Toward resolving the catalytic mechanism of dihydrofolate reductase using neutron and ultrahigh-resolution X-ray crystallography

Dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent reduction of dihydrofolate (DHF) to tetrahydrofolate (THF). An important step in the mechanism involves proton donation to the N5 atom of DHF. The inability to determine the protonation states of active site residues and substrate has led to a lack of consensus regarding the catalytic mechanism involved. To resolve this ambiguity, we conducted neutron and ultrahigh-resolution X-ray crystallographic studies of the pseudo-Michaelis ternary complex of Escherichia coli DHFR with folate and NADP⁺. The neutron data were collected to 2.0-Å resolution using a 3.6-mm³ crystal with the quasi-Laue technique. The structure reveals that the N3 atom of folate is protonated, whereas Asp27 is negatively charged. Previous mechanisms have proposed a keto-to-enol tautomerization of the substrate to facilitate protonation of the N5 atom. The structure supports the existence of the keto tautomer owing to protonation of the N3 atom, suggesting that tautomerization is unnecessary for catalysis. In the 1.05-Å resolution X-ray structure of the ternary complex, conformational disorder of the Met20 side chain is coupled to electron density for a partially occupied water within hydrogen-bonding distance of the N5 atom of folate; this suggests direct protonation of substrate by solvent. We propose a catalytic mechanism for DHFR that involves stabilization of the keto tautomer of the substrate, elevation of the pK_a value of the N5 atom of DHF by Asp27, and protonation of N5 by water that gains access to the active site through fluctuation of the Met20 side chain even though the Met20 loop is closed.Dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NADP⁺ oxidoreductase) (DHFR) is a housekeeping enzyme that catalyzes the NADPH-dependent reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8,-tetrahydrofolate (THF). Various redox states of THF are used in several one-carbon transfer reactions to generate thymidine, methionine, glycine, serine, and other molecules (1–3). Given its role in biosynthesis, DHFR is a target for anticancer, antimicrobial, and rheumatoid arthritis drugs, such as methotrexate (MTX) and trimethoprim (4–7).Although the kinetics, structure, and biophysical properties of Escherichia coli DHFR (ecDHFR) have been well characterized, unresolved questions with respect to its catalytic mechanism remain (1, 3, 8–13), as evidenced by the recent controversy over whether millisecond time-scale structural fluctuations can directly affect the chemical step in catalysis (14, 15). Folate is a poor substrate for DHFR, whereas DHF is reduced more efficiently (2). In addition, unlike DHF, folate cannot be further oxidized in solution. Thus, the abortive DHFR-folate-NADP⁺ complex is an excellent mimic of the DHFR-DHF-NADPH Michaelis complex (3, 16), and its stability makes it well suited for structural studies.During catalysis, a proton is donated to the N5 atom of the DHF pterin ring and a hydride equivalent is transferred from NADPH to the C6 atom of the pterin. With folate as a substrate, proton donation occurs at the N8 atom (10). The five intermediates in the catalytic cycle are E-NADPH, E-NADPH-DHF, E-NADP⁺-THF, E-THF, and E-NADPH-THF (3), with product release as the rate-limiting step at neutral pH. THF is released on binding of a new NADPH molecule. The enzyme displays pH dependence with a characteristic pK_a value of 6.5 (8).Previous crystallographic and NMR studies of the DHFR binary and ternary complexes have revealed the locations of the folate and nicotinamide cofactor optimal for hydride transfer and the juxtaposition of the substrate with respect to the catalytic Asp27, which forms hydrogen bonds with the N3 and NA2 atoms of folate (3, 12). The DHFR-folate-NADP⁺ complex structure is considered the closest mimic of the Michaelis complex and has been used as a reference model in studies of the molecular details required for proton donation and hydride transfer (2, 3, 14, 17). Although it is clear from the structure that the nicotinamide ring is optimally positioned for hydride transfer to the C6 atom of the DHF substrate, how a proton can be donated to the N5 atom is unclear, especially considering that the conserved Asp27 is almost 5 Å distant from it. Disagreement abounds as to the protonation state of the Asp27 during catalysis (10, 18, 19). The mutation of the other residues contacting the substrate diminishes but does not abrogate activity, suggesting that the enzyme is flexible and has built-in redundancies (9, 20).Several catalytic mechanisms have been proposed based on X-ray and NMR structures, molecular dynamics, enzyme kinetic measurements, and Raman spectroscopy studies (1, 17, 18, 21). According to Maharaj et al. (21), the pK_a of the N5 atom of DHF is 2.6 in solution. When bound in a binary complex to DHFR, its N5 pK_a remains strongly acidic. However, the pK_a is elevated from <4 in the binary complex to 6.5 in the catalytic mimic complex, where NADP⁺ is bound as well (1, 21). This value matches the pK_a describing the hydride transfer step (8), suggesting that the kinetic pK_a describes the level of N5 protonated substrate available. The accompanying article by Liu et