J. Dam et al., Complementation between dimeric mutants as a probe of dimer-dimer interactions in tetrameric dihydrofolate reductase encoded by R67 plasmid of E-coli, J MOL BIOL, 302(1), 2000, pp. 235-250
The effect of mutations on the interactions between dimers in R67 dihydrofo
late reductase (R67 DHFR), a tetrameric enzyme conferring resistance to tri
methoprim, was investigated by site-directed mutagenesis combined with phen
otypic, 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 be
tween pairs of dimeric mutated proteins resulted in the restoration of the
enzymatic activity and heterotetramer formation. A combinatorial approach w
as set up to create efficiently such heterotetramers and identify the compl
ementing pairs of mutations. A dozen of such pairs were found.
An accurate method was set up to measure the association of the complementi
ng dimers in a "quasi-isologous" heterotetramer and used to study the effec
ts of mutations and pH on the association. Thus, the pair of proteins beari
ng respectively the S59A and H62L mutations was shown to form heterotetrame
rs with catalytic properties close to those of the wild-type protein. Its a
ssociation 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 effect
s of the pH and of the molecular context on a subset of interactions betwee
n the R67 DHFR subunits. (C) 2000 Academic Press.