Structure and dynamics of the Ovarian Tumour Domain Protease from the Crimean-Congo Hemorrhagic Fever Virus by Nuclear Magnetic Resonance spectroscopy
Date
2016
Authors
Saran, Sagar
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Abstract
Crimean-Congo Hemorrhagic Fever Virus (CCHFV) is endemic to more than 30 countries and shows fatality rates among humans ranging from 30-50%. The CCHFV 169-residue L-segment deubiquitinase (DUB) is a member of the superfamily of Ovarian Tumour (OTU) ubiquitin thiolesterases that interfere with innate immune responses and hence is an attractive antiviral target. I report here the application of Nuclear Magnetic Resonance spectroscopy to probe the role of CCHFV OTU enzyme dynamics in the catalytic mechanism of the enzyme. 13C/15N triple-resonance experiments and an amino acid “unlabelling” scheme were used for backbone resonances assignments. NMR chemical shift analysis, NMR spin-relaxation experiments at two magnetic fields, Lipari-Szabo Model-free formalism, reduced spectral density mapping and Carr-Purcell-Meiboom-Gill Relaxation Dispersion experiments were done to obtain structure and dynamics data. The dynamics data suggested an unfolded C-terminus and a well-packed protein core. Relaxation dispersion measurements show that a significant number of residues undergo conformational exchange on the millisecond timescale. Some of these are near the active site and neighbouring segments and may represent a rate-limiting event along the proteolytic kinetic pathway. They may also play a role in determining the enzyme’s broad substrate specificity.
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Keywords
Crimean-Congo Hemorrhagic Fever, Enzyme, NMR spectroscopy, Protease, Protein, Protein dynamics, Protein structure, Virus,