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

Noto R, Randazzo L, Raccosta S, Caccia S, Moriconi C, Miranda E, Martorana V, Manno M - Sci Rep (2015)

Bottom Line: 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.Our results showed that deletion of the salt bridge leads to a moderate but clear reduction of the overall protein stability and activity.

View Article: PubMed Central - PubMed

Affiliation: National Research Council of Italy, Institute of Biophysics, Palermo, Italy.

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.

No MeSH data available.


Related in: MedlinePlus

The alchemic mutation of E289A NS in MD simulations.(a) Cartoon representation of native NS; the lateral chains of residues E289 and R362 are shown in bond representation. (b–d) Time evolution of the distance between the selected residues highlighted in the circular insets: (b) E289-R362 (black line) in WT NS; (c) A289-R362 (red line) in E289A NS; (d) E289-R362 (blue line) in WT NS after the back mutation A289E;
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f1: The alchemic mutation of E289A NS in MD simulations.(a) Cartoon representation of native NS; the lateral chains of residues E289 and R362 are shown in bond representation. (b–d) Time evolution of the distance between the selected residues highlighted in the circular insets: (b) E289-R362 (black line) in WT NS; (c) A289-R362 (red line) in E289A NS; (d) E289-R362 (blue line) in WT NS after the back mutation A289E;

Mentions: In our previous work23, we reported a long MD simulation of native NS, which showed the formation of a persistent salt bridge between the arginine Arg362 on the RCL and the glutamic acid Glu289 on strand s2C in the main protein core (Fig. 1a). Figure 1b shows that the distance between the acid or basic groups of of the two residues is below 3 Å for most of the simulation run. Such a structural detail remained undetected in previous studies of NS.


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

Noto R, Randazzo L, Raccosta S, Caccia S, Moriconi C, Miranda E, Martorana V, Manno M - Sci Rep (2015)

The alchemic mutation of E289A NS in MD simulations.(a) Cartoon representation of native NS; the lateral chains of residues E289 and R362 are shown in bond representation. (b–d) Time evolution of the distance between the selected residues highlighted in the circular insets: (b) E289-R362 (black line) in WT NS; (c) A289-R362 (red line) in E289A NS; (d) E289-R362 (blue line) in WT NS after the back mutation A289E;
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556959&req=5

f1: The alchemic mutation of E289A NS in MD simulations.(a) Cartoon representation of native NS; the lateral chains of residues E289 and R362 are shown in bond representation. (b–d) Time evolution of the distance between the selected residues highlighted in the circular insets: (b) E289-R362 (black line) in WT NS; (c) A289-R362 (red line) in E289A NS; (d) E289-R362 (blue line) in WT NS after the back mutation A289E;
Mentions: In our previous work23, we reported a long MD simulation of native NS, which showed the formation of a persistent salt bridge between the arginine Arg362 on the RCL and the glutamic acid Glu289 on strand s2C in the main protein core (Fig. 1a). Figure 1b shows that the distance between the acid or basic groups of of the two residues is below 3 Å for most of the simulation run. Such a structural detail remained undetected in previous studies of NS.

Bottom Line: 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.Our results showed that deletion of the salt bridge leads to a moderate but clear reduction of the overall protein stability and activity.

View Article: PubMed Central - PubMed

Affiliation: National Research Council of Italy, Institute of Biophysics, Palermo, Italy.

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.

No MeSH data available.


Related in: MedlinePlus