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PSD-95: structural aspects of PDZ domain binding
Published on July 31, 2022 13 min
Other Talks in the Series: Synapses, Neurotransmitters and Receptor Channels
Postsynaptic scaffold proteins in health and disease
- Dr. Jonathan Hanley
- University of Bristol, UK
PSD-95: dynamic localization of PSD-95 and glutamate receptors at postsynaptic sites
- Prof. Johannes Hell
- University of California, Davis, USA
Molecular basis of NMDA receptor functional diversity
- Prof. Jon Johnson
- University of Pittsburgh, USA
AMPA-receptors and fast synaptic transmission in the brain
- Prof. Stuart Cull-Candy
- University College London, UK
As I stated in the introduction to Part 1 of this talk on PSD-95, my name is Johannes Hell. I am a Professor of Pharmacology at the University of California, Davis, Vice-Chair for Academic Development and the Director of our T-32 training program in Pharmacology.
This second part will delineate our work on the structural details that mediate binding of the PDZ domains of PSD-95 to target recognition sites on binding partners, like the NMDAR receptor GluN2 subunits or the AMPAR receptor auxiliary TARP subunits.
Starting points for our modeling were binding assays with each peptide measuring fluorescence polarization upon addition of increasing amounts of individual purified PDZ domains, which we expressed as GST fusion proteins in E.coli. When fluorescent molecules are excited with polarized light, they will maintain the vector of the wave when emitting fluorescence, but tumbling will degrade the orientation of this vector. Small molecules tumble faster than large ones. When PDZ domains bind to our peptides, the tumbling rate will decrease and polarization will be better preserved. Thus, the increase in polarization will reflect the binding of PDZ domains to peptides. We determined the K_D values of a number of PDZ domains, which is given as half-maximal binding concentration of such titration curves, as shown here for PDZ1 and as shown here for PDZ1 and PDZ2 from PSD-95, and SAP102 against the peptide visible type sequence of GluN2B. These experiments show that PDZ2 domains have a significantly higher affinity than PDZ1 domains and that SAP102 has a higher affinity than PSD-95 for GluN2 subunits.