Cao Hongnan, Gao Mu, Zhou Hongyi, Skolnick Jeffrey
Center for the Study of Systems Biology, School of Biological Sciences, Georgia Institute of Technology, 950 Atlantic Drive, NW, Atlanta, GA 30332 USA.
Commun Biol. 2018 Dec 12;1:226. doi: 10.1038/s42003-018-0236-y. eCollection 2018.
Dihydrofolate reductase (DHFR) catalyzes the stereospecific reduction of 7,8-dihydrofolate (FH2) to (6s)-5,6,7,8-tetrahydrofolate (FH4) via hydride transfer from NADPH. The consensus DHFR mechanism involves conformational changes between closed and occluded states occurring during the rate-limiting product release step. Although the Protein Data Bank (PDB) contains over 250 DHFR structures, the FH4 complex structure responsible for rate-limiting product release is unknown. We report to our knowledge the first crystal structure of an . DHFR:FH4 complex at 1.03 Å resolution showing distinct stabilizing interactions absent in FH2 or related (6R)-5,10-dideaza-FH4 complexes. We discover the time course of decay of the co-purified endogenous FH4 during crystal growth, with conversion from FH4 to FH2 occurring in 2-3 days. We also determine another occluded complex structure of DHFR with a slow-onset nanomolar inhibitor that contrasts with the methotrexate complex, suggesting a plausible strategy for designing DHFR antibiotics by targeting FH4 product conformations.
二氢叶酸还原酶（DHFR）通过从烟酰胺腺嘌呤二核苷酸磷酸（NADPH）转移氢负离子，将7,8-二氢叶酸（FH2）立体定向还原为（6S）-5,6,7,8-四氢叶酸（FH4）。公认的DHFR机制涉及在限速产物释放步骤中发生的封闭状态和闭塞状态之间的构象变化。尽管蛋白质数据库（PDB）包含超过250种DHFR结构，但负责限速产物释放的FH4复合物结构尚不清楚。据我们所知，我们报道了首个分辨率为1.03Å的DHFR:FH4复合物晶体结构，该结构显示出在FH2或相关的（6R）-5,10-二脱氮-FH4复合物中不存在的独特稳定相互作用。我们发现了晶体生长过程中共纯化的内源性FH4的衰减时间进程，在2-3天内发生了从FH4到FH2的转化。我们还确定了DHFR与一种起效缓慢的纳摩尔抑制剂形成的另一种闭塞复合物结构，该结构与甲氨蝶呤复合物形成对比，这为通过靶向FH4产物构象设计DHFR抗生素提供了一种可行的策略。
