Effects of Local and Global Dynamics on the Supertertiary Organization of Postsynaptic Density Protein 95

Abstract

Postsynaptic density protein 95 (PSD-95) is a critical scaffolding protein in the excitatory postsynaptic density (PSD) with key functions in synaptic organization and regulation of synaptic strength. PSD-95 is a canonical multidomain scaffold protein comprised of three PDZ domains, an SH3 domain, and a guanylate “pseudokinase” (GuK) domain connected in series by flexible polypeptide linkers. The domains of PSD-95 show varying degrees of interaction and were suggested to partition into two independent supramodules. The first two PDZ domains display weak interactions but the third PDZ domain preferentially interacted with the SH3 and GuK domains. Despite a trove of structural studies, we do not understand how the interactions of PSD-95 with its diverse set of synaptic binding partners are regulated. PSD-95 exchanges between supertertiary configurations as part of its functional cycle. Interactions and posttranslational modifications can change the conformation with effects on function. This suggests that internal dynamics may regulate access to binding sites in PSD-95. In this work, we utilize a FRET network designed to probe all pairwise combinations of the five domains. We used multiparameter fluorescence detection and filtered fluorescence correlation spectroscopy to identify interdomain interactions and resolve dynamics from picoseconds to milliseconds. The labeling sites were on α-helices or surface loops. We distinguished locally rigid or flexible sites based on their relative contribution to FRET efficiencies and correlation amplitudes. Global analysis of the entire FRET network allowed comparison of the timescales associated with local and interdomain motions. Describing how local and interdomain dynamics drive the supertertiary organization of PSD-95 provides a basis for understanding how conformational changes regulate multidomain scaffold proteins.