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.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/36537 |
| Date | 12 June 2019 |
| Creators | Hu, Tony Ken |
| Contributors | Franzblau, Carl |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
| Rights | Attribution-NonCommercial-NoDerivatives 4.0 International, http://creativecommons.org/licenses/by-nc-nd/4.0/ |
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