MacDonald, JustinSandall, Christina F.2022-10-282022-10-282022-09-19Sandall, C. F. (2022). Structural dissection and catalytic properties of the NLRP (nucleotide-binding domain and leucine-rich repeat-containing gene family, pyrin domain containing) family of inflammatory proteins (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.http://hdl.handle.net/1880/115387https://dx.doi.org/10.11575/PRISM/40375Inflammasomes are high molecular weight hetero-oligomeric protein complexes nucleated by innate immune cytosolic pattern recognition receptors (PRRs) including the NOD-like receptors (NLRs). A subset of NLR proteins include those with N-terminal Pyrin domains (NLRPs) which play critical roles in the detection and response to both endogenous and exogenous danger signals. The NLRP3 inflammasome is the most highly studied and contributes to a multitude of inflammatory and autoimmune conditions. Thus, this work provides a more detailed understanding of NLRP3 activation mechanisms that will be essential for the rationalised development of future pharmacological interventions. First, the impact of orientation and linkage on NLRP3 capture with immobilised ATP demonstrates ATP binds this protein with an exposed phosphate tail and buried adenine ring. Decreased competitive recovery with free ATP indicated NLRP3 underwent a conformational change upon ATP binding. Additionally, P-linked ATP Sepharose provides a strategy for capture of the entire NLRP family and enrichment of NLRP3 containing samples for mass spectrometry analyses. Next, the ATP hydrolysis kinetics of NLRP proteins and hyperactive NLRP3 disease mutant R262W were evaluated with GFP nano-trap beads and a bioluminescent ATPase assay. NLRP proteins displayed distinct ATPase kinetic profiles, suggesting variable ATP sensitivity and kinetics of assembly for some NLRP proteins. Classical Michaelis-Menten kinetics were observed for NLRP1, 3 and 12, and positive Hill cooperativity was revealed for NLRP3R262W as well as NLRP6 and 7. Furthermore, NLRP3 inhibitors targeting ATPase activity demonstrated promising results in blocking inflammasome activation. Next, the impacts of NLRP3 phosphorylation at Ser295 on protein structure, ATP binding & inflammasome activation were evaluated in silico. These results suggest that modifications to the NACHT domain are conveyed globally and result in variable nucleotide hydrolysis and inflammasome activation. Finally, unique mechanisms of ATP and ADP binding and the detailed structural impacts were illuminated by molecular dynamics simulations with NLRP3. NLRP3-ADP simulations indicate high protein stability and few global rearrangements, while NLRP3-ATP binding was thermodynamically favourable, induced protein flexibility, and resulted in global structural rearrangements that were initiated from the NACHT domain. To conclude, a detailed mechanism of ATP induced structural changes provided a basis for rationale design of NLRP3 inhibitors, and a region of NLRP3 in a cleft between the HD2 and LRR domains was proposed for pharmacological targeting.engUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.InflammasomeNLRPNOD-like ReceptorsNucleotide-Binding Domain and Leucine-Rich Repeat-Containing Gene Family, Pyrin Domain ContainingNLRP3ATP BindingATP HydrolysisATPasePhosphorylationMolecular Dynamics SimulationsBioinformaticsImmunologyBiochemistrydoctoral thesis