Structural Modeling of PSD-95 and other MAGUKs using Single-Molecule FRET

Abstract

In order for information to be correctly propagated between cells, it is necessary for both the sending and receiving cells to have organized molecular machinery so that the transmitted signals can be accurately received and processed. By conjoining protein interaction domains, multidomain scaffold proteins form a framework for maintaining and modulating these junctional communications. Scaffold proteins have been traditionally conceptualized as “beads on a string” with unstructured linkers allowing each domain to be independently oriented, but recent data on multidomain supramodules suggests that the fundamental functional units of scaffold proteins may be larger multidomain complexes. Membrane-associated guanylate kinase (MAGUK) proteins are multidomain scaffold proteins that function at cellular junctions; most importantly at epithelial tight junctions and within the postsynaptic densities of neurons. MAGUK proteins contain both PSD-95/Dlg/ZO-1 (PDZ) and Src Homology (SH3) domains, as well as an enzymatically inactive guanylate kinase domain. While the high-resolution structures of the isolated domains of the canonical MAGUK, PSD-95, have been solved, the organization of these domains remains unknown. In combination with rigid body docking and all-atom molecular dynamics simulations, single-molecule FRET measurements between each of five domains allowed for the determination of the domain positioning in full-length PSD-95. The “ground” state configuration of PSD-95 was found to contain two stable multidomain subunits that while connected by a flexible linker do not appear to interact. A comparison of measurements within each of these partitions with homologous measurements in other MAGUKs showed that domain orientations can vary among this family of scaffold proteins. These findings represent the first unambiguous assignment of domain positioning in a full-length scaffold protein and provide insights into potential allosteric coupling between domains in MAGUK proteins.