Abstract
Intragenic complementation is a unique property of oligomeric enzymes with which to study subunit-subunit interactions. Complementation occurs when different subunits, each possessing distinct mutations that render the individual homomutant proteins inactive, interact to form a heteromutant protein with partial recovery of activity. In this paper, complementation events between human argininosuccinate lyase (ASL) and its homolog, duck delta2 crystallin, were characterized. Different active site mutants in delta2 crystallin complement by the regeneration of native-like active sites as reported previously for ASL. The complementarity of the ASL and delta2 crystallin subunit interfaces was illustrated by the in vivo formation of active hybrid tetramers from inactive ASL and inactive delta2 crystallin mutants. Subunits of both ASL and delta2 crystallin do not dissociate and reassociate in vitro at room temperature, even after 6 days of incubation, indicating that the multimerization interface is very strong. However, disruption of a salt bridge network in the tetrameric interface of delta2 crystallin caused a drastic acceleration of subunit dissociation. Double mutants combining these interface mutants with active site mutants of delta2 crystallin were able to dissociate and reassociate to form active tetramers in vitro within hours. These results suggest that exchange of subunits may occur without unfolding of the monomer. Intragenic complementation in these interface mutants occurs by reintroducing the native salt bridge interaction upon hetero-oligomerization. Our studies demonstrate the value of intragenic complementation as a tool for investigating subunit-subunit interactions in oligomeric proteins.
Highlights
The majority of naturally occurring proteins exist as oligomers
argininosuccinate lyase (ASL)/␦ Crystallin Subunit-Subunit Interactions mentation can occur between two stable active site mutants by the formation of one or more functional native-like active sites or when a stable mutant subunit and an unstable mutant subunit form a hybrid protein with enough overall stability for catalysis to occur
Production of ␦2 Crystallin Mutant Heterotetramers in Vivo—To determine whether ␦2 crystallin could exhibit intragenic complementation by the regeneration of native-like active sites, plasmids were constructed to coexpress within the same E. coli cell different pairs of ␦2 crystallin genes with the amino acid substitutions, D87N, N114D, T159D, and S281T
Summary
The majority of naturally occurring proteins exist as oligomers. While surveying the Escherichia coli genome, Goodsell and Olsen [1] found that about 80% of the proteins were at least dimers if not higher order multimers. Intragenic complementation is a phenomenon that can occur between different mutant subunits within a multimeric enzyme. Complementation occurs when certain combinations of mutant alleles produce an enzyme with greater catalytic activity than is observed in the homozygous state of either mutant. The occurrence of intragenic complementation in well characterized multimeric enzymes provides a valuable tool to study the subunit-subunit interactions of proteins. There is ϳ70% sequence identity between human ASL and the various ␦ crystallins [13]. Both ␦ crystallin and ASL function as homotetramers with four identical multisubunit active sites (14 –18). By characterizing the effects of different pairs of mutations in ASL, two general mechanisms by which intragenic complementation can occur have been identified [20]. ASL/␦ Crystallin Subunit-Subunit Interactions mentation can occur between two stable active site mutants by the formation of one or more functional native-like active sites or when a stable mutant subunit and an unstable mutant subunit form a hybrid protein with enough overall stability for catalysis to occur
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