Structural Characterization of Dihydrofolate Reductase Complexes by Top-Down Ultraviolet Photodissociation Mass Spectrometry
Structural Characterization of Dihydrofolate Reductase Complexes by Top-Down Ultraviolet Photodissociation Mass Spectrometry
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DOI：
10.1021/jacs.5b04628
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发表时间：
2015-07-22
影响因子：
15
通讯作者：
Brodbelt, Jennifer S.
中科院分区：
文献类型：
--
作者：
Cammarata, Michael B.;Thyer, Ross;Brodbelt, Jennifer S.
二氢叶酸逐步还原为四氢叶酸需要二氢叶酸还原酶（DHFR）的显著构象变化。含有辅因子NADPH、抑制剂甲氨蝶呤（MIX）或NADPH和MTX的DHFR二元和三元复合物的特征在于193 nm。紫外光解离（UVPD）质谱法。UVPD产生了超过80%的序列覆盖度的DHFR，并导致产生的碎片离子，揭示了DHFR和每个配体之间的相互作用。二元DHFR中心点NADPH和. DHFR中心点MTX复合物导致了前所未有数量的片段离子，其含有通过保留非共价相互作用仍与配体结合的N-或C-末端蛋白片段。此外，在三元DHFR中心点NADPH中心点MTX复合物的UVPD后观察到保留两种配体的全片段。广泛的卤素和载脂蛋白碎片离子的组合允许绘制NADPH和MTX配体的位置，其中NADPH与DHFR的腺苷结合结构域相关，MTX与环结构域相互作用。这些发现与以前的晶体学证据一致。比较载脂蛋白DHFR及其卤代对应物的骨架裂解倾向，发现UVPD的显著变化，预期在结合NADPH、MTX或两种配体后经历构象变化的区域中的片段化。特别是，子域旋转和环路运动，这被认为是发生在形成的过渡态的三元复合物，反映在UVPD质谱。UVPD光谱表明增强的骨架裂解的区域，变得更加灵活或显示抑制骨架裂解的配体屏蔽或参与新的分子内相互作用的那些区域。这项研究证实了193 nm UVPD质谱作为一种敏感的技术，跟踪酶循环，涉及构象重排的多功能性。
The stepwise reduction of dihydrofolate to tetrahydrofolate entails significant conformational changes of dihydrofolate reductase (DHFR). Binary and ternary complexes of DHFR containing cofactor NADPH, inhibitor methotrexate (MIX), or both NADPH and MTX were characterized by 193 nth. ultraviolet photodissociation (UVPD) mass spectrometry. UVPD yielded over 80% sequence coverage of DHFR and resulted in production of fragment ions that revealed the interactions between DHFR and each ligand. UVPD of the binary DHFR center dot NADPH and. DHFR center dot MTX complexes led to an unprecedented number of fragment ions containing either an N- or C-terminal protein fragment still bound to the ligand via retention of noncovalent interactions. In addition, holo-fragments retaining both ligands were observed upon UVPD of the ternary DHFR center dot NADPH center dot MTX complex. The combination of extensive halo and apo fragment ions allowed the locations of the NADPH and MTX ligands to be mapped, with NADPH associated with the adenosine binding domain of DHFR and MTX interacting with the loop domain. These findings are consistent with previous crystallographic evidence. Comparison of the backbone cleavage propensities for apo DHFR aria its halo counterparts revealed significant variations in UVPD,fragmentation in the regions expected to experience conformational changes upon binding NADPH, MTX, or both ligands. In particular, the subdomain rotation arid loop movements, which are believed to occur upon formation of the transition state of the ternary complex, are reflected in the UVPD mass spectra. The UVPD spectra indicate enhanced backbone cleavages in regions that become more flexible or show suppressed backbone cleavages for those regions either shielded by the ligand or involved in new intramolecular interactions. This study corroborates the versatility of 193 nm UVPD mass spectrometry as a sensitive technique to track enzymatic cycles that involve conformational rearrangements.