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

María Ángeles Pajares,Gabino Francisco Sánchez-Pérez

doi:10.3390/ijms222413206

Abstract

Catalytic MATα1 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 Å) at the rat MAT I dimer–dimer interface were disrupted by site-directed mutagenesis. Heterologous expression rendered decreased soluble mutant MATα1 levels that appeared mostly as dimers. Substitutions at the B1–B2 or B3–C1 β-strand loops, or changes in charge on helix α2 located behind, induced either MAT III or MAT I accumulation. Notably, double mutants combining neutral changes on helix α2 with substitutions at either β-strand loop further increased MAT III content. Mutations had negligible impact on secondary or tertiary protein structure, but induced changes of 5–10 °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 Vmax and methionine affinities correlating with the association state induced by the corresponding mutations. In conclusion, polar interactions control MATα1 tetramerization and kinetics, diverse effects being induced by changes on opposite β-sheet loops putatively leading to subtle variations in central domain β-sheet orientation.

Highlights

S-adenosylmethionine (AdoMet) is a promiscuous compound able to participate in a large variety of reactions that are estimated to be as numerous as those involving ATP
Q81, E112 and N106 established two such polar interactions each (Table 1). All these residues concentrated into the 29–116 stretch, which is extremely conserved between rat and human MATα1 sequences (Figure 1E), and that together with the 255–289 segment, constitute the central domain of the monomer [15]
Comparison with SSRL activity data showed that only the Q81T mutant attained similar AdoMet synthesis levels, whereas reductions above 75% were exhibited by the rest of the mutants. These results suggested the preservation of AdoMet synthesis in all the mutants, the real impact of the substitutions could not be ascertained in these calculations which used the total protein content

Summary

Introduction

S-adenosylmethionine (AdoMet) is a promiscuous compound able to participate in a large variety of reactions that are estimated to be as numerous as those involving ATP. Liberation of the methyl donor from the active site requires PPPi hydrolysis into pyrophosphate and inorganic phosphate These two activities can be independently measured, a fact that has enabled the characterization of some mutations in the MAT family, leading to proteins with decreased or erased AdoMet synthesis activity, while tripolyphosphatase (PPPase) activity is preserved [4,5,6]. Some of these mutants were identified in the 1970s during newborn screenings for persistent hypermethioninemia (>43 μM methionine in plasma), named Mudd’s disease [4]

Methods

Results

Conclusion