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.
Highlights
Human neuroserpin (NS) is a member of the serpin family (SERine Protease INhibitor)[1]
In our previous work[23], we reported a long Molecular Dynamics (MD) simulation of native NS, which showed the formation of a persistent salt bridge between the arginine Arg[362] on the reactive centre loop (RCL) and the glutamic acid Glu[289] on strand s2C in the main protein core (Fig. 1a)
We highlight a structural detail, not previously reported, that emerged from MD and was not resolved in crystallographic structures
Summary
Human neuroserpin (NS) is a member of the serpin family (SERine Protease INhibitor)[1]. The RCL is a key structural element of all inhibitory serpins It acts as a bait for the target protease, which cleaves the RCL forming an acyl-enzyme complex and triggering the insertion of the RCL as a new strand of the main central β-sheet. This mousetrap-like mechanism causes the translocation of the protease to the opposite side of the serpin and its disruption[24]. The salt bridge between Glu[289] and Arg[362] was not observed in the available crystal structures of native NS, due to the intrinsic flexibility of the RCL. In α1-antitrypsin, a salt bridge links Glu[223] on strand s3C to Arg[354] on the RCL, near to the clevage site (P1-P1’) at residues Met358-Ser35925
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