Polar Interactions at the Dimer-Dimer Interface of Methionine Adenosyltransferase MAT I Control Tetramerization

Catalytic MAT alpha 1 subunits associate into kinetically distinct homo-dimers (MAT III) and homo-tetramers (MAT I) that synthesize S-adenosylmethionine in the adult liver. Pathological reductions in S-adenosylmethionine levels correlate with MAT III accumulation; thus, it is important to know the determinants of dimer-dimer associations. Here, polar interactions (<3.5 angstrom) at the rat MAT I dimer-dimer interface were disrupted by site-directed mutagenesis. Heterologous expression rendered decreased soluble mutant MAT alpha 1 levels that appeared mostly as dimers. Substitutions at the B1-B2 or B3-C1 beta-strand loops, or changes in charge on helix alpha 2 located behind, induced either MAT III or MAT I accumulation. Notably, double mutants combining neutral changes on helix alpha 2 with substitutions at either beta-strand loop further increased MAT III content. Mutations had negligible impact on secondary or tertiary protein structure, but induced changes of 5-10 degrees C in thermal stability. All mutants preserved tripolyphosphatase activity, although AdoMet synthesis was only detected in single mutants. Kinetic parameters were altered in all purified proteins, their AdoMet synthesis V-max and methionine affinities correlating with the association state induced by the corresponding mutations. In conclusion, polar interactions control MAT alpha 1 tetramerization and kinetics, diverse effects being induced by changes on opposite beta-sheet loops putatively leading to subtle variations in central domain beta-sheet orientation.