In the frequently studied construct of dengue virus protease, the protease domain of NS3 links covalently to NS2B via Gly4-Ser-Gly4 linker1. Its NMR spectra display severe line broadening, indicating conformational exchange. A previous crystal structure without inhibitor had shown the C-terminal of NS2B in an open conformation2. Pseudocontact shifts studies established that NS2B readily assumes closed conformation3, which agreed with a subsequent crystal structure4. Recent NMR studies of unlinked NS2B-NS3pro suggested that open conformation could be an artifact of the glycine linker5.
Here we present a new construct of unlinked NS2B-NS3pro obtained by autoproteolysis. NS2B was found to be in the closed conformation defined by PCSs generated by lanthanide tags. At high ionic strength, however, most peaks of the C-terminal part of NS2B vanish, indicating extreme line broadening due to conformational exchange and, hence, destabilization of the NS2B structure. Ser85 is the only residue of C-terminal b-hairpin of NS2B for which a 15N-HSQC cross-peak could still be observed in the presence of 300 mM NaCl. Its PCS agrees with closed conformation, indicating little, if any, population of open conformations even under destabilizing conditions.