Biophysical analysis and NMR structural characterization of the binding between peptidomimetic drug CN2097 and scaffolding protein PSD-95
Hu, Tony Ken
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BACKGROUND: At the postsynaptic membrane of neurons there is a dense network of proteins called the postsynaptic density (PSD). One such protein is the postsynaptic density protein 95 (PSD-95), which functions as a molecular scaffold for forming protein complexes at the PSD. PSD-95 is composed of three PDZ domains, which studies have shown to be sequentially and structurally similar. Studies have shown that PSD-95 plays a role in regulating signaling of glutamatergic neurons, as well as the induction of longterm potentiation through an association with TrkB receptors. PSD-95 may be a promising target for treatment of a number of neurological disorders such as depression, epilepsy, and cognitive dysfunction. The cyclic peptidomimetic drug CN2097 was designed based on the PDZ-binding motif of the CRIPT protein that binds to PDZ3. While CN2097 has been shown to affect the binding of PSD-95 to different synaptic proteins, no NMR studies have been performed to characterize the binding of CN2097 to PDZ3. Furthermore, few studies have characterized the inter-domain interactions between PDZ domains or whether the binding of calmodulin (CaM) to the N-terminal region of PSD-95 has any effect on the binding between the PDZ domains and CN2097. OBJECTIVE: To use isothermal titration calorimetry and nuclear magnetic resonance spectroscopy to analyze and characterize how CN2097 binds to PDZ domains and whether inter-domain interactions exist between PDZ domains. METHODS: The gene sequences for the PDZ domains were inserted into the pET28a(+) vector by subcloning. E. coli bacteria were then transformed with the different PDZ plasmids. The bacterial cells were grown and induced to express the proteins of interest, followed by lysis and purification using affinity chromatography and fast protein liquid chromatography (FPLC). Isothermal titration calorimetry (ITC) was used to measure the dissociation constant and thermodynamic binding parameters between the peptidomimetic drug CN2097 and each isolated PDZ domain. Nuclear magnetic resonance (NMR) spectroscopy was used to study how CN2097 binds to PDZ1 and PDZ3, and how the PDZ domains interact with each other. RESULTS: The ITC data showed the dissociation constant between CN2097 and PDZ3 to be 5.12 ± (1.65) μM, and that of PDZ2+3 to be 42.63 ± (6.11)μM. ITC data on other domains was inconclusive. The NMR data showed no interaction between N-terminal region and PDZ1, and between PDZ2 and PDZ3 but significant interaction was seen between PDZ1 and PDZ2, as well as between PDZ3 and the inter-domain linker connecting it to PDZ2. NMR data showed that CN2097 binding perturbs PDZ3 more strongly than PDZ1 and that CN2097 does not bind to PDZ1 in the presence of CaM. Significant NMR chemical shift perturbations are seen on the second α-helix, second β- sheet, and β2-β3 loop. CONCLUSIONS: There are no significant contacts between the N-terminal α-helix and PDZ1. There is inter-domain conformational exchange and interaction between PDZ1 and PDZ2. PDZ3 interacts with the second inter-domain linker. CN2097 binds tighter to PDZ3 than to PDZ1, and does not bind to PDZ1 in the presence of CaM. The β2-β3 loop is a prime target for future development of CN2097.
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