Domain Orientation in the N-Terminal PDZ Tandem from PSD-95 Is Maintained in the Full-Length Protein

McCann, James J.; Zheng, Liqiang; Chiantia, Salvatore; Bowen, Mark E.

doi:10.1016/j.str.2011.02.017

Cited by 40 publications

(58 citation statements)

References 51 publications

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“…If local structure existed, we would have expected a dispersion of FRET values (37). Instead, the results showed minimal positional dependence and instead the FRET scaled linearly with the fluorophore separation.…”

Section: Discussionmentioning

confidence: 89%

Effect of Src Kinase Phosphorylation on Disordered C-terminal Domain of N-Methyl-d-aspartic Acid (NMDA) Receptor Subunit GluN2B Protein

Choi

Xiao

Wollmuth

et al. 2011

Journal of Biological Chemistry

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NMDA receptors are ligand-gated ion channels with a regulatory intracellular C-terminal domain (CTD). In GluN2B, the CTD is the largest domain in the protein but is intrinsically disordered. The GluN2B subunit is the major tyrosine-phosphorylated protein in synapses. Src kinase phosphorylates the GluN2B CTD, but it is unknown how this affects channel activity. In disordered proteins, phosphorylation can tip the balance between order and disorder. Transitions can occur in both directions, so it is not currently possible to predict the effects of phosphorylation. We used single molecule fluorescence to characterize the effects of Src phosphorylation on GluN2B. Scanning fluorescent labeling sites throughout the domain showed no positional dependence of the energy transfer. Instead, efficiency only scaled with the separation between labeling sites suggestive of a relatively featureless conformational energy landscape. Src phosphorylation led to a general expansion of the polypeptide, which would result in greater exposure of known protein-binding sites and increase the physical separation between contiguous sites. Phosphorylation makes the CTD more like a random coil leaving open the question of how Src exerts its effects on the NMDA receptor.

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Section: Discussionmentioning

confidence: 89%

Effect of Src Kinase Phosphorylation on Disordered C-terminal Domain of N-Methyl-d-aspartic Acid (NMDA) Receptor Subunit GluN2B Protein

Choi

Xiao

Wollmuth

et al. 2011

Journal of Biological Chemistry

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“…The narrow smFRET peak widths and lack of signal variance suggest rapid dynamics across the PDZ2-3 linker (13). Measurements between PDZ1 and PDZ2 was measured previously (44). VMF characterizes replicate measurements using different labeling sites to oversample the domain position.…”

Section: Resultsmentioning

confidence: 99%

“…Recombinant protein samples were purified and fluorescently labeled with a mixture of Alexa Fluor 555 C 2 maleimide and Alexa Fluor 647 C 2 maleimide as described previously (44). PSD-95 was encapsulated in 100 nm liposomes, which were immobilized to a quartz slide for Total Internal Reflection illumination with alternating laser excitation at 532 nm (approximately 7.5 mW before prism) and 633 nm (approximately 3 mW before prism).…”

Section: Methodsmentioning

confidence: 99%

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Supertertiary structure of the synaptic MAGuK scaffold proteins is conserved

McCann

Zheng

Rohrbeck

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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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). ...

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“…Ligands approaching from the same direction seem to be preferably bound by the PDZ domains oriented in parallel. However, measurements based on single-molecule fluorescence suggest an antiparallel orientation of binding sites favouring the interaction with ligands from opposite directions (McCann et al, 2011). A more recent transmission electron microscopy (TEM) based study showed that PSD-95 and cytoplasmic domains of Kir2.1 build up 1:1 complexes, which are able to tetramerize into self-enclosed structures (Fomina et al, 2011).…”

Section: Discussionmentioning

confidence: 99%

Visualization of the dynamics of PSD-95 and Kir2.1 interaction by fluorescence lifetime-based resonance energy transfer imaging

et al. 2015

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Domain Orientation in the N-Terminal PDZ Tandem from PSD-95 Is Maintained in the Full-Length Protein

Cited by 40 publications

References 51 publications

Effect of Src Kinase Phosphorylation on Disordered C-terminal Domain of N-Methyl-d-aspartic Acid (NMDA) Receptor Subunit GluN2B Protein

Effect of Src Kinase Phosphorylation on Disordered C-terminal Domain of N-Methyl-d-aspartic Acid (NMDA) Receptor Subunit GluN2B Protein

Supertertiary structure of the synaptic MAGuK scaffold proteins is conserved

Visualization of the dynamics of PSD-95 and Kir2.1 interaction by fluorescence lifetime-based resonance energy transfer imaging

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