title: Binding of low molecular weight inhibitors promotes large conformational changes in the dengue virus NS2B-NS3 protease: fold analysis by pseudocontact shifts.
abstract: The two-component dengue virus NS2B-NS3 protease (DEN NS2B-NS3pro) is an established drug target, but inhibitor design is hampered by the lack of a crystal structure of the protease in its fully active form. In solution and without inhibitors, the functionally important C-terminal segment of the NS2B cofactor is dissociated from DEN NS3pro ("open state"), necessitating a large structural change to produce the "closed state" thought to underpin activity. We analyzed the fold of DEN NS2B-NS3pro in solution with and without bound inhibitor by nuclear magnetic resonance (NMR) spectroscopy. Multiple paramagnetic lanthanide tags were attached to different sites to generate pseudocontact shifts (PCS). In the face of severe spectral overlap and broadening of many signals by conformational exchange, methods for assignment of (15)N-HSQC cross-peaks included selective mutation, combinatorial isotope labeling, and comparison of experimental PCSs and PCSs back-calculated for a structural model of the closed conformation built by using the structure of the related West Nile virus (WNV) protease as a template. The PCSs show that, in the presence of a positively charged low-molecular weight inhibitor, the enzyme assumes a closed state that is very similar to the closed state previously observed for the WNV protease. Therefore, a model of the protease built on the closed conformation of the WNV protease is a better template for rational drug design than available crystal structures, at least for positively charged inhibitors. To assess the open state, we created a binding site for a Gd(3+) complex and measured paramagnetic relaxation enhancements. The results show that the specific open conformation displayed in the crystal of DEN NS2B-NS3pro is barely populated in solution. The techniques used open an avenue to the fold analysis of proteins that yield poor NMR spectra, as PCSs from multiple sites in combination with model building generate powerful information even from incompletely assigned (15)N-HSQC spectra.
authors: Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia.
authors: Laura
authors: L
authors: de la Cruz
authors: Thi Hoang Duong
authors: TH
authors: Nguyen
authors: Kiyoshi
authors: K
authors: Ozawa
authors: James
authors: J
authors: Shin
authors: Bim
authors: B
authors: Graham
authors: Thomas
authors: T
authors: Huber
authors: Gottfried
authors: G
authors: Otting
mesh_terms: D001665:Binding Sites / Q000187:drug effects; D003716:Dengue Virus / Q000201:enzymology*; D008958:Models, Molecular; D008968:Molecular Conformation; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D017510:Protein Folding / Q000187:drug effects; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000378:metabolism*; D000085242:Viral Proteases
chemical_list: D011480:Protease Inhibitors; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C080102:NS2B protein, flavivirus; C504449:NS3 protease, dengue virus; D000085242:Viral Proteases