Improved affinity at the cost of decreased specificity: a recurring theme in PDZ-peptide interactions.
The E6 protein from human papillomavirus (HPV) plays an important role during productive infection and is a potential drug target. We have previously designed a high affinity bivalent protein binder for the E6 protein, a fusion between a helix from the E6 associated protein and PDZØ9, an engineered variant (L391F/K392M) of the second PDZ domain from synapse associated protein 97 (SAP97 PDZ2). How the substitutions improve the affinity of SAP97 PDZ2 for HPV E6 is not clear and it is not known to what extent they affect the specificity for cellular targets. Here, we explore the specificity of wild type SAP97 PDZ2 and PDZØ9 through proteomic peptide phage display. In addition, we employ a double mutant cycle of SAP97 PDZ2 in which the binding kinetics for nine identified potential cellular peptide ligands are measured and compared with those for the C-terminal E6 peptide. The results demonstrate that PDZØ9 has an increased affinity for all peptides, but at the cost of specificity. Furthermore, there is a peptide dependent coupling free energy between the side chains at positions 391 and 392. This corroborates our previous allosteric model for PDZ domains, involving sampling of intramolecular energetic pathways.
Department of Medical Biochemistry and Microbiology, Uppsala University, BMC Box 582, SE-75123 Uppsala, Sweden.
O Andreas
OA
Karlsson
Department of Chemistry-BMC, Uppsala University, Box 576, SE-751 23 Uppsala, Sweden.
Gustav N
GN
Sundell
Department of Medical Biochemistry and Microbiology, Uppsala University, BMC Box 582, SE-75123 Uppsala, Sweden.
Eva
E
Andersson
Department of Chemistry-BMC, Uppsala University, Box 576, SE-751 23 Uppsala, Sweden.
Ylva
Y
Ivarsson
Department of Medical Biochemistry and Microbiology, Uppsala University, BMC Box 582, SE-75123 Uppsala, Sweden.
Per
P
Jemth
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