Functional and dysfunctional conformers of human neuroserpin characterized by optical spectroscopies and Molecular Dynamics

Abstract

Neuroserpin (NS) is a serine protease inhibitor (SERPIN) involved in different neurological pathologies, including the Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB), related to the aberrant polymerization of NS mutants. Here we present an in vitro and in silico characterization of native neuroserpin and its dysfunctional conformation isoforms: the proteolytically cleaved conformer, the inactive latent conformer, and the polymeric species. Based on circular dichroism and fluorescence spectroscopy, we present an experimental validation of the latent model and highlight the main structural features of the different conformers. In particular, emission spectra of aromatic residues yield distinct conformational fingerprints, that provide a novel and simple spectroscopic tool for selecting serpin conformers in vitro. Based on the structural relationship between cleaved and latent serpins, we propose a structural model for latent NS, for which an experimental crystallographic structure is lacking. Molecular Dynamics simulations suggest that NS conformational stability and flexibility arise from a spatial distribution of intramolecular salt-bridges and hydrogen bonds.

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

“…Interestingly, many of the main HBs that stabilised β -sheet A between strands s3A and s5A were lost in the mutant form of NS. As studied in our previous work 23 , the free-energy gain obtained in the conformational change from native to latent or cleaved NS is largely controlled by an increased number of HBs. Thus, we may argue that E289A NS may present a lower stability towards the non-native conformations that require the insertion of RCL as a strand in β -sheet A.…”

“…In our previous work 23 , 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 ).…”

“…After the determination of the crystal structure of the native and cleaved conformers of NS 9 10 , other studies have revealed the main structural features of monomeric and polymeric NS 20 21 22 23 . In a recent work 23 , we performed Molecular Dynamics (MD) simulations of different NS isoforms, finding that the conformational stability and flexibility of NS arise from a characteristic spatial distribution of intramolecular salt bridges and hydrogen bonds. The MD trajectory for native NS revealed the presence of a persistent salt bridge between the glutamic acid Glu289 on strand s2C in the main protein core and the arginine Arg362 located in the reactive centre loop (RCL), close to the cleavage site (P1-P1’).…”

Interactions between N‐linked glycosylation and polymerisation of neuroserpin within the endoplasmic reticulum

“…For the calculation of the RMSF of the wild type NS, we used the same MD trajectory described in . The reported RMSF are averages over the last 20 ns of the simulation and were mapped to a representative structure using Chimera .…”

“…The reported RMSF are averages over the last 20 ns of the simulation and were mapped to a representative structure using Chimera . For comparative analysis of the G392E and G392R variants, two MD trajectories of 50 ns each were generated using the namd 2 package and the Charm22 force field using the protocol reported in . The two variants were generated by using the vmd package , starting from a configuration obtained from a previous simulation of wild type NS .…”