Ligand-induced conformational changes in the crystal structures of Pneumocystis carinii dihydrofolate reductase complexes with folate and NADP+
Ligand-induced conformational changes in the crystal structures of Pneumocystis carinii dihydrofolate reductase complexes with folate and NADP+
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DOI：
10.1021/bi982728m
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发表时间：
1999-04-06
期刊：
影响因子：
2.9
通讯作者：
Queener, SF
中科院分区：
文献类型：
--
作者：
Cody, V;Galitsky, N;Queener, SF
来自叶酸（FA）二元复合物的两种独立晶体形式（P2（1）2（1）2（1）和P2（1））以及来自具有氧化型辅酶、NADP（+）和重组卡氏肺孢子虫二氢叶酸还原酶（pcDHFR）的三元复合物的结构数据精确到平均2.15埃分辨率，显示了配体诱导的pcDHFR结构构象变化的第一个证据。这些数据也比较了氨甲喋呤（MTX）与NADPH和pcDHFR的单斜晶格的数据，以2.5埃分辨率的三元复合物的晶体结构。pcDHFR的FA二元复合物的数据与三元结构的数据的比较揭示了显著的差异，其中残基23附近的环区域的>7埃移动导致二元复合物的新的“瓣打开”位置和三元复合物中的“闭合”位置，类似于大肠杆菌（ec）DHFR复合物所报道的。在二元FA pcDHFR复合物的正交晶格中，残基47附近的短螺旋区域也存在解旋，该区域将疏水残基Phe-46和Phe-49朝向外表面放置，这是通过分子间包装接触稳定的构象。与MTX-NADPH-pcDHFR三元复合物中观察到的相比，三元叶酸pcDHFR复合物中NADP+的焦磷酸盐部分在构象上显示出显著差异。此外，这四个pcDHFR结构之间的构象比较揭示了与辅因子结合状态相关的亚结构域运动的证据。在二元FA复合物的新的“瓣-开放”环23构象中较大的结合位点接近与催化期间辅因子从产物复合物的快速释放以及底物产物从二元复合物的更快速释放一致，这是由于与ecDHFR相比，闭环23构象的较弱接触。
Structural data from two independent crystal forms (P2(1)2(1)2(1) and P2(1)) of the folate (FA) binary complex and from the ternary complex with the oxidized coenzyme, NADP(+), and recombinant Pneumocystis carinii dihydrofolate reductase (pcDHFR) refined to an average of 2.15 Angstrom resolution, show the first evidence of ligand-induced conformational changes in the structure of pcDHFR. These data are also compared with the crystal structure of the ternary complex of methotrexate (MTX) with NADPH and pcDHFR in the monoclinic lattice with data to 2.5 Angstrom resolution. Comparison of the data for the FA binary complex of pcDHFR with those for the ternary structures reveals significant differences, with a >7 Angstrom movement of the loop region near residue 23 that results in a new "flap-open" position for the binary complex, and a "closed" position in the ternary complexes, similar to that reported for Escherichia coli (ec) DHFR complexes. In the orthorhombic lattice for the binary FA pcDHFR complex, there is also an unwinding of a short helical region near residue 47 that places hydrophobic residues Phe-46 and Phe-49 toward the outer surface, a conformation that is stabilized by intermolecular packing contacts. The pyrophosphate moiety of NADP+ in the ternary folate pcDHFR complexes shows significant differences in conformation compared with that observed in the MTX-NADPH-pcDHFR ternary complex. Additionally, comparison of the conformations among these four pcDHFR structures reveals evidence for subdomain movement that correlates with cofactor binding states. The larger binding site access in the new "flap-open" loop 23 conformation of the binary FA complex is consistent with the rapid release of cofactor from the product complex during catalysis as well as the more rapid release of substrate product from the binary complex as a result of the weaker contacts of the closed loop 23 conformation, compared to ecDHFR.