The necessary chances of a thermodynamically metastable protein: inactivation and polymeritzation of human neuroserpin

Serpins are a wide class of proteins with high structural similarity, characterized by a unique substrate-like inhibitory mechanism that resembles a "molecular mousetrap". The active serpin is characterized by a main 5-stranded β-sheet and an exposed Reactive Centre Loop, which acts as a bait for the target protease. The cleavage of the loop by the protease triggers the insertion of the loop into the β-sheet as a strand and the disruptive translocation of the protease. This peculiar conformational mobility is achievable since serpins fold into a metastable native conformation. This feature gives a selective advantage to the serpin family to develop inhibitory activities, but leaves these proteins labile to misfolding and dysfunctional mutations, which cause a class of diseases known as serpinopathies. Indeed, the thermodynamically stable non-functional conformation can be reached without cleavage by intramolecular loop insertion (latent state) or by intermolecular insertion, leading to polymerization. Neuroserpin is a an inhibitor of tissue-type plasminogen activator that has a role in many pathologies, such as ischemia, Alzheimer disease, and FENIB (Familial Encephalopathy with Neuroserpin Inclusion Body). It is particularly suited to study the molecular basis of serpin inactivation and polymerization, since it may form latent conformers and polymers by thermal activation even in the wild type form. Here, we study the mechanism of neuroserpin unfolding and polymerization by different experimental techniques (static and dynamic light scattering, liquid chromatography, Fourier transform infrared spectroscopy) and by the support of Molecular Dynamics simulations. Our results show that at intermediate temperatures (45-55 °C) neuroserpin forms flexible polymers with a size from a few tens to a few hundreds of nanometers. At high temperatures, above 80 °C, our results reveal a different polymeric form, reached through an analogous loop-sheet mechanism, with considerably larger size and higher chemical stability. These observations highlight the fact that serpin polymerization is context-dependent, and that polymers accumulated in serpinopathies under physiological conditions may be different for different serpin and even for different pathological mutants of the same serpin.