Structure determination of Murine Norovirus NS6 proteases with C-terminal extensions designed to probe protease-substrate interactions

PeerJ
Humberto FernandesStephen Curry

Abstract

Noroviruses are positive-sense single-stranded RNA viruses. They encode an NS6 protease that cleaves a viral polyprotein at specific sites to produce mature viral proteins. In an earlier study we obtained crystals of murine norovirus (MNV) NS6 protease in which crystal contacts were mediated by specific insertion of the C-terminus of one protein (which contains residues P5-P1 of the NS6-7 cleavage junction) into the peptide binding site of an adjacent molecule, forming an adventitious protease-product complex. We sought to reproduce this crystal form to investigate protease-substrate complexes by extending the C-terminus of NS6 construct to include residues on the C-terminal (P') side of the cleavage junction. We report the crystallization and crystal structure determination of inactive mutants of murine norovirus NS6 protease with C-terminal extensions of one, two and four residues from the N-terminus of the adjacent NS7 protein (NS6 1', NS6 2', NS6 4'). We also determined the structure of a chimeric extended NS6 protease in which the P4-P4' sequence of the NS6-7 cleavage site was replaced with the corresponding sequence from the NS2-3 cleavage junction (NS6 4' 2|3).The constructs NS6 1' and NS6 2' yielded crystals that diffra...Continue Reading

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Citations

Apr 6, 2016·Journal of Interferon & Cytokine Research : the Official Journal of the International Society for Interferon and Cytokine Research·Soroush T SarvestaniJason M Mackenzie
Apr 26, 2016·Expert Opinion on Drug Discovery·Sahani WeerasekaraDuy H Hua
Aug 4, 2020·Journal of Structural Biology: X·Jingxu GuoFrank von Delft

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Methods Mentioned

BETA
NMR
X-ray
nuclear magnetic resonance

Software Mentioned

Coot
Phenix refine
REFMAC
Phaser
CCP4
PyMOL

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