The role of lysine 55 in determining the specificity of the purine repressor for its operators through minor groove interactions
The role of lysine 55 in determining the specificity of the purine repressor for its operators through minor groove interactions
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DOI：
10.1006/jmbi.1999.2946
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发表时间：
1999-08-13
影响因子：
5.6
通讯作者：
Brennan, RG
中科院分区：
文献类型：
--
作者：
Glasfeld, A;Koehler, AN;Brennan, RG
二聚体大肠杆菌嘌呤阻遏物（PurR）与其同源序列的相互作用导致在朝向大沟的中央CpG碱基步骤处的45度至50度扭结，因为二联体相关的亮氨酸侧链在这些碱基之间从小沟交错。由此产生的小沟加宽增加了六个中心碱基对对与PurR残基的小沟相互作用的可及性。研究表明，PurR的赖氨酸55与高亲和力purF操纵基因序列中中央CpG碱基对步骤5'端的腺嘌呤碱基（Ade 8）直接接触。我们已经研究了这种相互作用的重要性，野生型PurR（WT）的特异性和亲和力，其运营商，我们已经研究了突变体的PurR中，赖氨酸55被替换为丙氨酸（K55 A）。WT和K55 A与含有在位置8变化的pur操纵子序列的双链体DNA的复合物进行了晶体学研究，并使用荧光各向异性进行了结合研究。的蛋白质-DNA复合物的结构揭示了一个相对不受干扰的全球构象，无论在位置8或残基55的碱基对的身份。在所有的结构中，更高的分辨率和purF回文操作位点的组合允许观察到几个新的PurR DNA相互作用，包括Thr 15，Thr 16和His 20的接触。Lys 55的侧链与第8位的腺嘌呤、胸腺嘧啶或胞嘧啶碱基产生了富有成效的相互作用，尽管相互作用各不相同，但其平衡解离常数分别为2.6 nM、10 nM和35 nM。然而，赖氨酸侧链的大部分明显地阻断了操作者与位置8处的鸟嘌呤的高亲和力结合（Kd 620 nM）。此外，高亲和力结合构象似乎被阻断，因为WT与在位置8处具有鸟嘌呤的DNA结合的晶体不能生长。在含有K55 A的复合物中，丙氨酸侧链离得太远而不能与操纵子进行货车范德华相互作用，并且随着磷酸骨架和赖氨酸的铵基团之间的一般静电相互作用的丧失，K55 A与每个操纵子的结合很弱。然而，该突变导致PurR对位置8处的碱基的特异性交换，K55 A表现出对鸟嘌呤超过腺嘌呤的两倍偏好。除了定义Lys 55在PurR小沟结合中的作用外，这些研究还提供了对其他LacI/GalR家族成员的小沟结合特异性的结构洞察，这些家族成员在可比位置具有丙氨酸（例如LacI，GalR，CcpA）或碱性残基（例如RafR，ScrR，RbtR）。(C)北京：科学出版社.
The interaction of the dimeric Escherichia coli purine repressor (PurR) with its cognate sequences leads to a 45 degrees to 50 degrees kink at a central CpG base step towards the major groove, as dyad-related leucine side-chains interdigitate between these bases from the minor groove. The resulting broadening of the minor groove increases the accessibility of the six central base-pairs towards minor groove interactions with residues from PurR. It has been shown that lysine 55 of PurR makes a direct contact with the adenine base (Ade8) directly 5' to the central CpG base-pair step in the high-affinity purF operator sequence. We have investigated the importance of this interaction in the specificity and affinity of wild-type PurR (WT) for its operators and we have studied a mutant of PurR in which Lys55 is replaced with alanine (K55A). Complexes of WT and K55A with duplex DNA containing pur operator sequences varied at position 8 were investigated crystallographically, and binding studies were performed using fluorescence anisotropy. The structures of the protein-DNA complexes reveal a relatively unperturbed global conformation regardless of the identity of the base-pair at position 8 or residue 55. Ln all structures the combination of higher resolution and a palindromic purF operator site allowed several new PurR DNA interactions to be observed, including contacts by Thr15, Thr16 and His20. The side-chain of Lys55 makes productive, though varying, interactions with the adenine, thymine or cytosine base at position 8 that result in equilibrium dissociation constants of 2.6 nM, 10 nM and 35 nM, respectively. However, the bulk of the lysine side-chain apparently blocks high-affinity binding of operators with guanine at position 8 (K-d 620 nM). Also, the high-affinity binding conformation appears blocked, as crystals of WT bound to DNA with guanine at position 8 could not be grown. Ln complexes containing K55A, the alanine side-chain is too far removed to engage in van der Waals interactions with the operator, and, with the loss of the general electrostatic interaction between the phosphate backbone and the ammonium group of lysine, K55A binds each operator weakly. However, the mutation leads to a swap of specificity of PurR for the base at position 8, with K55A exhibiting a twofold preference for guanine over adenine. In addition to defining the role of Lys55 in PurR minor groove binding, these studies provide structural insight into the minor groove binding specificities of other LacI/GalR family members that have either alanine (e.g. LacI, GalR, CcpA) or a basic residue (e.g. RafR, ScrR, RbtR) at the comparable position. (C) 1999 Academic Press.