The stability and activity of human neuroserpin are modulated by a salt bridge that stabilises the reactive centre loop.
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.
National Research Council of Italy, Institute of Biophysics, Palermo, Italy.
Rosina
R
Noto
National Research Council of Italy, Institute of Biophysics, Palermo, Italy.
Loredana
L
Randazzo
National Research Council of Italy, Institute of Biophysics, Palermo, Italy.
Samuele
S
Raccosta
Department of Medical Biotechnologies and Translational Medicine, University of Milan, Milan, Italy.
Sonia
S
Caccia
Department of Biology and Biotechnologies "Charles Darwin" and Pasteur Institute - Cenci Bolognetti Foundation, Sapienza University of Rome, Rome, Italy.
Claudia
C
Moriconi
Department of Biology and Biotechnologies "Charles Darwin" and Pasteur Institute - Cenci Bolognetti Foundation, Sapienza University of Rome, Rome, Italy.
Elena
E
Miranda
National Research Council of Italy, Institute of Biophysics, Palermo, Italy.
Vincenzo
V
Martorana
National Research Council of Italy, Institute of Biophysics, Palermo, Italy.
Mauro
M
Manno
D003198:Computer Simulation; D006801:Humans; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics; D009479:Neuropeptides / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D058105:Polymerization; D055550:Protein Stability; D017433:Protein Structure, Secondary; D015203:Reproducibility of Results; D012492:Salts / Q000378:metabolism*; D015843:Serpins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D000097522:Neuroserpin
D009479:Neuropeptides; D012492:Salts; D015843:Serpins; D000097522:Neuroserpin