Primary and Secondary Structure Prediction

Target identification

Primary structure analyses of all the Janus kinases (JAK) proteins were done using ExPASy-ProtParam. It was found that the aliphatic index of all the selected target proteins were comparatively high, which correspond to high thermodynamic stability of the protein molecules. The negative values for GRAVY (Grand average of hydropathicity), which supports the hydrophilic nature of proteins. The secondary structure prediction carried out using SOPMA(Geourjon and Deleage; 1995), which point out whether a given amino acid lies in a helix, strand or coil. It shows that the proteins were structurally stable because of the high percentage of α-Helix and low percentage of β-turn. The quality of each of the proteins was evaluated using Ramachandran plot obtained from MOE 2014.0901 (Lovell et al., 2003).

Primary and secondary structure prediction

The primary and secondary structure prediction results are shown in Table S1. On the detailed comparison and examinations of these result, in this study we selected four proteins such as 3LXK, 3PJC, 4HVG and 4RIO; 3IO7, 3TJD, 4F08, and 4FVP; 3EYH, 4K77, 4EI4 and 4E4N as the target proteins for the molecular docking studies of JAK3, JAK2 and JAK1 respectively. All these proteins have comparatively good resolution, having single cofactor with single chain.

The ProtParam analysis shows that theoretical pI of 3LXK, 3PJC, 4HVG and 4RIO are 6.78, 6.63, 6.63 and 6.43 respectively. The half-life estimated for these selected proteins were 30, 30, 1.2, and 30 hours respectively. And the SOPMA analysis gives α-Helix (%) values 43.43, 43.17, 43.31 and 47.78 respectively and the β-turn (%) values are 11.62, 10.48, 10.51 and 9.90 respectively for the selected proteins of JAK3. The SOPMA analysis also gives the extended strand and random coil values also.

The ProtParam analysis of 3IO7, 3TJD, 4F08, and 4FVP shows the theoretical pI values 8.16, 6.79, 6.79 and 6.77 respectively. The half-life for these selected proteins were 30, 30, 30 and 100 hours respectively. The α-Helix (%) values 39.94, 41.61, 40.73 and 38.41 and the β-turn (%) values 11.82, 10.74, 11.59 and 11.76 respectively. The extended strand and random coil values for these proteins were 17.89, 18.79, 18.54 and 17.30; 30.35, 28.86, 29.14 and 32.53 respectively.

Similarly the ProtParam analysis of JAK1 target proteins, 3EYH, 4K77, 4EI4 and 4E4N shows the theoretical pI values 7.18, 6.78, 6.16 and 6.16 respectively with half-life periods 100, 30, 30 and 30 hours. The percentage of α-Helix, β-turn, extended strand and random coil values were found to be 35.52, 9.66, 21.72, and 33.10 respectively for 3EYH and for the other proteins the values are similar, that is 33.77, 9.93, 21.85 and 34.44 respectively.

The Ramachandran plots of these selected proteins were shown in Supplementary Material S2. (Ramachandran et al., 1963) The result shows that the residues are mostly in the favored regions. For the protein 3LXK, the number of residues in the core region is 263 (95.64%). The number of residues in the allowed region is 11 (4%) and only one amino acid residue that is ARG 944 is present in the outlier region. In the protein 3PJC, there is no outlier residue. The number of residues in core region is 282 (95.39%), and the number of residues in allowed region is 13 (4.61%). For the protein 4HVG has three outlier amino acids ASN 832, ASP 967 and PRO 1080. The number of residues in the core region is 255 (92.73%), in the allowed region is 17 (6.18%) and in the outlier region is 3(1.09%). Similarly 4RIO shows the amino acid residue ASP 967 in the outlier region. The number of residues in the core region is 262 (92.58%), in the allowed region is 20 (7.08 %) and in the outlier region is 1 (0.35 %).

