Figure 1.
Production of recombinant proteins and purification steps.
The upper part of the figure shows a scheme of the purification steps followed to prepare the three types of hetero-oligomers obtained in this work. The numbers indicate the corresponding lane of the representative stained gels shown below. Panel A shows a representative SDS-PAGE gel used for molecular mass estimation, including two sets of standards as indicated in the Materials and Methods section. Samples (30 µl) of key purification steps to produce wild type (B), mutated (C) and truncated MAT II (D) were prepared under standard reducing conditions for SDS-PAGE electrophoresis. Gel lanes correspond to: (1) refolded α2 (10 µg); (2) Q-Sepharose collected peak (10 µg); (3) concentrated Q-Sepharose peak (25 µg); (4) standards; (5) purified hetero-oligomer (10 µg); and (6) purified wild type (10 µg), mutant (5 µg) or truncated β subunit (10 µg). Panels B–D show only the relevant sections of the stained gels for each type of purification; the positions for the 45 and 31 kDa protein standards are indicated on the side of the gels. Dots indicate places where gel lanes have been cropped for clarity.
Figure 2.
MAT α2 sequence identified by peptide mass fingerprint using LC-MS/MS.
The figure shows the amino acid sequence of human MAT α2, where the peptides identified by mass spectrometry are indicated. Blue squares highlight the two mutations detected in the ORF (underlined). Pink squares indicate the peptides identified with high scores in both α2 and α2′ bands. The green square indicates a peptide identified in both bands, but showing higher score for α2′ than for the α2 band. The sequence coverage is ∼50% of the total protein sequence.
Table 1.
Results of peptide mass fingerprint of MAT α2 bands.
Figure 3.
Analytical gel filtration chromatography of MAT proteins.
The figure shows gel filtration chromatography profiles of purified MAT forms followed by activity measurements (•) and densitometric scanning of Dot Blots incubated with anti-MATα2 (▪). The elution behavior of the α2 subunit is depicted in panels A (50 µg) and B (200 µg), and that of the MAT II hetero-oligomer including the wild type β subunit appears in panel C. Panel D shows a representative regression line for the standards used in column calibration [log Mr = (−2.085×KAV)+5.903]. The elution volume of representative standards is indicated by a vertical bar and specified in the Materials and Methods section.
Table 2.
MAT activity data of α2 homo- and hetero-oligomers.
Table 3.
Methionine kinetics of α2 homo- and hetero-oligomers.
Table 4.
ATP kinetics for α2 homo- and hetero-oligomers.
Figure 4.
Gel filtration chromatography of the β subunit.
The purified wild type β subunit was analyzed on a Superdex 200 16/60 gel filtration column and the elution followed by A280. Vertical bars indicate the elution position of relevant standards. The elution volume of all the markers used was: dextran blue (2000 kDa) 39.4 ml; apoferritin (443 kDa) 52.3 ml; β-amylase (200 kDa) 59.5 ml; alcohol dehydrogenase (150 kDa) 64.3 ml; bovine serum albumin (66.2 kDa) 71.6 ml; and cytochrome c (12.4 kDa) 93 ml.
Figure 5.
Comparison of the structures of human β subunits in free and NADP+ bound states.
The figure depicts the structure of free β subunits (white; 2YDY) and that of the NADP+ bound state (red; 2YDX). Cofactor and resveratrol molecules are shown as sticks with coloured carbon (black), nitrogen (red), phosphorus (orange) and oxygen (blue) atoms. Three regions not visible in the apoenzyme and ordered upon NADP+ binding are highlighted: F60-A77 (A), A95-N113 (B), and D325-F333 (C); only A and B regions are linked to NADP+. On the right the surface of the β dimer is shown as found in the NADP+ bound crystal structure, illustrating location of A and C regions in the dimer interface.
Figure 6.
Isothermal titration calorimetry of MAT subunits in the presence or absence of NADP+.
The figure shows representative titration experiments carried out with the wild type regulatory β subunit to characterize binary and tertiary binding with α2 subunits and/or NADP+. Panel A depicts titration of α2 (13.4 µM in the cell) and β subunits (190 µM in the syringe). Panel B shows titration of the β subunit (9.8 µM in the cell) and NADP+ (155 µM in the syringe); the insets show titrations using the β subunit (20 µM) and 300 µM NAD+ (top) and the Y159F/K163A-β subunit (20 µM) and 330 µM NADP+ (bottom). The very low affinity observed for the mutant precluded a precise estimation of the binding affinity, and hence only a lower limit for the dissociation constant could be determined. Panel C illustrates titration of α2 (4.3 µM in the cell) and β subunits (62.4 µM in the syringe) in the presence of NADP+ (300 µM in both the cell and the syringe). All measurements were performed at 25°C as described in the Materials and Methods section.
Table 5.
Thermodynamic parameters for MAT II interactions.
Figure 7.
Sequence comparison of the β subunit (V1 form) with relevant members of the RED family.
The figure shows an alignment of sequences for Dtdp-6-deoxy-L-lyxo-4-hexulose reductase (1N2S), Dtdp-glucose 4,6-dehydratase (1BXK) and the β regulatory subunit of MAT II. Conserved residues of the GXXGXXG phosphate binding motif and the catalytic triad (SXNYXXXK) appear in blue.
Figure 8.
The NADP+ binding site in the β subunit.
The figure shows the structure of the NADP+ site with the protein residues shown as wheat sticks. Colour codes are as in Figure 4. Panel A depicts recognition of the phosphate moieties, showing that segment A is involved in NADP+/NAD+ discrimination by linking the additional phosphate group. This phosphate is linked to R62 (region A) which establishes a strong network of atomic interactions that connects to R97 (region B), conforming the NADP+ binding site. Panel B shows a detail of the residues that bind NADP+ and have been mutated in this work.
Figure 9.
Structural model of the MAT II trimer.
A putative model of the MAT II hetero-oligomer was obtained with ClusPro using available coordinates for α2 (2P02) and β (2YDX) subunits. The α2 subunits in the dimer are shown as cream and black cartoons. Each α2 monomer consists of N-terminal, central and C-terminal domains, both subunits being related by a 2-fold symmetry axis. The β subunit is represented as a green surface, with A, B and C regions highlighted in red. The hetero-oligomer interface is formed by central domains of both α2 monomers and regions A, B and C from the regulatory β subunit. Only models displaying β interacting to α2 central domains, therefore located on its 2-fold axis, would be consistent with a 2∶1 stoichiometry in the hetero-oligomer.
Figure 10.
Sequence alignment of regulatory subunit splicing forms.
Sequences of the four splicing forms reported to date for the regulatory β subunit of MAT II in hepatoma cells were aligned against the most abundant V1 form (334 residues). The figure shows underlined the residues truncated in the ΔS16 protein used in this study. In addition, several differences among splicing forms are also indicated: the N-terminal differences (orange), the sequence lacking in V2a (pink) and V2b (green), and the putative NADP+ binding motifs (blue).