McrBC is a conserved modification-dependent restriction system that in Escherichia coli specifically targets foreign DNA containing methylated cytosines. Crystallographic data show that the N-terminal domain of Escherichia coli McrB binds substrates via a base flipping mechanism. This region is poorly conserved among the plethora of McrB homologs, suggesting that other species may use alternative binding strategies and/or recognize different targets. Here we present the crystal structure of the N-terminal domain from Stayphlothermus marinus McrB (Sm3-180) at 1.92 Å, which adopts a PUA-like EVE fold that is closely related to the YTH and ASCH RNA binding domains. Unlike most PUA-like domains, Sm3-180 binds DNA and can associate with different modified substrates. We find the canonical ‘aromatic cage’ binding pocket that confers specificity for methylated bases in other EVE/YTH domains is degenerate and occluded in Sm3-180, which may contribute to its promiscuity in target recognition. Further structural comparison between different PUA-like domains identifies motifs and conformational variations that correlate with the preference for binding either DNA or RNA. Together these data have important implications for PUA-like domain specificity and suggest a broader biological versatility for the McrBC family than previously described.

McrBC 是一种保守的修饰依赖性限制系统，在大肠杆菌中专门针对含有甲基化胞嘧啶的外源 DNA。晶体学数据显示，大肠杆菌McrB的 N 端结构域通过碱基翻转机制结合底物。该区域在大量 McrB 同源物中的保守性较差，这表明其他物种可能使用替代结合策略和/或识别不同的目标。在这里，我们展示了来自海洋海藻的 N 端结构域的晶体结构McrB (Sm3-180) 在 1.92 Å，采用与 YTH 和 ASCH RNA 结合域密切相关的 PUA 样 EVE 折叠。与大多数类似 PUA 的结构域不同，Sm3-180 可结合 DNA 并可与不同的修饰底物结合。我们发现赋予其他 EVE/YTH 域中甲基化碱基特异性的典型“芳香笼”结合口袋在 Sm3-180 中退化并被封闭，这可能有助于其在目标识别中的混杂。不同 PUA 样域之间的进一步结构比较确定了与结合 DNA 或 RNA 的偏好相关的基序和构象变异。这些数据一起对 PUA 样域特异性具有重要意义，并表明 McrBC 家族比以前描述的具有更广泛的生物学多功能性。