Supertertiary structure of the synaptic MAGuK scaffold proteins is conserved

Abstract

Scaffold proteins form a framework to organize signal transduction by binding multiple partners within a signaling pathway. This shapes the output of signal responses as well as providing specificity and localization. The Membrane Associated Guanylate Kinases (MAGuKs) are scaffold proteins at cellular junctions that localize cell surface receptors and link them to downstream signaling enzymes. Scaffold proteins often contain protein-binding domains that are connected in series by disordered linkers. The tertiary structure of the folded domains is well understood, but describing the dynamic inter-domain interactions (the superteritary structure) of such multidomain proteins remains a challenge to structural biology. We used 65 distance restraints from singlemolecule fluorescence resonance energy transfer (smFRET) to describe the superteritary structure of the canonical MAGuK scaffold protein PSD-95. By combining multiple fluorescence techniques, the conformational dynamics of PSD-95 could be characterized across the biologically relevant timescales for protein domain motions. Relying only on a qualitative interpretation of FRET data, we were able to distinguish stable interdomain interactions from freely orienting domains. This revealed that the five domains in PSD-95 partitioned into two independent supramodules: PDZ1-PDZ2 and PDZ3-SH3-GuK. We used our smFRET data for hybrid structural refinement to model the PDZ3-SH3-GuK supramodule and include explicit dye simulations to provide complete characterization of potential uncertainties inherent to quantitative interpretation of FRET as distance. Comparative structural analysis of synaptic MAGuK homologues showed a conservation of this supertertiary structure. Our approach represents a general solution to describing the supertertiary structure of multidomain proteins.intrinsic disorder | protein structure | single molecule fluorescence | fluorescence lifetime | fluorescence correlation spectroscopy N ature relies on scaffold proteins to provide the physical constraints necessary for efficient signal transduction. Scaffold proteins interact with multiple pathway components to hold the signal transduction machinery in close proximity. Scaffolds are often composed of modular, protein-binding domains linked together in series by intrinsically disordered linkers (1). The presence of disorder may be a defining feature for scaffolds and other proteins that interact with multiple binding partners (2).The high effective concentrations brought about by domain tethering can give rise to unexpected interactions between the protein-binding domains. In some cases, canonical protein-binding domains fold together into an inseparable structural supramodule (3). While much has been learned by studying truncated fragments, we need to put the pieces back together. Such questions are difficult to address because disorder presents a fundamental challenge to structural biology.The Membrane-Associated Guanylate Kinase (MAGuK) scaffold proteins regulate signaling at cellular junctions (4). ...

Modulating the Intrinsic Disorder in the Cytoplasmic Domain Alters the Biological Activity of the N-Methyl-d-aspartate-sensitive Glutamate Receptor

“…The increased volume allows IDPs to function as entropic barriers in the nuclear pore (39). Disordered linkers can modulate supertertiary structure in multidomain proteins (40) and have been proposed to enable fly-casting (41), wherein IDPs "search" a large volume for potential ligands. In such instances, variation in compaction could regulate the biological activity at the level of individual proteins without invoking a folded state.…”

Structure of Crumbs tail in complex with the PALS1 PDZ–SH3–GK tandem reveals a highly specific assembly mechanism for the apical Crumbs complex

“…All MAGUKs, except for MAGI, contain a PDZ-SH3-GK core element (Fig. 6A), raising a possibility that the three domains in the tandem may function synergistically in binding to their respective targets (34,38,39). Earlier, the SH3 domain of ZO1-PSG had been shown to enhance the PDZ domain-mediated binding to connexin45 (34).…”

Multisteric Regulation by Structural Disorder in Modular Signaling Proteins: An Extension of the Concept of Allostery

“…48 It also enabled to address the "supertertiary" structure of the complex regulatory protein PSD95. 164 Atomic-force microscopy (AFM) has also been recently applied for studying the structure and structural changes of the complex motor protein myosin V with disordered linkers 165 and the structural ensemble and unfolding patterns of α-synuclein. 50 …”