Enzyme aging: Structural insights from NTE and patatin

Neuropathy target esterase (NTE) is an integral membrane protein whose covalent modification by organophosphorus compounds (OPs) results in a delayed axonopathy (called OP-induced delayed neuropathy (OPIDN)) whose mechanisms of initiation and progression are unknown. The current model for OPIDN states that inhibition of NTE by a neuropathic OP followed by a post-inhibitory aging reaction results in NTE gaining a new function that is detrimental to the survival of the axon. Thus, we hypothesized that if NTE gains a new function, it would also need to experience a change in its structure. To this end, we employed molecular modeling along with x-ray crystallography of NTE's plant homologue patatin-17 (pat17) to investigate conformational changes in tertiary structure associated with inhibition and aging of a neuropathic OP-pat17 conjugate. Analysis of NTE's catalytic (patatin-homology) domain (PNTE) revealed it to have 30% sequence homology and 18% identity to pat17, allowing us to build a homology model for PNTE using the crystal structure of pat17 as a template. This model revealed that contrary to previously published data, NTE has a Ser966-Asp1086 catalytic dyad with the Asp1086 acting as the general base during catalysis. Simulated inhibition (SI) and simulated aging (SA) work undertaken with this model using the neuropathic OP diisopropylphosphorofluoridate (DFP) indicated the absence of significant conformational changes following inhibition (Calpha root mean square deviation (RMSD): 1.4A) and aging (RMSD: 1.6A), suggesting that the current model for OPIDN 's pathogenesis may be flawed. Furthermore, biochemical analysis of pat17 revealed that it could be inhibited and aged by OPs like DFP (IC50: 181muM; t 1/2 aging: 11.4 min). We also showed that pat17 could be inhibited by methyl arachidonyl fluorophosphonate (MAFP) (IC50: 116nM). The crystal structures of pat17 aged with DFP along with that of pat17 inhibited with MAFP were solved via molecular replacement in space-group P4 3212. Analysis of these crystal structures confirmed our SI and SA results, indicating no significant conformational change associated with inhibition by MAFP (RMSD: 1.07A) and aging by DFP (0.98A). In conclusion, a new model for the initiation and progression of OPIDN, based on disruption of membrane lipids is presented.