Structural basis for the regulation of human 5,10-methylenetetrahydrofolate reductase by phosphorylation and S-adenosylmethionine inhibition

Abstract

The folate and methionine cycles are crucial for biosynthesis of lipids, nucleotides and proteins, and production of the methyl donor S-adenosylmethionine (SAM). 5,10-methylenetetrahydrofolate reductase (MTHFR) represents a key regulatory connection between these cycles, generating 5-methyltetrahydrofolate for initiation of the methionine cycle, and undergoing allosteric inhibition by its end product SAM. Our 2.5 Å resolution crystal structure of human MTHFR reveals a unique architecture, appending the well-conserved catalytic TIM-barrel to a eukaryote-only SAM-binding domain. The latter domain of novel fold provides the predominant interface for MTHFR homo-dimerization, positioning the N-terminal serine-rich phosphorylation region near the C-terminal SAM-binding domain. This explains how MTHFR phosphorylation, identified on 11 N-terminal residues (16 in total), increases sensitivity to SAM binding and inhibition. Finally, we demonstrate that the 25-amino-acid inter-domain linker enables conformational plasticity and propose it to be a key mediator of SAM regulation. Together, these results provide insight into the molecular regulation of MTHFR.

Fig. 1

Schematic representation of MTHFR. Domain organization of MTHFR orthologs across evolution. Numbers given represent approximate amino acid boundaries in human MTHFR corresponding to NP_005948. In brackets is shown representative species within each category

Fig. 2

Phosphorylation status of HsMTHFR_1–656 and HsMTHFR_38–644. a Phosphorylation mapping of HsMTHFR_1–656. The protein sequence is given as amino acids in single letter code, including the C-terminal His/flag-tag (underlined). Black font represents amino acids identified by the mass spectrometer (covered), blue font represents amino acids not identified (non-covered), red font represents phosphorylated amino acids. Domains are coloured as in Fig. 1. b Dephosphorylation of HsMTHFR_1–656 following treatment with CIP. Treatment time at 37 °C is given. Large number above peaks represents number of phosphate groups attached. Proteins were analysed by denaturing mass spectrometry. amu, atomic mass units. c Native mass spectrometry analysis of HsMTHFR_1–656 before and after treatment with CIP. Upper panel: as purified (untreated) protein. Monomer represents protein bound to 1 FAD plus 10 phosphate groups (expected mass: 76831.16 amu); dimer represents protein bound to 2 FADs and 1 SAM plus 20 phosphate groups (expected mass: 154060.74 amu). Lower panel: protein following 180 min treatment with CIP. Monomer represents protein bound to 1 FAD (expected mass: 76031.16 amu); dimer represents protein bound to 2 FADs and 1 SAH (expected mass: 152446.74 amu). Expected sizes: protein without first methionine, 75245.6 amu; FAD, 785.56 amu; SAM, 398.44 amu; SAH, 384.42 amu, phosphate, 80.00 amu. * indicates a truncated protein representing amino acids 353–663 (expected mass: 36136.6 amu). d HsMTHFR_38–644 before and after treatment with CIP. Treatment time at 37 °C is given. Protein was analysed by denaturing mass spectrometry. e Native mass spectrometry of HsMTHFR_1–656 identifying sequential binding of SAM or SAH. Graphs represent areas zoomed in on dimeric protein. Upper panel: As purified (untreated) protein. Middle panel: control protein (heated in assay buffer for 180 min without CIP). Bottom panel: treated protein (heated in assay buffer for 180 min with CIP). Expected size of protein with 2 FAD bound and 20 phosphates: 153662.3. Expected size of protein with 2 FAD bound and 0 phosphates: 152062.32. Expected size of SAM: 398.44, SAH: 384.41

Fig. 3

SAM inhibition of HsMTHFR_1–656 and HsMTHFR_38–644. Inhibition of MTHFR catalytic activity following pre-incubation with various concentrations of SAM. Remaining activity represents percentage of activity compared to MTHFR incubated without SAM. Inset: Replot of percent activity remaining against SAM concentration transformed by log10 to reveal differences between truncated (HsMTHFR_38–644) and dephosphorylated full-length (HsMTHFR_1–656 CIP) protein with phosphorylated full-length protein (HsMTHFR_1–656; HsMTHFR_1–656 mock). Inhibitory constants (K_i) for SAM calculated from this graph were calculated as described in Methods and are provided in Table 1. Each value represents the results of at least three separate experiments and is given as ±S.D

Fig. 4

Structural overview of HsMTHFR. a Orthogonal views of HsMTHFR showing the catalytic domain (cyan), the linker (red) and the regulatory domain (yellow). Bound FAD (green) and SAM (pink) are shown in sticks. Dotted lines indicate disordered regions that are not modelled in the structure. α-Helices and β-sheets are labelled, and correspond to the multiple sequence alignment in Supplementary Figs. 13 and 15 and the topology depicted in Supplementary Fig. 6. b Homodimer of HsMTHFR as seen in the crystal. Chain A represents the more ordered protomer. In chain B, the part of the catalytic domain that was poorly ordered is represented in dark blue and only the main chain was modelled. The FAD in this subunit was also partially disordered. c Juxtaposition of the N-terminus of HsMTHFR protomer towards both regulatory domains of the homodimer. The first residue observed in the structure, Glu40 of chain A, is shown in black spheres. Other coloured features are as described for panel a

Fig. 5

SAXS analysis of HsMTHFR_38–644 and HsMTHFR_1–656 phosphorylated and dephosphorylated. SAXS analysis of HsMTHFR_38–644. Experimental scattering profile is shown in black, theoretical scattering curve of the HsMTHFR_38–644 dimer observed in the crystal is in green and that of the rigid body modelling by CORAL is in red. Chi² was determined by CRYSOL

Fig. 6

Structural examination of the HsMTHFR_38–644 catalytic domain. a Structural alignment of HsMTHFR_38–644 (cyan) with EcMTHFR (grey) and ScMET12_1–301. Four sites of important differences are indicated by arrows (1–4). α-helices of HsMTHFR_38–644 are indicated for orientation. b Binding pocket of FAD. FAD is shown in green sticks, residues contributing to FAD binding are labelled and shown in black sticks. c Binding pocket of NAD(P)H. NADH is taken from an overlay of EcMTHFR (PDB: IZRQ) with HsMTHFR_38–644 but for clarity EcMTHFR is not shown. FAD is shown in green sticks, NADH in brown sticks, residues expected to contribute to NADH binding are labelled and shown in black sticks. d Binding pocket of CH₃–THF. CH₃–THF is taken from an overlay of EcMTHFR (PDB: 2FMN) with HsMTHFR_38–644 but for clarity EcMTHFR is not shown. FAD is shown in green sticks, CH₃-THF in yellow sticks, residues expected to contribute to CH₃-THF binding are labelled and shown in black sticks

Fig. 7

SAH/SAM binding and conformational change. a The SAH binding site. Amino acids that contribute to binding are labelled and shown in black sticks. SAH is shown in green sticks. b Size exclusion chromatography of HsMTHFR with various N-terminal truncations following incubation with SAM (dashed lines), SAH (dotted lines), or buffer (apo; solid line). For each N-terminally truncated construct, the corresponding structure is shown. c Size exclusion chromatography of HsMTHFR_348–656 proteins without (wt) or with (wt-SAM) pre-incubation with SAM. Mutated HsMTHFR_348–656 proteins were pre-incubated with SAM