The secreted form of the enzyme nicotinamide phosphoribosyltransferase (NAMPT), which catalyzes a key reaction in intracellular NAD biosynthesis, acts as a damage-associated molecular pattern triggering Toll-like receptor 4 (TLR4)mediated inflammatory responses. However, the precise mechanism of interaction is unclear. Using an integrated approach combining bioinformatics and functional and structural analyses, we investigated the interaction between NAMPT and TLR4 at the molecular level. Starting from previous evidence that the bacterial ortholog of NAMPT cannot elicit the inflammatory response, despite a high degree of structural conservation, two positively charged areas unique to the human enzyme (the α1-α2 and β1-β2 loops) were identified as likely candidates for TLR4 binding. However, alanine substitution of the positively charged residues within these loops did not affect either the oligomeric state or the catalytic efficiency of the enzyme. The kinetics of the binding of wildtype and mutated NAMPT to biosensor-tethered TLR4 was analyzed. We found that mutations in the α1-α2 loop strongly decreased the association rate, increasing the KD value from 18 nM, as determined for the wildtype, to 1.3 μM. In addition, mutations in the β1-β2 loop or its deletion increased the dissociation rate, yielding KD values of 0.63 and 0.22 μM, respectively. Mutations also impaired the ability of NAMPT to trigger the NF-κB inflammatory signaling pathway in human cultured macrophages. Finally, the involvement of the two loops in receptor binding was supported by NAMPT-TLR4 docking simulations. This study paves the way for future development of compounds that selectively target eNAMPT/TLR4 signaling in inflammatory disorders.
Molecular insights into the interaction between human nicotinamide phosphoribosyltransferase and Toll-like receptor 4 / Gasparrini, M.; Mazzola, F.; Cuccioloni, M.; Sorci, L.; Audrito, V.; Zamporlini, F.; Fortunato, C.; Amici, A.; Cianci, M.; Deaglio, S.; Angeletti, M.; Raffaelli, N.. - In: THE JOURNAL OF BIOLOGICAL CHEMISTRY. - ISSN 0021-9258. - 298:3(2022), p. 101669. [10.1016/j.jbc.2022.101669]