Protein-protein interaction: a genetic selection for compensating mutations at the barnase-barstar interface.

Barnase and barstar are trivial names of the extracellular RNase and its intracellular inhibitor produced by Bacillus amyloliquefaciens. Inhibition involves the formation of a very tight one-to-one complex of the two proteins. With the crystallographic solution of the structure of the barnase-barstar complex and the development of methods for measuring the free energy of binding, the pair can be used to study protein-protein recognition in detail. In this report, we describe the isolation of suppressor mutations in barstar that compensate for the loss in interaction energy caused by a mutation in barnase. Our suppressor search is based on in vivo selection for barstar variants that are able to protect host cells against the RNAse activity of those barnase mutants not properly inhibited by wild-type barstar. This approach utilizes a plasmid system in which barnase expression is tightly controlled to keep the mutant barnase gene silent. When expression of barnase is turned on, failure to form a complex between the mutant barnase and barstar has a lethal effect on host cells unless overcome by substitution of the wild-type barstar by a functional suppressor derivative. A set of barstar suppressors has been identified for barnase mutants with substitutions in two amino acid positions (residues 102 and 59), which are critically involved in both RNase activity and barstar binding. The mutations selected as suppressors could not have been predicted on the basis of the known protein structures. The single barstar mutation with the highest information content for inhibition of barnase (H102K) has the substitution Y30W. The reduction in binding caused by the R59E mutation in barnase can be partly reversed by changing Glu-76 of barstar, which forms a salt bridge with the Arg-59 in the wild-type complex, to arginine, thus completing an interchange of the two charges.