域，其氨基酸序列与编码序列与3-7位残基几乎完全一致。基于上述实验结果，我们构建了基因32蛋白与单链核酸协同结合的作用模型。