[NMR paper] NMR analysis of a novel enzymatically-active unlinked Dengue NS2B-NS3 protease complex.

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NMR analysis of a novel enzymatically-active unlinked Dengue NS2B-NS3 protease complex.

Related Articles NMR analysis of a novel enzymatically-active unlinked Dengue NS2B-NS3 protease complex.

J Biol Chem. 2013 Mar 19;

Authors: Kim YM, Gayen S, Kang C, Joy J, Huang Q, Chen AS, Wee JL, Ang MJ, Lim HA, Hung AW, Li R, Noble CG, Lee LT, Yip A, Wang QY, Chia CS, Hill J, Shi PY, Keller TH

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 a cofactor region from the nonstructural (NS) protein 2B and the protease domain from NS3 (NS3pro). The NS2B-NS3pro 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-S-Gly4 linker (linked protease), X-ray crystal structures indicate 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. In order to obtain a protease construct with a resolved NMR spectrum, we expressed and purified an unlinked protease complex containing a 50-residue region of NS2B cofactor region and NS3pro without the glycine linker using a co-expression system. This unlinked protease complex is catalytically active at neutral pH in the absence of glycerol and produced dispersed cross-peaks in an 1H-15N- HSQC 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 (PRE) 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 the DENV.


PMID: 23511634 [PubMed - as supplied by publisher]



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