Paramagnetic Studies of Autoproteolytic Dengue Virus Protease — ASN Events
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Paramagnetic Studies of Autoproteolytic Dengue Virus Protease (#110)

Wanna Chen 1 , Laura de la Cruz 1 , Gottfried Otting 1
  1. Research School of Chemistry, Australian National University, Canberra, ACT, Australia

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.

  1. Leung et al. (2001) J. Biol. Chem. 276, 45762-45771.
  2. Erbel et al. (2006) Nat. Struct. Mol. Biol. 13, 372-373.
  3. de la Cruz et al. (2011) J. Am. Chem. Soc. 133, 19205-19215.
  4. Noble et al. (2012) J. Virol. 86, 438-446.
  5. Kim et al. (2013) J. Biol. Chem. 288, 12891-12900.