Computational and Functional Characterization of Angiogenin Mutations, and Correlation with Amyotrophic Lateral Sclerosis

<div><p>The Angiogenin (<i>ANG</i>) gene is frequently mutated in patients suffering from the neurodegenerative disease - amyotrophic lateral sclerosis (ALS). Most of the ALS-causing mutations in Angiogenin affect either its ribonucleolytic or nuclear translocation activity. Here we report the functional characterization of two previously uncharacterized missense mutations in Angiogenin - D22G and L35P. We predict the nature of loss-of-function(s) in these mutants through our previously established Molecular Dynamics (MD) simulation extended to 100 ns, and show that the predictions are entirely validated through biochemical studies with wild-type and mutated proteins. Based on our studies, we provide a biological explanation for the loss-of-function of D22G-Angiogenin leading to ALS, and suggest that the L35P-Angiogenin mutation would probably cause ALS symptoms in individuals harboring this mutation. Our study thus highlights the strength of MD simulation-based predictions, and suggests that this method can be used for correlating mutations in Angiogenin or other effector proteins with ALS symptoms.</p></div

Cartoon representation of human Angiogenin showing its functional sites and D22G, L35P mutations.

<p>The D22G and L35P mutations in Angiogenin (PDB ID: 1B1I) are labeled and represented as stick models (red). The three key functional sites of human Angiogenin (catalytic triad, nuclear localization signal and receptor binding site) are also shown. The color scheme is as follows - Angiogenin protein: yellow, nuclear localization signal: green, receptor-binding site: violet and catalytic triad of Angiogenin: yellow and blue stick models.</p

Snapshots extracted from MD simulations for D22G and L35P Angiogenin mutants.

<p><b>A)</b> Stable and native conformation of catalytic residue His114 from MD simulations of wild-type Angiogenin <b>B)</b> Conformational switching of His114 observed from the MD simulation of ALS associated D22G-Angiogenin mutant <b>C)</b> L35P-Angiogenin mutant showing conformational switching of His114 by 99° during the simulations. <b>D)</b> Wild-type Angiogenin showing an open packing and loose conformation of nuclear localization signal residues ³¹RRR³³ from MD simulations <b>E)</b> D22G mutant shows a similar conformation of ³¹RRR³³ residues compared to wild-type Angiogenin, suggesting no loss of nuclear translocation activity, while <b>F)</b> a closed and tight packing of ³¹RRR³³ residues observed from the MD simulation of L35P-Angiogenin, suggesting loss of nuclear translocation activity <b>G)</b> Control plots showing the RMSD profiles of the backbone atoms from the equilibrated conformation for wild-type Angiogenin and D22G, L35P-Angiogenin mutants during the MD simulations. RMSD time profiles for wild-type Angiogenin, D22G and L35P are shown in black, green and brown respectively.</p

Purification, secondary structure depiction and ribonucleolytic activity of wild-type Angiogenin-GST and mutants.

<p><b>A)</b> Coomassie stained SDS-PAGE gel showing wild-type Angiogenin-GST (lane 2), D22G (lane 3) and L35P (lane 4) proteins after Ni-NTA affinity purification. Lane 1 contains molecular weight markers. The Angiogenin proteins (14.1 kDa) are all tagged with Glutathione S-transferase (GST, ∼26 kDa) for soluble expression in <i>E. coli</i>. <b>B)</b> Plot showing CD spectra for wild-type (black), D22G (green), and L35P (brown) Angiogenin-GST proteins. Samples were diluted in PBS to yield a concentration of 0.4 mg/ml; three spectra were recorded, averaged and plotted after subtracting the buffer baseline for each sample. <b>C)</b> Ribonucleolytic activity of wild-type (black), D22G (green) and L35P (red) Angiogenin-GST proteins measured using yeast tRNA as substrate. The proteins, at concentrations of 0.05 to 0.5 mg/ml, were incubated with yeast tRNA (2 mg/ml) at 37°C for 2 hours. Undigested tRNA was precipitated by addition of ice-cold perchloric acid, and the absorbance of the supernatents was measured at 260 nm; data were collected from three independent experiments for each protein concentration. Student’s <i>t</i>-test of three independent experiments shows that the difference between wild-type and each of the three mutant protein is significant (n = 3; p<0.05). <b>D)</b> The loss of ribonucleolytic activity of D22G and L35P mutants compared to wild-type Angiogenin-GST. The amount of protein required to generate 1.0 optical density (OD) is compared with wild-type Angiogenin-GST to generate same OD unit for mutants.</p

Nuclear translocation activity of wild-type Angiogenin-GST and mutants.

<p>HeLa cells, untreated or incubated with 2 µg/ml of wild-type Angiogenin-GST, or the D22G, L35P mutants were fixed, permeabilized and stained with mouse anti-Angiogenin monoclonal antibody and Alexa Fluor 555 goat anti-mouse IgG, while the nuclei were counter-stained with 4′,6-diamidino-2-phenylindole (DAPI) dihydrochloride. Individual channels, as well as a merge, are shown. The magnification in all cases is 200X. The images are representative of cells from at least three areas (each area containing 35–50 cells) from two independent experiments.</p

Hydrogen bond interaction network for L35P mutant.

<p>The hydrogen bond interactions between contiguous amino acid residues based on a 3.2 Å cut-off has been presented here. The paths leading from the site of mutation Pro35 to catalytic residue His114 have been shown in blue square boxes. Other hydrogen bond interactions are shown in grey circles. The bond length is given on the edge between the nodes of amino acid residues. One of the path is mediating through Leu115 which plays a role in His114 conformational switching and the other path is mediating through Thr44 similar to that of WT-ANG.</p

Comparison of AutoDock and ParDOCK binding energy scores for NCI-65828 complexes with WT-ANG and ANG mutants.

<p>*(ANG in which there is no conformational change of the catalytic residue His114).</p

Percentage hydrogen bonding occupancy between Thr44-Thr80 and Asp116-Ser118.

<p>Percentage hydrogen bonding occupancy between Thr44-Thr80 and Asp116-Ser118.</p

RMSD and RMSF profile for WT-ANG and mutants.

<p>(A) Control plots representing the stability of the models during the molecular dynamics run. The root mean square deviation (RMSD) of the backbone atoms from the equilibrated conformation (0 ns) is presented as a function of time. The RMSD time profiles for WT-ANG, K17I, S28N, P112L, L35P, K60E and V113I are shown in black, red, dark green, blue, orange, pink, and light green, respectively. (B) Root mean square fluctuation (RMSF) values of atomic positions computed for the backbone atoms are shown as a function of residue number. The RMSF values for WT-ANG, K17I, S28N, P112L, L35P, K60E and V113I are shown in black, red, dark green, blue, orange, pink, and light green, respectively.</p

Cartoon representation of X-ray structure for Human Angiogenin (PDB code: 1B1I).

<p>Cartoon representation of the structure of Human Angiogenin (PDB entry 1B1I) showing its functional sites; catalytic triad residues are represented as stick models, nuclear localization signal is represented in magenta color and receptor binding site is represented in orange color. Figure produced using PyMOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032479#pone.0032479-DeLano1" target="_blank">[53]</a>.</p