Elucidating molecular connetion between IAHSP onset and Alsin protein by means of Homology Modelling and Molecular Dynamics Marcello Miceli, 1 Cecile Exertier, 2 Vallone Beatrice, 2 Marco Cavaglià, 1 Marco A. Deriu 1 1 PolitoBIOMedLab, Department of Mechanical and Aerospace Engineering, Politecnico di Torino,Turin; 2 Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”, Sapienza, Università di Roma, Rome, Italy Abstract The Infantile-onset Ascending Hereditary Spastic Paralysis (IAHSP) is an incurable rare neurodegerative disease related to a mutation-driven aberrant behaviour of the Alsin protein. The lack of information on Alsin atomic structure limits a complete understanding on pathology mechanisms. In this work, molecular mod- elling techniques have been applied to shed lights on Alsin folding dynamics and mis- function induced by aberrant mutations. Introduction Alsin is a key protein involved in the onset of the IAHSP, a rare pathology char- acterized by the degeneration of upper motor neurons of the pyramidal tract. The Alsin protein, encoded by the Als2 gene, 1 is composed of 1657 amino acids and also expressed in the spinal cord and brain. 2 Alsin is predicted as formed by three struc- tured domains, i.e.: i) the RCC1-like domain (RLD) in N-terminal position, ii) the central B cell lymphomahomology (DH) and pleckstrin-homology (PH) domain, and iii) the C-terminal vacuolar protein sorting 9 (VPS9) domain. In-vitro studies have proposed that a crucial step for the Alsin physiological pathway is the self- tetramerization and has highlighted how single point mutations in the sequence of VPS9 structured domain are correlated to a reduced tendency to oligomerise 3 and thus to the onset of the pathology. Nowadays, any resolved protein model for the Alsin is available, limiting computational studies and computer-aided drug discovery investi- gations. For this reason, the current research will be focused on the development of a homology model for the VPS9 domain of Alsin. The point mutation R1611W in VPS9, i.e. amino acid change in position 1611, Arginine to Tryptophane, altering Alsin function, will be investigated. Models developed will be employed to understand the effect of single point mutation on the stability of the structured domain and to suggest possible alteration on the self- oligomerization process. Materials and Methods To predict the secondary and tertiary structure, the VPS9 sequence obtained from the Uniprot database (Q96Q42) has been employed. Homology modelling has been performed through the I-Tasser and the quality of the model has been assessed with PROCHECK software. Subsequently, the obtained model of the VPS9 Wild Type (WT) has been mutated to obtain a model for the single point mutant R1611W (MUT). Three replicas of 1.5 μs long Molecular Dynamics (MD) simulations have been carried out both for the WT and MUT systems. All systems’ topology has been modelled through AMBER99sb-ildn forcefield. Systems were placed in a dodec- ahedron box filled with explicit water (TIP3P) and neutralized with Na + and Cl - ions added at a physiological concentration of 0.15 M. The engine employed for MD simulations and analysis was GROMACS 2020.3. Firstly temperature (298 K) and pressure (1 bar) have been equilibrated restraining protein positions. After remov- ing restraints the MD production was per- formed at constant temperature (298 K) and pressure (1 bar). The radius of gyration (Rg) has been employed as a measure of the compactness of the structure. Results have been obtained as the time average concate- nating the last 500 ns of simulations. Results The quality of VPS9 homology model (Figure 1A) has been evaluated through I- Tasser scores, i.e. the Confidence Score (C- Score) and Template Modelling Score (TM- Score) respectively equal to 1.76 and 0.96 ± 0.05. Moreover, the stereochemistry has been validated via the Ramachandran plot, which reported 95,6 % of residues in the most favoured regions and 4,6 % in the additional allowed regions (Figure 1B). Molecular dynamics simulation both on the WT and the MUT models have been done to understand the consequences of the mutation on the protein function. Figure 2 reports the probability distribution of Rg for both WT and MUT VPS9 models. Both systems show the same mean value for the distribution of Rg, but with remarkably different standard deviations (WT 15.4±0.42 Å, MUT 15.1±0.13 Å), showing a greater ability of the MUT sys- tem to explore a wider range of values com- pared to the WT. Discussion and Conclusions The current study represents the first step on a better comprehension of the Alsin protein both developing the first homology model for the VPS9 domain and study its dynamics via Molecular Modelling tech- niques. Computational results pointed attention to conformational modifications and flexibility changes in protein dynamics, induced by the R1611W mutation. Indeed, as observed in vitro, mutated forms of Alsin shows a reduced tendency to oligomerize. Being the Rg a measure of protein compact- ness, results suggest that the Wild Type (WT) can explore a broader range of con- formations than the Mutated (MUT), sug- Correspondence: Marcello Miceli, PolitoBIOMedLab, Department of Mechanical and Aerospace Engineering, Politecnico di Torino,Turin, Italy. E-mail: marcello.miceli@polito.it Key words: Molecular dynamics; homology modelling; neurodegenerative; IAHSP; rare pathologies. Acknowledgments: This work has been financed in the framework of the Telethon project CRYSTAL (GA: GSP20005_PAsIAHSP007). Disclosures: The authors have declared no conflicts of interest Conference presentation: This paper was pre- sented at the Third Centro 3R Annual Meeting - L’era delle 3R: modelli in silico, in vitro e in vivo per promuovere la ricerca traslazionale - 30 September - 1 October 2021, Evento online organizzato dal Politecnico di Torino. Received for publication: 9 July 2021. Accepted for publication: 7 September 2021. This work is licensed under a Creative Commons Attribution NonCommercial 4.0 License (CC BY-NC 4.0). ©Copyright: the Author(s), 2021 Licensee PAGEPress, Italy Biomedical Science and Engineering 2021; 4(s1):183 doi:10.4081/bse.2021.183 Biomedical Science and Engineering 2021; volume 4(s1):183 [page 88] [Biomedical Science and Engineering 2021; 4(s1):183] Non commercial use only