For the protein 3LXK, the number of residues in the core region is 264 (94.96%), the number of residues in allowed region is 13 (4.68) and only one amino acid residue that is LYS 945 in the outlier region. For the target protein 3TJD, the number of residues in the core region is 267 (94.68%), the number of residue in the allowed region is 14 (4.96%) and only one that is THR 888 amino acid in the outlier region. In protein 4F08, the number of residues in core region is 277 (96.18%), and the number of residues in allowed region is 10 (3.47%) with only one that is PRO 870 amino acid in the outlier region. In 4FVP, there is no outlier residue. The number of residues in core region is 260 (95.59%), and the number of residues in allowed region is 12 (4.41%).

The Ramachandran plot analysis of JAK1 proteins was also performed. The plot analysis of 3EYH shows 270 (95.41%) amino acid residues in the core region, 11 (3.89%) amino acids residues in the allowed region and two amino acids such as ASP 1031 and ASN 1119 in the outlier region. The plot analysis of 4E4N shows that there is no outlier residue. The number of residues in core and allowed region are 276 (96.17%) and 11 (3.83%) respectively. For the protein 4EI4, the number of residues in the core and allowed region are 275 (96.49%), and 9 (3.16%) respectively with the amino acid ASP 1031 as the outlier residue. Similarly for the protein 4K77, the number of residues in the core region are 260 (94.20%), in the allowed region are 13 (4.71%) and has 3 outlier residues, that is the amino acids such as LYS 974, ASP 1003 and ASP 1031 are in the outlier region. Such percentage distribution of the protein residues shows that the protein structures are of good quality.

[1]C. Geourjon, G. Deleage, SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments, Comput. Appl. Biosci. 11 (1995) 681-684.

[2]S.C. Lovell, I.W. Davis, W.B. Arendall, P.I. de Bakker, J.M. Word, M.G. Prisant, J.S. Richardson, D.C. Richardson, Structure validation by C alpha geometry: phi psi and C beta deviation, Proteins 50 (2003) 437-50.

[3]Ramachandran, G.N.; Ramakrishnan, C.; Sasisekharan, V. (1963). "Stereochemistry of polypeptide chain configurations". Journal of Molecular Biology. 7: 95–9.

Table S1. Primary and secondary structure prediction results

PDB ID / pI / Half-life (Hrs) / Instability index / Aliphatic / GRAVY / α-Helix(%) / β-turn(%) / Extended strand(%) / Random coil(%) / Resolution
JAK3 / 3LXK / 6.78 / 30 / 54.09 / 81.74 / -0.357 / 43.43 / 11.62 / 16.21 / 28.75 / 2.00
3PJC / 6.63 / 30 / 53.87 / 82.98 / -0.327 / 43.17 / 10.48 / 16.51 / 29.84 / 2.20
4HVG / 6.63 / 1.2 / 54.38 / 83.25 / -0.335 / 43.31 / 10.51 / 16.56 / 29.62 / 2.75
4RIO / 6.43 / 30 / 58.85 / 87.54 / -0.302 / 47.78 / 9.90 / 13.70 / 28.42 / 2.69
JAK2 / 3IO7 / 8.16 / 30 / 50.18 / 79.04 / -0.635 / 39.94 / 11.82 / 17.89 / 30.35 / 2.60
3TJD / 6.79 / 30 / 51.14 / 80.74 / -0.641 / 41.61 / 10.74 / 18.79 / 28.86 / 2.90
4F08 / 6.79 / 30 / 50.32 / 80.00 / -0.621 / 40.73 / 11.59 / 18.54 / 29.14 / 2.82
4FVP / 6.77 / 100 / 43.70 / 91.76 / -0.378 / 38.41 / 11.76 / 17.30 / 32.53 / 2.01
JAK1 / 3EYH / 7.18 / 100 / 44.82 / 80.97 / -0.507 / 35.52 / 9.66 / 21.72 / 33.10 / 2.00
4K77 / 6.78 / 30 / 44.82 / 80.33 / -0.525 / 33.77 / 9.93 / 21.85 / 34.44 / 2.40
4EI4 / 6.16 / 30 / 45.67 / 79.66 / -0.545 / 33.77 / 9.93 / 21.85 / 34.44 / 2.22
4E4N / 6.16 / 30 / 45.36 / 79.04 / -0.558 / 33.77 / 9.93 / 21.85 / 34.44 / 1.90

Fifure S1: Ramachandran plot