Guiding a docking mode by phage display: selection of correlated mutations at the staphylokinase-plasmin interface

• Published in: Journal of molecular biology 1999-07, Vol.290 (2), p.471-479
• Main Authors: Jespers, Laurent, Lijnen, H.Roger, Vanwetswinkel, Sophie, Van Hoef, Berthe, Brepoels, Katleen, Collen, Désiré, De Maeyer, Marc
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Bacteria - enzymology; Bacteriophages - genetics; Binding Sites; Catalysis; Catalytic Domain; correlated mutations; dead-end elimination; Fibrinolysin - chemistry; Fibrinolysin - genetics; Fibrinolysin - metabolism; Humans; Inhibitory Concentration 50; Kinetics; Metalloendopeptidases - chemistry; Metalloendopeptidases - genetics; Metalloendopeptidases - metabolism; Models, Molecular; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Fragments - metabolism; Peptide Library; phage display; Plasminogen - chemistry; Plasminogen - genetics; Plasminogen - metabolism; plasminogen activation; Plasminogen Activators - chemistry; Plasminogen Activators - genetics; Plasminogen Activators - metabolism; staphylokinase; Static Electricity; Suppression, Genetic - genetics; Thermodynamics
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.1999.2887

During co-evolution of interacting proteins, functionally disruptive mutations on one side of the interface may be compensated by local amino acid changes on the other to restore binding affinity. This information can be useful for geometry-based docking approaches by reducing the translational and rotational space available to the proteins. Here, we demonstrate that correlated 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 interacting partner. This approach was used to deduce the binding mode of staphylokinase (Sak), a 15.5 kDa “indirect” plasminogen activator on microplasmin (μPli), the 28 kDa serine protease domain of plasmin. Biopanning indicated that residues Arg94 and Gly174 in μPli are located in close proximity to Glu75 and the Glu88:Ile128 pair in Sak, respectively. The coupled mutations 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 ability of the mutated complexes were partially restored. These findings suggested a unique docking mode of Sak at the western rim of the active-site cleft of μPli, that is in agreement with the structure of the Sak-μPli complex as recently derived by other methods.