Abstract
Cell scaffolding and signaling are governed by protein-protein interactions. Although a particular interaction is often defined by two specific domains binding to each other, this interaction often occurs in the context of other domains in multidomain proteins. How such adjacent domains form supertertiary structures and modulate protein-protein interactions has only recently been addressed and is incompletely understood. The postsynaptic density protein PSD-95 contains a three-domain supramodule, denoted PSG, which consists of PDZ, Src homology 3 (SH3), and guanylate kinase-like domains. The PDZ domain binds to the C terminus of its proposed natural ligand, CXXC repeat-containing interactor of PDZ3 domain (CRIPT), and results from previous experiments using only the isolated PDZ domain are consistent with the simplest scenario for a protein-protein interaction; namely, a two-state mechanism. Here we analyzed the binding kinetics of the PSG supramodule with CRIPT. We show that PSG binds CRIPT via a more complex mechanism involving two conformational states interconverting on the second timescale. Both conformational states bound a CRIPT peptide with similar affinities but with different rates, and the distribution of the two conformational states was slightly shifted upon CRIPT binding. Our results are consistent with recent structural findings of conformational changes in PSD-95 and demonstrate how conformational transitions in supertertiary structures can shape the ligand-binding energy landscape and modulate protein-protein interactions.
Highlights
Cell scaffolding and signaling are governed by protein– protein interactions
The postsynaptic density protein PSD-95 contains a three-domain supramodule, denoted PSG, which consists of PDZ, Src homology 3 (SH3), and guanylate kinase-like domains
The PDZ domain binds to the C terminus of its proposed natural ligand, CXXC repeat– containing interactor of PDZ3 domain (CRIPT), and results from previous experiments using only the isolated PDZ domain are consistent with the simplest scenario for a protein–protein interaction; namely, a two-state mechanism
Summary
The binding kinetics of PSG and a six-amino-acid CRIPT peptide displayed biphasic behavior. All remaining dissociation traces (delay times between 24 ms and 200 s) were fitted simultaneously to a double-exponential function with shared values for k app off and koff2app, either locked to the average values reported in Fig. 3B or free-fitted (Fig. 6B) This is possible because the final conditions under which kinetics were measured were identical for each double-jump experiment. The double-exponential fit yielded koffapp values (B) and their associated amplitudes (C, normalized), which were plotted versus CRIPT concentration. The kinetic phase k app off decreases, whereas the phase k app off should increase (the latter is not as clear as the former because of scatter in the data) This reflects the redistribution of conformations in the bound complex occurring over 1–10 s with a net flow from PSGA: D-CRIPT6 to PSGB:D-CRIPT6 (Fig. 5). Regardless of whether D-CRIPT15 binds to PSGB, this conformation is present in equilibrium with PSGA, as monitored by the smaller D-CRIPT6, which can likely access a more narrow binding pocket present in PSGB
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