Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan.
Arid Land Research Center, Tottori University, Tottori, Japan.
PLoS One. 2020 May 29;15(5):e0233689. doi: 10.1371/journal.pone.0233689. eCollection 2020.
Nucleoside monophosphate kinases play crucial roles in biosynthesis and regeneration of nucleotides. These are bi-substrate enzymes that catalyze reversible transfers of a phosphoryl group between ATP and nucleoside monophosphate. These enzymes are comprised of the CORE domain, the NMP-binding domain, and the LID domain. Large conformational rearrangement of the three domains occurs during the catalytic cycle. Although many structures of CMP kinase have been determined, only limited structural information has been available on the conformational changes along the reaction pathway. We determined five crystal structures of CMP kinase of Thermus thermophilus HB8 in ligand-free form and the CMP "open", CMP "closed", ADP-CDP-Gd3+-, and CDP-bound forms at resolutions of 1.7, 2.2, 1.5, 1.6, and 1.7 Å, respectively. The ligand-free form was in an open conformation, whereas the structures of the CMP "closed", ADP-CDP-Gd3+-, and CDP-bound forms were in a closed conformation, in which the shift of the NMP-binding domain and LID domain caused closure of the substrate-binding cleft. Interestingly, the CMP "open" form was in an open conformation even with CMP bound, implying intrinsic conformational fluctuation. The structure of the ADP-CDP complex is the first structure of CMP kinase with a phosphoryl group donor and an acceptor. Upon simultaneous binding of ADP and CDP, the side chains of several residues in the LID domain moved toward the nucleotides without global open-closed conformational changes compared to those in the CMP "closed" and CDP complexes. These global and local conformational changes may be crucial for the substrate recognition and catalysis. The terminal phosphate groups of ADP and CDP had similar geometry to those of two ADP in AMP kinase, suggesting common catalytic mechanisms to other nucleoside monophosphate kinases. Our findings are expected to contribute to detailed understanding of the reaction mechanism of CMP kinase.
核苷酸单磷酸激酶在核苷酸的生物合成和再生中起着至关重要的作用。这些是双底物酶，可催化 ATP 和核苷酸单磷酸之间磷酸基的可逆转移。这些酶由核心结构域、NMP 结合结构域和 LID 结构域组成。在催化循环中，三个结构域会发生大的构象重排。尽管已经确定了许多 CMP 激酶的结构，但沿反应途径的构象变化的结构信息有限。我们确定了 Thermus thermophilus HB8 的 CMP 激酶的五个晶体结构，分别为无配体形式和 CMP“打开”、CMP“关闭”、ADP-CDP-Gd3+、和 CDP 结合形式，分辨率分别为 1.7、2.2、1.5、1.6 和 1.7 Å。无配体形式呈开放构象，而 CMP“关闭”、ADP-CDP-Gd3+和 CDP 结合形式呈封闭构象，其中 NMP 结合结构域和 LID 结构域的移位导致底物结合裂隙的闭合。有趣的是，即使结合了 CMP，CMP“打开”形式也呈开放构象，这表明存在固有构象波动。ADP-CDP 复合物的结构是第一个具有磷酸供体和受体的 CMP 激酶结构。在 ADP 和 CDP 同时结合时，与 CMP“关闭”和 CDP 复合物相比，LID 结构域中的几个残基的侧链向核苷酸移动，而没有全局打开-关闭构象变化。这些全局和局部构象变化可能对底物识别和催化至关重要。ADP 和 CDP 的末端磷酸基团的几何形状与 AMP 激酶中两个 ADP 的相似，这表明与其他核苷酸单磷酸激酶具有共同的催化机制。我们的发现有望有助于详细了解 CMP 激酶的反应机制。
