Amyotrophic Lateral Sclerosis Type 20 - In Silico Analysis and Molecular Dynamics Simulation of hnRNPA1.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that affects the upper and lower motor neurons. 5-10% of cases are genetically inherited, including ALS type 20, which is caused by mutations in the hnRNPA1 gene. The goals of this work are to analyze the effects of non-synonymous single nucleotide polymorphisms (nsSNPs) on hnRNPA1 protein function, to model the complete tridimensional structure of the protein using computational methods and to assess structural and functional differences between the wild type and its variants through Molecular Dynamics simulations. nsSNP, PhD-SNP, Polyphen2, SIFT, SNAP, SNPs&GO, SNPeffect and PROVEAN were used to predict the functional effects of nsSNPs. Ab initio modeling of hnRNPA1 was made using Rosetta and refined using KoBaMIN. The structure was validated by PROCHECK, Rampage, ERRAT, Verify3D, ProSA and Qmean. TM-align was used for the structural alignment. FoldIndex, DICHOT, ELM, D2P2, Disopred and DisEMBL were used to predict disordered regions within the protein. Amino acid conservation analysis was assessed by Consurf, and the molecular dynamics simulations were performed using GROMACS. Mutations D314V and D314N were predicted to increase amyloid propensity, and predicted as deleterious by at least three algorithms, while mutation N73S was predicted as neutral by all the algorithms. D314N and D314V occur in a highly conserved amino acid. The Molecular Dynamics results indicate that all mutations increase protein stability when compared to the wild type. Mutants D314N and N319S showed higher overall dimensions and accessible surface when compared to the wild type. The flexibility level of the C-terminal residues of hnRNPA1 is affected by all mutations, which may affect protein function, especially regarding the protein ability to interact with other proteins

Schematic representation of the domains found on hnRNPA1.

<p>The two RNA recognition motifs (RRM 1 and 2) are represented in blue, the glycine-rich domain is represented in purple, the RNA-binding box is represented in green, and the nuclear localization signal M9 is represented in pink. The red arrows indicate the location where the four known mutations occur: position 73 (mutation N73S), position 314 (mutations D314V and D314N) and position 319 (mutation N319S).</p

Validation of the <i>in silico</i> modeled hnRNPA1 structure.

<p>The modeled structure was validated by PROCHECK, Rampage and ERRAT. (A) PROCHECK’s Ramachandran plot indicates that 89.9% of residues lie in most favored regions, 8.2% in additional allowed regions, 1.5% in generously allowed regions and 0.4% in disallowed regions. (B) Rampage’s Ramachandran plot shows 95.7% of residues in favored regions, 2.7% in allowed regions and 1.6% in outlier regions. (C) According to ERRAT, the structure obtained an 89.011 overall quality factor.</p

Representation of hnRNPA1 colored according to B-factor.

<p>Warm colors indicate high B-factor values, whereas cold colors indicate low B-factor values. (A) Wild type protein. (B) Mutant N73S. (C) Mutant D314V. (D) Mutant D314N. (E) Mutant N319S.</p

Radius of gyration (Rg) of Cα atoms as a function of time.

<p>The Radius of gyration of Cα atoms of the wild type and the mutants during the MD trajectory is shown. (A) The wild type is represented in black, and mutant N73S in red. (B) The wild type is represented in black, and mutant D314V in green. (C) The wild type is represented in black, and mutant D314N in purple. (D) The wild type is represented in black, and mutant N319S in pink.</p

RMSF for each residue of hnRNPA1.

<p>The Root-mean-square Fluctuation for each residue of hnRNPA1 is shown. (A) The wild type is represented in black, and mutant N73S in red. (B) The wild type is represented in black, and mutant D314V in green. (C) The wild type is represented in black, and mutant D314N in purple. (D) The wild type is represented in black, and mutant N319S in pink.</p

Number of “deleterious” and “neutral” predictions of each hnRNPA1 mutation.

<p>The four known mutations were analyzed by non-synonymous single nucleotide polymorphism (nsSNP) prediction algorithms. The graph indicates how many algorithms predicted each mutation as having a deleterious effect or a neutral effect on hnRNPA1. Blue bars indicate neutral predictions, and purple bars indicate deleterious predictions.</p

Functional effect prediction of hnRNPA1 natural variants by different SNP prediction algorithms.

<p>Functional effect prediction of hnRNPA1 natural variants by different SNP prediction algorithms.</p

Backbone RMSD as a function of time.

<p>The RMSD for the backbone atoms of the wild type and the mutants are shown as a function of time. Wild type is represented in black, mutant N73S in red, mutant D314V in green, mutant D314N in purple, and mutant N319S in pink.</p

Conservation analysis of each hnRNPA1 amino acid.

<p>HnRNPA1 represented as a space-filling model with the conservation grades color-coded onto each amino acid, viewed from three different angles. As the color-coding bar shows, bordeaux indicates highly conserved amino acids, while turquoise indicates variable positions. Amino acids colored in yellow did not receive a conservation score due to insufficiency of data. According to Consurf analysis, position 73 is variable, position 314 is highly conserved and position 319 is conserved.</p