Molecular Neuropharmacology and Genetics Laboratory, Department of Neuroscience, Faculty of Health and Medical Sciences, The Panum Institute, University of Copenhagen, Copenhagen, Denmark; Structural Biology and NMR Laboratory, Department of Neuroscience, University of Copenhagen, Copenhagen, Denmark
Thor Seneca Thorsen
Molecular Neuropharmacology and Genetics Laboratory, Department of Neuroscience, Faculty of Health and Medical Sciences, The Panum Institute, University of Copenhagen, Copenhagen, Denmark
Georges Vauquelin
Molecular and Biochemical Pharmacology, Department of Biotechnology, Free University Brussels (VUB), Brussels, Belgium
Ina Ammendrup-Johnsen
Molecular Neuropharmacology and Genetics Laboratory, Department of Neuroscience, Faculty of Health and Medical Sciences, The Panum Institute, University of Copenhagen, Copenhagen, Denmark
Volker Wirth
Bionanotechnology and Nanomedicine Laboratory, Department of Chemistry, Nano-science Center, University of Copenhagen, Copenhagen, Denmark
Karen L Martinez
Bionanotechnology and Nanomedicine Laboratory, Department of Chemistry, Nano-science Center, University of Copenhagen, Copenhagen, Denmark
Structural Biology and NMR Laboratory, Department of Neuroscience, University of Copenhagen, Copenhagen, Denmark
Ulrik Gether
Molecular Neuropharmacology and Genetics Laboratory, Department of Neuroscience, Faculty of Health and Medical Sciences, The Panum Institute, University of Copenhagen, Copenhagen, Denmark
Molecular Neuropharmacology and Genetics Laboratory, Department of Neuroscience, Faculty of Health and Medical Sciences, The Panum Institute, University of Copenhagen, Copenhagen, Denmark
PDZ domain scaffold proteins are molecular modules orchestrating cellular signalling in space and time. Here, we investigate assembly of PDZ scaffolds using supported cell membrane sheets, a unique experimental setup enabling direct access to the intracellular face of the cell membrane. Our data demonstrate how multivalent protein-protein and protein-lipid interactions provide critical avidity for the strong binding between the PDZ domain scaffold proteins, PICK1 and PSD-95, and their cognate transmembrane binding partners. The kinetics of the binding were remarkably slow and binding strength two-three orders of magnitude higher than the intrinsic affinity for the isolated PDZ interaction. Interestingly, discrete changes in the intrinsic PICK1 PDZ affinity did not affect overall binding strength but instead revealed dual scaffold modes for PICK1. Our data supported by simulations suggest that intrinsic PDZ domain affinities are finely tuned and encode specific cellular responses, enabling multiplexed cellular functions of PDZ scaffolds.
