Guiding a docking mode by phage display: Selection of correlated mutationsat the staphylokinase-plasmin interface

During co-evolution of interacting proteins, functionally disruptive mutati ons on one side of the interface may be compensated by local amino acid cha nges on the other to restore binding affinity. This information can be usef ul for geometry-based docking approaches by reducing the translational and rotational space available to the proteins. Here, we demonstrate that corre lated mutations at a protein-protein interface can be rapidly identified by selecting a phage-displayed library of a randomly mutated component of the complex for complementation of mutations that decreased binding in the int eracting partner. This approach was used to deduce the binding mode of stap hylokinase (Sak), a 15.5 kDa "indirect" plasminogen activator on microplasm in (mu Pli), the 28 kDa serine protease domain of plasmin. Biopanning indic ated that residues Arg94 and Gly174 in mu Pli are located in close proximit y to Glu75 and the Glu88:Ile128 pair in Sak, respectively. The coupled muta tions Glu94 <----> Lys75 reversed and Gly174 <----> Lys88:Val128 introduced a salt bridge, whereby the binding affinities (with coupling energies of 1 .8 to 2.3 kcal mol(-1), respectively) and the plasminogen activation abilit y of the mutated complexes were partially restored. These findings suggeste d a unique docking mode of Sak at the western rim of the active-site cleft of mu Pli, that is in agreement with the structure of the Sak-mu Pli comple x as recently derived by other methods. (C) 1999 Academic Press.