Molecular dynamics and protein frustration analysis of human fused in Sarcoma protein variants in Amyotrophic Lateral Sclerosis type 6: An In Silico approach

Abstract

Amyotrophic lateral sclerosis (ALS) is the most frequent adult-onset motor neuron disorder. The disease is characterized by degeneration of upper and lower motor neurons, leading to death usually within five years after the onset of symptoms. While most cases are sporadic, 5%-10% of cases can be associated with familial inheritance, including ALS type 6, which is associated with mutations in the Fused in Sarcoma (FUS) gene. This work aimed to evaluate how the most frequent ALS-related mutations in FUS, R521C, R521H, and P525L affect the protein structure and function. We used prediction algorithms to analyze the effects of the non-synonymous single nucleotide polymorphisms and performed evolutionary conservation analysis, protein frustration analysis, and molecular dynamics simulations. Most of the prediction algorithms classified the three mutations as deleterious. All three mutations were predicted to reduce protein stability, especially the mutation R521C, which was also predicted to increase chaperone binding tendency. The protein frustration analysis showed an increase in frustration in the interactions involving the mutated residue 521C. Evolutionary conservation analysis showed that residues 521 and 525 of human FUS are highly conserved sites. The molecular dynamics results indicate that protein stability could be compromised in all three mutations. They also affected the exposed surface area and protein compactness. The analyzed mutations also displayed high flexibility in most residues in all variants, most notably in the interaction site with the nuclear import protein of FUS.

Fig 1. Graphic representation of the domains that compose the FUS protein.

LC represents the low complexity, prion-like domain; RGG1 represents the first R-G-G pattern-rich domain; RRM represents the RNA-recognition motif; RGG2 represents the second R-G-G pattern-rich domain; ZnF represents the zinc-finger domain; RGG3 represents the third R-G-G pattern-rich domain, and NLS represents the nuclear localization signal.

Fig 2. Mutational frustration analysis.

Levels of mutational frustration by each residue-residue interaction. The colored scale represents the levels of frustration, in which blue represents minimal frustration, gray represents neutral frustration, and yellow represents high frustration. A) Wild-type FUS. B) R521C variant. C) R521H variant. D) P525L variant.

Fig 3. Evolutionary conservation analysis.

Amino acid conservation analysis of wild-type FUS protein fragment (residue 507 to 526) represented in a space-filling model and colored according to conservation scores. Conservation scores of the residues where R521C, R521H, and P525L mutations occur are highlighted in the table. CONSURF color scheme representing conservation levels. Cyan represents more variable positions, while bordeaux represents more conserved positions.

Fig 4. Root mean square deviation (RMSD) of the backbone atoms of the protein as a function of time.

The wild-type FUS protein [PDB ID: 4FDD] is represented in black, variant R521C is represented in blue, variant R521H is represented in green, variant P525L is represented in red, and variant P525L-PDB [ID: 7CYL] is represented in yellow.

Fig 5. Root mean square fluctuation (RMSF) of the backbone atoms of the protein as a function of each amino acid residue.

The wild-type FUS protein is represented in black, variant R521C is represented in blue, variant 521H is represented in green, variant P525L is represented in red, and variant P525L-PDB [ID: 7CYL] is represented in yellow.

Fig 6. Radius of gyration (RG) of the backbone atoms of the protein as a function of time.

The wild-type FUS protein is represented in black, variant R521C is represented in blue, variant 521H is represented in green, variant P525L is represented in red, and variant P525L-PDB [ID: 7CYL] is represented in yellow.

Fig 7. Solvent-accessible surface area (SASA) of the protein as a function of time.

The wild-type FUS protein is represented in black, variant R521C is represented in blue, variant 521H is represented in green, variant P525L is represented in red, and variant P525L-PDB [ID: 7CYL] is represented in yellow.