Complementation between dimeric mutants as a probe of dimer-dimer interactions in tetrameric dihydrofolate reductase encoded by R67 plasmid of E. coli.

The effect of mutations on the interactions between dimers in R67 dihydrofolate reductase (R67 DHFR), a tetrameric enzyme conferring resistance to trimethoprim, was investigated by site-directed mutagenesis combined with phenotypic, enzymatic, and biochemical analysis. Some 14 mutants at two positions involved in a hydrogen bond between dimers were constructed. All were shown to be dimers. However, complementation between pairs of dimeric mutated proteins resulted in the restoration of the enzymatic activity and heterotetramer formation. A combinatorial approach was set up to create efficiently such heterotetramers and identify the complementing pairs of mutations. A dozen of such pairs were found. An accurate method was set up to measure the association of the complementing dimers in a "quasi-isologous" heterotetramer and used to study the effects of mutations and pH on the association. Thus, the pair of proteins bearing respectively the S59A and H62L mutations was shown to form heterotetramers with catalytic properties close to those of the wild-type protein. Its association was as strong as that of the wild-type protein at cytoplasmic pH (6. 5), and was more stable at lower pH values.A double-mutant protein bearing simultaneously the S59A and H62L mutations was produced and analyzed. Its association was weakened by 1.2 kcal/mol as compared to the wild-type enzyme at pH 6.5 but was insensitive to pH. Comparing the energy of association between dimers in the wild-type protein, the heterotetramer and the double mutant allowed us to dissect the effects of the pH and of the molecular context on a subset of interactions between the R67 DHFR subunits.