Biophysical Society Thematic Meeting | Trieste 2024

Emerging Theoretical Approaches to Complement Single-Particle Cryo-EM

Poster Abstracts

16-POS Board 16 UNDERSTANDING THE MOLECULAR CONSEQUENCES OF THE R1611W MUTATION IN ALSIN'S VPS9 DOMAIN: EXPERIMENTAL AND COMPUTATIONAL PERSPECTIVES Marcello Miceli 1 ; Elena Gugole 2 ; Exertier Cécile 2 ; Lorenzo Pallante 1 ; Beatrice Vallone 2 ; Marco Agostino Deriu 1 ; 1 Politecnico di Torino, Department of Mechanical and Aerospace engineering, Turin, Italy 2 Università La Sapienza , Department of Biochemical Sciences " Alessandro Rossi Fanelli," Rome, Italy The expression of mutated forms of the Alsin protein has been linked to Infantile Onset Ascending Hereditary Spastic Paraplegia (IAHSP), a rare neurodegenerative disease. One of the mutations identified as pathological is located in Alsin’s Vacuolar Protein Sorting 9 (Vps9). The Alsin’s Vps9 domain works as an exchange factor (GEF) for Rab5, and evidence suggests its involvement in the process of Alsin-mediated endosome formation. More in detail, it was observed that the missense mutation that produces the expression of a tryptophan instead of an asparagine (R1611W), in the Vps9 domain alters the oligomeric state of Alsin and its GEF functions affecting vesicular trafficking. An understanding of the molecular structure of the Vps9 domain in the Wild Type (WT) and the mutated case may help to understand the mechanisms involved in the onset of the disease. In this work, a complementary in-vitro/in-silico approach was implemented to shed light on the VPS9 domain secondary structure and understand the effects of the R1611W mutation on the isolated VPS9 domain of Alsin. Expression and purification of WT and Mutant VPS9 constructs were performed in the E. coli expression system. The circular dichroism and the size exclusion chromatography showed altered folding and a different oligomerisation behaviour because of the single-point mutation. Structure prediction and multiscale molecular dynamics, both all-atom and coarse-grained, were employed to elucidate the structure-to-function altered behaviour observed in the experiments. The mutation appears to induce conformational changes that alter the protein's structure and its ability to oligomerise. This study lays the groundwork for understanding how R1611W alters the function of the VPS9 domain and for developing an experimental protocol to obtain stable constructs for experimental structure determination experiments such as cryo-electron microscopy.

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