Deciphering the ATP-binding mechanism(s) in NLRP-NACHT 3D models using structural bioinformatics approaches

Abstract

Nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs), the first line of defense, are the cytosolic pattern recognition receptors (PRRs) that regulate the inflammatory activity in response to invading pathogens. NLRs are the members of AAA+ ATPase superfamily that comprises of N-terminal EBD(s), a centrally positioned NOD/NACHT and varying range of LRRs towards the C-terminal end. Due to the lack of structural data, the functional aspects of NLRP-signaling mechanism, which includes pathogen recognition, nucleotide-binding, and sensor-adaptor-effector interactions, are not fully understood. In this study, we implemented structural bioinformatics approaches including protein modeling, docking, and molecular dynamics simulations to explore the structural-dynamic features of ADP-/ATP-Mg2+ binding in NLRPNACHT models. Our results indicate a similar mode of ATP-Mg2+ binding in all NLRPNACHT models and the interacting residues are found consistent with reported mutagenesis data. Accompanied by the key amino acids (proposed to be crucial for ATP-Mg2+ coordination), we further have noticed that some additional conserved residues (including ‘Trp’ of the PhhCW motif, and ‘Phe’ and ‘Tyr’ of the GFxxxxRxxYF motif) are potentially interacting with ATP during dynamics; which require further experimentation for legitimacy. Overall, this study will help in understanding the ADP-/ATP-Mg2+ binding mechanisms in NLRPs in a broader perspective and the proposed ATP-binding pocket will aid in designing novel inhibitors for the regulation of inflammasome activity.

ATP-Binding and Hydrolysis in Inflammasome Activation

“…The reports on NLRP1, NLRP3, and NLRP7 support a critical role for the binding of ATP in oligomerization and inflammasome activation. Maharana and colleagues observed a similar mode of ADP- and ATP-binding by in silico modelling of the NACHT domain from NLRP1-14 with molecular dynamic simulations [ 47 ]. These structural models revealed a similar spatial arrangement of nucleotide-binding domain (NBD), helical domain 1 (HD1) and winged helix domain (WHD) for the entire NLRP family.…”

“…Conservation of key functional modules within the family suggest that certain mechanisms of action could be shared amongst NLR member proteins, and these structural and functional linkages will be reviewed in upcoming sections. With NTP binding, the NACHT domain is presumed to undergo a conformational shift into the active state; however, the specific relationship between NTP binding and hydrolysis and the NLR protein conformational change that leads to inflammasome assembly is not fully understood [ 46 , 47 ]. For the NLRP members, activation following NTP-binding and/or hydrolysis provides the opportunity for homotypic PYD-PYD binding between the NLRP and adaptor protein ASC, which then recruits pro-caspase-1 via CARD-CARD interactions.…”

“…There is a paucity of studies that examine the importance of the NACHT domain in NLRP2 inflammasome assembly and downstream signaling. While structural modeling supports the ability of NLRP2 to bind ATP [ 47 ], NLRP2 was unique amongst the NLRPs in displaying low elution efficiency from immobilized ATP-Sepharose resin. This could indicate lower comparative efficacy in ATP binding [ 80 ].…”

“…NLRP9 could be recovered from immobilized ATP-Sepharose resin [ 80 ], which suggests effective ATP-binding. Moreover, 3D modeling of the NACHT domain suggests stable coordination of ADP and ATP in the binding pocket of NLRP9, although comparatively fewer H-bonds were identified with molecular dynamic simulations using a manually-docked complex [ 47 ]. Finally, structures of the PYD from the human NLRP9 gene reveal a novel orientation for the domain [ 146 , 147 ] which may reflect differences in oligomerization and ATP-dependent inflammasome formation.…”

“…In contrast, Maharana and colleagues failed to display the opening mechanism of ATP-bound NOD1/NOD2-NACHT domains in a zebrafish model for 50 ns of MD simulation [ 46 ]. In longer duration, 200 ns simulations with NLRP1-, NLRP3- and NLRC4-NACHT-ATP-Mg 2+ bound homodimeric complexes modeled on the NBD-HD1 of NLRC4, no significant rigid body movement was detected between HD1 and WHD when compared against ADP-bound models [ 47 ]. The authors concluded the interaction of ATP-Mg 2+ might not be the only component to drive the switching mechanism in NLRs and suggested PAMP/DAMP recognition to play a more significant role than nucleotide exchange.…”