title: Reconstitution of multivalent PDZ domain binding to the scaffold protein PSD-95 reveals ternary-complex specificity of combinatorial inhibition.
abstract: Multidomain scaffold proteins serve as hubs in the signal transduction network. By physically colocalizing sequential steps in a transduction pathway, scaffolds catalyze and direct incoming signals. Much is known about binary interactions with individual domains, but it is unknown whether "scaffolding activity" is predictable from pairwise affinities. Here, we characterized multivalent binding to PSD-95, a scaffold protein containing three PDZ domains connected in series by disordered linkers. We used single molecule fluorescence to watch soluble PSD-95 recruit diffusing proteins to a surface-attached receptor cytoplasmic domain. Different ternary complexes showed unique concentration dependence for scaffolding despite similar pairwise affinity. The concentration dependence of scaffolding activity was not predictable based on binary interactions. PSD-95 did not stabilize specific complexes, but rather increased the frequency of transient binding events. Our results suggest that PSD-95 maintains a loosely connected pleomorphic ensemble rather than forming a stereospecific complex containing all components.
authors.affiliation: Department of Physiology & Biophysics, Stony Brook University, Stony Brook, NY 11794, USA.
authors.forename: James J
authors.initials: JJ
authors.lastname: McCann
authors.affiliation: Department of Physiology & Biophysics, Stony Brook University, Stony Brook, NY 11794, USA.
authors.forename: Ucheor B
authors.initials: UB
authors.lastname: Choi
authors.affiliation: Department of Physiology & Biophysics, Stony Brook University, Stony Brook, NY 11794, USA. Electronic address: mark.bowen@stonybrook.edu.
authors.forename: Mark E
authors.initials: ME
authors.lastname: Bowen
mesh_terms: D000076122:Disks Large Homolog 4 Protein; D005454:Fluorescence Polarization; D020128:Inhibitory Concentration 50; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I / Q000737:chemistry / Q000378:metabolism; D054731:PDZ Domains / Q000502:physiology*; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry / Q000378:metabolism; D013269:Stochastic Processes
chemical_list: C501444:DLG4 protein, human; D000076122:Disks Large Homolog 4 Protein; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D046912:Multiprotein Complexes; C121001:NR2B NMDA receptor; D016194:Receptors, N-Methyl-D-Aspartate; D052248:Nitric Oxide Synthase Type I