UPMC Université Paris 06, Laboratoire des BioMolécules UMR 7203, 4 place Jussieu, 75005 Paris, France.
J Struct Biol. 2012 Dec;180(3):577-84. doi: 10.1016/j.jsb.2012.09.005. Epub 2012 Sep 17.
The bacterial Uup protein belongs to the REG subfamily of soluble ATP-binding cassette (ABC) ATPases, and is implicated in precise excision of transposons. In Escherichia coli, the uup gene encodes a 72 kDa polypeptide that comprises two ABC domains, separated by a linker region, and a 12kDa C-terminal domain (CTD). Uup binds double-stranded DNA with no sequence specificity, and we previously demonstrated that the CTD domain is a crucial region that participates in DNA-binding activity. We report herein the NMR structure of Uup CTD, consisting of an intramolecular antiparallel two-stranded coiled coil motif. Structural comparison with analogous coiled coil domains reveals that Uup CTD contains an atypical 3(10)-helix in the α-hairpin region that contributes to the hydrophobic core. Using NMR titration experiments, we identified residues of the CTD domain involved in the binding to double-stranded DNA. These residues are located on two opposite surfaces at the base of the coiled coil, formed by the N- and C-terminal extremities, where a strictly conserved proline residue induces an overwinding of the coiled coil. Finally, preliminary analysis of NMR spectra recorded on distinct Uup constructs precludes a fully flexible positioning of the CTD domain in full-length Uup. These structural data are the first reported for a non-ATPase domain within ABC REG subfamily.
细菌 Uup 蛋白属于可溶性 ATP 结合盒 (ABC) ATP 酶的 REG 亚家族，与转座子的精确切除有关。在大肠杆菌中，uup 基因编码一个 72kDa 的多肽，它由两个 ABC 结构域组成，由一个连接区隔开，还有一个 12kDa 的 C 端结构域 (CTD)。Uup 与双链 DNA 结合，没有序列特异性，我们之前证明 CTD 结构域是一个关键区域，参与 DNA 结合活性。我们在此报告 Uup CTD 的 NMR 结构，由一个分子内反平行的双股螺旋卷曲螺旋模体组成。与类似的卷曲螺旋结构域的结构比较表明，Uup CTD 在α发夹区含有一个非典型的 3(10)-螺旋，有助于形成疏水区核心。通过 NMR 滴定实验，我们确定了 CTD 结构域中与双链 DNA 结合的残基。这些残基位于卷曲螺旋底部的两个相对面上，由 N 端和 C 端的极端组成，其中一个严格保守的脯氨酸残基导致卷曲螺旋的过度缠绕。最后，对不同 Uup 构建体记录的 NMR 谱的初步分析排除了 CTD 结构域在全长 Uup 中的完全灵活定位。这些结构数据是 ABC REG 亚家族中非 ATP 酶结构域的首次报道。
