Molecular Dynamics of the Alsin DH/PH domain toward a better understanding of Infantile-onset Ascending Hereditary Spastic Paraplegia

Infantile-onset ascending hereditary spastic paralysis (IAHSP) is rare neurodegenerative disease characterized by onset of spasticity to lower limbs within the second year of life and progression towards spastic tetraparesis. This disorder is associated with mutations at the Amyotrophic Lateral Sclerosis type 2 (ALS2) gene, which encodes for Alsin, a protein composed by 1657 amino acids organized in multiple domains. Several studies on transgenic mice have highlighted its crucial role in vesicular trafficking, neuronal development, and homeostasis by virtue of its ability to interact with two guanosine triphosphatases, Rac1 and Rab5. In particular, evidence suggest that Rac1 can bind Alsin central region, composed by two structured domains i.e. a Dbl Homology (DH) domain followed by a Pleckstrin Homology (PH) domain. In vitro experiments have shown that this interaction is necessary for the subsequent activation of Rab5 through Alsin C-terminal region, leading to the maturation and fusion of different types of vesicles. However, as far as we know, the three-dimensional structure of Alsin protein and its relationship with specific functions are still unknown. Computational Molecular Modelling is an elective tool to study nanoscale level biological systems, both allowing to model the 3D structure and the dynamics of proteins. In this work, the first homology model of Alsin DH/PH domain was developed and studied through Molecular Dynamics both in presence and in absence of its binding partner, Rac1. As a proof of the results robustness, the employed experimental setup was first validated replicating MD studies on homologues DH/PH domains reported in literature. Regarding Alsin DH/PH essential dynamics, it consisted in a collective motion of PH region independently of Rac1 interaction. Due to different conformations of DH domain, the presence of Rac1 seems to stabilize an open state of the protein, while absence of its binding partner results in closed conformations. Furthermore, Rac1 interaction was able to reduce the fluctuations in the second conserved region of DH motif, which may be involved in the formation of a homodimer. Moreover, the dynamics of DH/PH was described through a Markov State Model to study the pathways linking the open and closed states. In conclusion, this work provided the first all atom model for DH/PH domain of Alsin protein, moreover, MD investigations suggested underlying molecular mechanisms in the signal transduction between Rac1 and Alsin, providing the basis for a deeper understanding of the whole structure-function relationship for the Alsin protein.