al. (22) further explores the kinetic pH profile for ecDHFR and its relationship to the hydride transfer step, as well as the sequential order of the mechanism. Outstanding questions remain, including, but not limited to, the following: (i) When a catalytically competent complex is present, how does the active site environment so drastically increase the N5 pK_a to promote protonation? (ii) What is the protonation state of Asp27 throughout catalysis? And (iii) what is the source and mechanism of proton donation to N5?An oft-proposed general mechanism based on several crystallography and Raman spectroscopy studies invokes a keto-enol tautomerization of the pterin substrate, initiating at the Asp27 and triggering a proton shuttle that ultimately results in a reduction of N5 (9, 10, 23). Two versions of this mechanism have been proposed, the major differences being the protonation state of Asp27 in the ground state and the ultimate proton source for reduction of N5 (SI Materials and Methods). The caveat regarding this mechanism is that keto-enol tautomerization as a critical step in the DHFR catalytic cycle remains a major point of ambiguity (24). Blakley et al. (25) have challenged the idea that substrate undergoes tautomerization during catalysis based on the NMR finding of a persistent substrate in an N3 imino-C4 keto tautomer across a pH range.Alternative catalytic mechanisms propose the direct involvement of water molecules in the proton transfer step. These mechanisms notably omit the necessity for a substrate tautomerization event and the requirement for protonation of Asp27 at some point in the catalytic cycle (17). Asp27 is mainly responsible for binding the substrate in a catalytically favorable conformation and maintaining a negative electrostatic field in the active site, which would be negated if its carboxylate were protonated even transiently. A recent study revealed that Met20 loop dynamics are critical for solvent access to N5, and proposed a mechanism involving direct solvent protonation of the substrate (22). There has been only one previous structural observation of a solvent molecule within hydrogen-bonding distance of the N5 atom, in a crystal structure of E. coli DHFR bound to folate and NADP⁺ (1RA2). It should be noted that the Met20 loop adopts an open conformation in this structure, likely because of crystal packing effects (3). A solvent molecule is typically modeled in this position in crystal structures with only substrate bound (2).A barrier to experimentally testing most proposed enzyme mechanisms is the inability to directly visualize the positions of important catalytic protons. The initial models used for most theoretical calculations are derived from X-ray structures, and determining the location of hydrogen atoms using X-rays is difficult even at atomic resolution (1.2 Å) (26). Neutron crystallography (NC) has a proven ability to determine the positions of hydrogen atoms or ions (protons) essential for catalysis (27–30). In fact, NC defines unique positions of hydrogen atoms within ordered water molecules (31), and H₃O⁺ molecules crucial for catalysis in xylose isomerase were recently identified (32). By virtue of the need to perform hydrogen/deuterium exchange (HDX) on crystals before data collection, NC can accurately identify hydrogen atom positions even at modest resolution. Deuterium coherently scatters neutrons with lengths similar to carbon and nitrogen, whereas hydrogen coherently scatters neutrons with negative lengths, rendering them invisible in positively contoured nuclear density maps. In the past, the determination of NC structures was hindered by the limited number of data collection facilities, low beam fluxes, and the requirement for extremely large crystals (>1 mm³ in volume). Recently, new spallation sources, enhanced deuterium labeling of samples, and improved detectors have allowed the collection of high-quality data from crystals of smaller volume, leading to a dramatic increase in the number of neutron structures deposited in the Protein Data Bank (PDB).In a previous NC study, we resolved a question pertaining to the protonation state of the classical antifolate inhibitor MTX and Asp27 when MTX binds DHFR (28). The DHFR-MTX neutron structure demonstrates that Asp27 is negatively charged, whereas the N1 atom of MTX is protonated and thus positively charged. After our initial success with the DHFR-MTX complex, we conducted NC studies of a DHFR pseudo-Michaelis complex to identify the protonation state of Asp27 in a catalytic mimic complex, the source of protonation for N5, as well as the presence (or absence) of a substrate keto-enol tautomerization event. Here we report the neutron diffraction structure of the DHFR-folate-NADP⁺ complex and complementary ultrahigh-resolution X-ray structures at three different temperatures. Refinement of the neutron structure allowed determination of the positions of crucial protons on the folate substrate and the ionization state of Asp27. Furthermore, our comprehensive map of backbone HDX sheds light on the dynamically driven changes in solvent accessibility of crystalline DHFR. The ultrahigh-resolution X-ray structures provide molecular details of Met20 loop fluctuations required for the entry of solvent, identifying a water molecule possibly involved in proton donation to N5.