The crystal structure of a tetrahydrofolate-bound dihydrofolate reductase reveals the origin of slow product release

Hongnan Cao,Hongyi Zhou,Mu Gao,Jeffrey Skolnick

doi:10.1038/s42003-018-0236-y

Hongnan Cao, Hongyi Zhou + Show 2 more

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https://doi.org/10.1038/s42003-018-0236-y

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Journal: Communications Biology	Publication Date: Dec 1, 2018
Citations: 26	License type: open-access

Affiliation: Georgia Institute of Technology

Abstract

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 Escherichia coli 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 E. coli. 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 E. coli 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.

Highlights

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 structure of the Escherichia coli DHFR (eDHFR):FH4 binary complex was determined by molecular replacement using the eDHFR:Folate:NADP+ closed ternary complex (PDB ID: 7DFR)[50]
We have determined the structural basis for the rate-limiting product release of the eDHFR:FH4 binary complex

Summary

Introduction

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. 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 determine another occluded complex structure of E. coli 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. We captured a distinct occluded conformation of eDHFR in complex with AMPQD, which to our knowledge is a novel slow-onset nanomolar inhibitor[46] This contrasts with eDHFR’s complex with the FDA-approved antifolate drug methotrexate, where eDHFR predominantly adopts the closed conformation[24,47,48,49]. We propose a strategy to explore alternative potent inhibitors of DHFR enzymes by targeting their FH4 related, occluded conformational states

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