Improved affinity at the cost of decreased specificity: a recurring theme in PDZ-peptide interactions.

O Andreas Karlsson,Per Jemth,Eva Andersson,Ylva Ivarsson,Gustav N Sundell

doi:10.1038/srep34269

Abstract

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.

Highlights

Persistent infection by high-risk human papillomavirus (HPV) can lead to cancer[1]
We focus on the second PDZ domain of SAP97 that has a preference for RETxV containing peptides[15,21], which matches the C-terminal motif of the HPV18 E6 C-terminus (RRRETQV)
We used the precision of stopped-flow spectroscopy to address (i) the specificity for the selected peptides benchmarked with a peptide corresponding to the C-terminus of HPV18 E6, and (ii) the presence of energetic coupling (‘communication’) between the residues at positions 391 and 392 in PDZØ9

Summary

Introduction

Persistent infection by high-risk human papillomavirus (HPV) can lead to cancer[1]. This is an indirect consequence of the combined actions of the HPV E6 and E7 proteins that are expressed during productive infection[2]. We focus on the second PDZ domain of SAP97 that has a preference for RETxV containing peptides[15,21], which matches the C-terminal motif of the HPV18 E6 C-terminus (RRRETQV). The L391F mutation is located in the p0 hydrophobic binding pocket, and is expected to make direct contacts with bound peptide This position is known to contribute to the specificity of PDZ domains for the p0 residue[25], while the significance of the K392 mutation is less obvious. A double mutant cycle analysis on PDZØ9 reveals a peptide-dependent energetic coupling between the two mutated residues, situated in the second alpha helix of PDZØ9 These results are in line with previous experiments suggesting dynamic, rather than conserved energetic networks in the PDZ domain family[26,27]

Methods

Results

Conclusion