By Toni Giorgino
Agel amyloidosis (also known as Finnish-type) is a rare hereditary disease caused by the abnormal aggregation and accumulation of fragments of the gelsolin protein. The fragments form fibrillar aggregates and affect the cornea, facial nerves, skin, and kidneys. A number of mutations in the G2 domain of the protein have been so far identified as disease-causing. While several mutated forms of G2 have been crystallized in the past, providing insights on the molecular etiology of fibrillation and possible therapeutic pathways, the D187N has long remained elusive, probably due to a shifted order-disorder equilibrium.
A collaboration between researchers at the National Research Council of Italy, the Mario Negri Institute, the University of Milan, the Scripps Institute, and Ghent University, applied a novel crystallization strategy, employing a nanobody (a small portion of llama-specific antibodies) raised against the WT protein, to stabilize the elusive D187N mutant and finally obtain its crystal structure. Yet, the success raised further provocative questions about the precise causal relationship between the genetic defect and the disease. Above all, two questions became pressing: (a) why does the mutation cause aggregation, given that the mutant has an overall arrangement largely similar to the WT? And, on the other hand, (b) how does the nanobody exert its protective effect, given that it is bound to an entirely different interface with respect to the one involved in the aberrant, disease-causing proteolysis?
Molecular dynamics simulations (built with HTMD and run with ACEMD) were central to resolve the structurally puzzling questions. First,long-timescale simulations of the dynamics of the four systems (WT and mutant; with and without bound nanobody) demonstrated a destabilization effect of the mutation in the “elbow” region, which shifted the equilibrium of the C-terminal loop towards a more dynamic state and enabling the exposure of the hydrophobic core of the protein. Conversely, the nanobody bound to the elbow region and largely restored the compact character of the domain by restricting the conformational space accessible to the open linker. Thus, the modulation due to the mutation and the nanobody were in large part kinetic, and were made visible in all-atom MD simulations.
Of note, simulations showed that the nanobody functioned as a “pharmacological chaperone” (pharmacoperone), an uncommon class of drugs whose therapeutic potential is exerted by preventing misfolding, e.g. stabilizing native folds. The drug-like potential was confirmed in vivo with C. elegans-based toxicity assays, a further testimony of the potential of MD in drug discovery efforts.
Toni Giorgino, Davide Mattioni, Amal Hassan, Mario Milani, Eloise Mastrangelo, Alberto Barbiroli, Adriaan Verhelle, Jan Gettemans, Maria Monica Barzago, Luisa Diomede, Matteo de Rosa, Nanobody interaction unveils structure, dynamics and proteotoxicity of the Finnish-type amyloidogenic gelsolin variant, Biochimica et Biophysica Acta (BBA) – Molecular Basis of Disease. Volume 1865, Issue 3, 1 March 2019, Pages 648-660, doi:10.1016/j.bbadis.2019.01.010. arXiv:1903.07308