Abstract
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.
Highlights
It is of fundamental importance for cell function to organize signaling processes in space and time
By quantitatively illuminating binding kinetics of PDZ domain protein scaffolds on a cell membrane, our results reveal novel principles for how cellular scaffold proteins can operate to ensure specificity and selectivity in cellular signaling networks, and how these principles change our current understanding of cellular binding equilibria and mechanisms underlying the function of scaffold proteins in general
The apparent affinity (Kd*) calculated from the binding curve was 67 ± 6 nM, which, remarkably, is ~100 fold higher than the low micromolar intrinsic affinity (Kdint)determined for binding of the GluA2 C-terminus to the PICK1 PDZ domain using an in-solution based assay (Erlendsson et al, 2014)
Summary
It is of fundamental importance for cell function to organize signaling processes in space and time. Our current understanding of the dynamics and kinetics underlying scaffold interactions relies mostly on in vitro assays of single domains isolated form their native membrane environment (Vincentelli et al, 2015; Stiffler et al, 2007; Long et al, 2003; Ivarsson, 2012). Such approaches reduce the complexity and makes binding assays simpler to both perform and analyze. The presence of several interaction domains in one protein, together with possible formation of higher order structures of both scaffold proteins and transmembrane interaction partners (Long et al, 2003; Ivarsson, 2012), strongly suggest that measurements in vitro cannot replicate
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