Computational Modeling of NLRP3 Identifies Enhanced ATP Binding and Multimerization in Cryopyrin-Associated Periodic Syndromes

Abstract

Cyropyrin-associated periodic syndromes (CAPS) are clinically distinct syndromes that encompass a phenotypic spectrum yet are caused by alterations in the same gene, NLRP3. Many CAPS cases and other NLRP3-autoinflammatory diseases (NLRP3-AIDs) are directly attributed to protein-coding alterations in NLRP3 and the subsequent dysregulation of the NLRP3 inflammasome leading to IL-1β-mediated inflammatory states. Here, we used bioinformatics tools, computational modeling, and computational assessments to explore the proteomic consequences of NLRP3 mutations, which potentially drive NLRP3 inflammasome dysregulation. We analyzed 177 mutations derived from familial cold autoinflammatory syndrome (FCAS), Muckle-Wells Syndrome (MWS), and the non-hereditary chronic infantile neurologic cutaneous and articular syndrome, also known as neonatal-onset multisystem inflammatory disease (CINCA/NOMID), as well as other NLRP3-AIDs. We found an inverse relationship between clinical severity and the severity of predicted structure changes resulting from mutations in NLRP3. Bioinformatics tools and computational modeling revealed that NLRP3 mutations that are predicted to be structurally severely-disruptive localize around the ATP binding pocket and that specific proteo-structural changes to the ATP binding pocket lead to enhanced ATP binding affinity by altering hydrogen-bond and charge interactions. Furthermore, we demonstrated that NLRP3 mutations that are predicted to be structurally mildly- or moderately-disruptive affect protein-protein interactions, such as NLRP3-ASC binding and NLRP3-NLRP3 multimerization, enhancing inflammasome formation and complex stability. Taken together, we provide evidence that proteo-structural mechanisms can explain multiple mechanisms of inflammasome activation in NLRP3-AID.

ATP-binding and hydrolysis of human NLRP3

“…Decreasing intracellular ATP levels have been correlated with NLRP3 inflammasome activation 32 . A recent computational analysis of NLRP1 to NLRP14 identified amino acid residues that may be involved in nucleotide binding 33 and modeling of NLRP3 suggested enhanced ATP-binding and multimerization in disease related cryopyrin-associated periodic syndrome (CAPS) mutations 34 . Molecular dynamic simulations examined the active site of ADP-and ATP-bound NLRP3 models supporting distinctions in nucleotide-binding domain topology that disseminate on to the global protein structure 35,36 .…”

Differential Binding of NLRP3 to non-oxidized and Ox-mtDNA mediates NLRP3 Inflammasome Activation

“…Activation occurs with diverse agents, including bacterial toxins, microcrystalline substances, ATP, monosodium urate (MSU), calcium pyrophosphate dihydrate (CPPD), silica, asbestos, alum, and hydroxyapatite (HA). Humans harboring one of many NLRP3 point mutations 8 suffer from diseases that result in Familial cold-induced autoinflammatory syndrome (FCAS), Muckle-Wells Syndrome (MWS), Chronic infantile neurologic cutaneous articular (CINCA) syndrome/Neonatal-onset multisystem inflammatory disease (NOMID), which are collectively called cryopyrin-associated periodic syndromes (CAPS) due to constitutively active NLRP3 9 , are more sensitive to activating stimuli. Since none of the agents that activate NLRP3 were shown to bind NLRP3 and are very dissimilar in structure, it is suggested that they trigger a common cellular event that leads to NLRP3 activation.…”

A detailed Molecular Network Map and Model of the NLRP3 Inflammasome

“…In addition, specific phosphorylation at Ser295 blocks NLRP3’s ATPase activity [8], and several mutations in the vicinity of this phosphorylation site is found in Cryopyrin-associated periodic syndromes (CAPS) [23], which are associated with spontaneous NLRP3- dependent inflammation. The CAPS mutations in the nucleotide binding domain (NBD) appear to have a higher affinity for ATP and thereby to stabilise the open, ATP-bound conformation [125]. For example, the R262W mutation increases speck formation [124], consistent with the predicted increase in ATP-binding affinity [125].…”

“…The CAPS mutations in the nucleotide binding domain (NBD) appear to have a higher affinity for ATP and thereby to stabilise the open, ATP-bound conformation [125]. For example, the R262W mutation increases speck formation [124], consistent with the predicted increase in ATP-binding affinity [125]. However, most NLRP3 mutations that decrease the ATPase activity prevent speck formation [124].…”