title: NMR analysis of a novel enzymatically active unlinked dengue NS2B-NS3 protease complex.
abstract: The dengue virus (DENV) is a mosquito-borne pathogen responsible for an estimated 100 million human infections annually. The viral genome encodes a two-component trypsin-like protease that contains the cofactor region from the nonstructural protein NS2B and the protease domain from NS3 (NS3pro). The NS2B-NS3pro complex plays a crucial role in viral maturation and has been identified as a potential drug target. Using a DENV protease construct containing NS2B covalently linked to NS3pro via a Gly4-Ser-Gly4 linker ("linked protease"), previous x-ray crystal structures show that the C-terminal fragment of NS2B is remote from NS3pro and exists in an open state in the absence of an inhibitor; however, in the presence of an inhibitor, NS2B complexes with NS3pro to form a closed state. This linked enzyme produced NMR spectra with severe signal overlap and line broadening. To obtain a protease construct with a resolved NMR spectrum, we expressed and purified an unlinked protease complex containing a 50-residue segment of the NS2B cofactor region and NS3pro without the glycine linker using a coexpression system. This unlinked protease complex was catalytically active at neutral pH in the absence of glycerol and produced dispersed cross-peaks in a (1)H-(15)N heteronuclear single quantum correlation spectrum that enabled us to conduct backbone assignments using conventional techniques. In addition, titration with an active-site peptide aldehyde inhibitor and paramagnetic relaxation enhancement studies demonstrated that the unlinked DENV protease exists predominantly in a closed conformation in solution. This protease complex can serve as a useful tool for drug discovery against DENV.
authors.affiliation: Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore 138669.
authors.forename: Young Mee
authors.initials: YM
authors.lastname: Kim
authors.forename: Shovanlal
authors.initials: S
authors.lastname: Gayen
authors.forename: CongBao
authors.initials: C
authors.lastname: Kang
authors.forename: Joma
authors.initials: J
authors.lastname: Joy
authors.forename: Qiwei
authors.initials: Q
authors.lastname: Huang
authors.forename: Angela Shuyi
authors.initials: AS
authors.lastname: Chen
authors.forename: John Liang Kuan
authors.initials: JL
authors.lastname: Wee
authors.forename: Melgious Jin Yan
authors.initials: MJ
authors.lastname: Ang
authors.forename: Huichang Annie
authors.initials: HA
authors.lastname: Lim
authors.forename: Alvin W
authors.initials: AW
authors.lastname: Hung
authors.forename: Rong
authors.initials: R
authors.lastname: Li
authors.forename: Christian G
authors.initials: CG
authors.lastname: Noble
authors.forename: Le Tian
authors.initials: le T
authors.lastname: Lee
authors.forename: Andy
authors.initials: A
authors.lastname: Yip
authors.forename: Qing-Yin
authors.initials: QY
authors.lastname: Wang
authors.forename: Cheng San Brian
authors.initials: CS
authors.lastname: Chia
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Hill
authors.forename: Pei-Yong
authors.initials: PY
authors.lastname: Shi
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Keller
mesh_terms: D018360:Crystallography, X-Ray; D003716:Dengue Virus / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D020365:RNA Helicases / Q000737:chemistry / Q000235:genetics; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D009097:Multienzyme Complexes; D020365:RNA Helicases; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C080102:NS2B protein, flavivirus; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
keywords: Drug Discovery; Flaviviruses; NMR; Protease; Protease Inhibitor