The stability and activity of human neuroserpin are modulated by a salt bridge that stabilises the reactive centre loop

Scientific Reports
Rosina NotoMauro Manno

Abstract

Neuroserpin (NS) is an inhibitory protein belonging to the serpin family and involved in several pathologies, including the dementia Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB), a genetic neurodegenerative disease caused by accumulation of NS polymers. Our Molecular Dynamics simulations revealed the formation of a persistent salt bridge between Glu289 on strand s2C and Arg362 on the Reactive Centre Loop (RCL), a region important for the inhibitory activity of NS. Here, we validated this structural feature by simulating the Glu289Ala mutant, where the salt bridge is not present. Further, MD predictions were tested in vitro by purifying recombinant Glu289Ala NS from E. coli. The thermal and chemical stability along with the polymerisation propensity of both Wild Type and Glu289Ala NS were characterised by circular dichroism, emission spectroscopy and non-denaturant gel electrophoresis, respectively. The activity of both variants against the main target protease, tissue-type plasminogen activator (tPA), was assessed by SDS-PAGE and chromogenic kinetic assay. Our results showed that deletion of the salt bridge leads to a moderate but clear reduction of the overall protein stability and activity.

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Citations

Jun 20, 2019·Journal of Biomolecular Structure & Dynamics·Prapasiri PongprayoonSathaporn Jittapalapong
Jan 10, 2021·International Journal of Molecular Sciences·Sonia ÁguilaIrene Martínez-Martínez
Aug 19, 2021·Cellular and Molecular Life Sciences : CMLS·Emanuela D'AcuntoElena Miranda

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Methods Mentioned

BETA
electrophoresis
circular dichroism
X-ray
NMR
gel filtration
dynamic light scattering

Software Mentioned

NACCESS
GelAnalyzer
NAMD2
VMD
Wordom

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