Cell Stress and Chaperones
Cell Stress and Chaperones
Bridget A. Culleton et al. “A role for the Parkinson’s Disease protein DJ-1 as a chaperone and antioxidant in the anhydrobiotic nematode Panagrolaimus superbus”.
Corresponding author: Ann M. Burnell, Biology Department, National University of Ireland Maynooth, Maynooth Co. Kildare, Ireland. Email
Homology Modelling & Molecular Dynamics
Supplementary Material Comprising Supplementary Text, Supplementary Figs. 2 & 3 and Supplementary Tables 3 & 4.
Using the crystal structure of human DJ-1 (PDB:2R1U) an homology model (HM) of the Panagrolaimus superbus homologue protein PsuDJ-1.1 was developed using the Modeller software implemented in Discovery Studio 3.5 [Fig. 4 a, b (main text); Supplementary Fig. 2]. To examine whether the 3D conformation of the homology model was stable it was subjected to 30ns aqueous solvent molecular dynamics (MD) simulation.
A number of quality assessment and validation tools tools, accessed at (http://swift.cmbi.kun.nl/WIWWWI), were used to validate our developed homology models for PsuDJ-1.1 The results of these validation assays are presented in Supplementary Table 3. The PsuDJ-1.1 model selected for MD simulation had yielded the overall best performance across the validation tools tested. ERRAT (Colovos and Yeates 1993) counts the number of non-bonded interactions between atoms (CC, CN, CO, NN, NO, and OO) within a cutoff distance of 3.5 Å and yields an overall quality factor for a protein structure, which is expressed as the percentage of the protein for which the calculated error value falls below a 95% rejection limit. Normally accepted model structures produce values above 50, with a higher score indicating that the model has a better ratio of non-bonded interactions. In this analysis the human DJ-1 (hDJ-1) template had a high score of 92.676%. The ERRAT score of the PsuDJ-1.1 homology model, while remaining acceptable, was lower at 74.64% (Supplementary Fig. 3) The introduction of protein flexibility through the MD simulations significantly improved the ERRAT quality score, yielding a score of 95.015% for the PsuDJ-1.1 HM after MD. Profiles-3D Verify Score checks the validity of a protein structure by measuring the compatibility score of each residue in the given 3D environment (Bowie et al. 1991). For the hDJ-1 template and both PsuDJ-1.1 models, this score was > 99.7% (Supplementary Table 3). PROCHECK (Laskowski et al. 1996) was employed to check stereochemical quality of the protein models. PROCHECK classifies every residue as having a favoured, additionally allowed, generously allowed, or disallowed conformation. Both the hDJ-1 2R1U template and the PsuDJ-1.1 HM had a high percentage of residues in the core favoured region at 94.9% for 2R1U and 92.3% for the PsuDJ-1.1HM and 89.3 % for the PsuDJ-1.1HM following MD. All structures had a low percentage of residues in the disallowed category, and hence the PsuDJ-1.1 structural models exhibited excellent stereochemistry. MD simulations are often used both as assessment and refinement tools for homology models of proteins. Our validation analyses using the Profiles 3D Verify Score and ERRAT tools show that the conformational flexibility facilitated by MD has resulted in an improvement in the observed non-bonded interactions and general protein structure for the PsuDJ-1.1 MD HM.
A representative snapshot of the simulated PsuDJ1.1 protein was overlaid on the original homology model with a root mean square deviation (RMSD) of 1.776Å (Supplementary Fig. 2a). This overlay shows that the secondary structure and overall 3D conformation of the protein has remained stable over the simulation with more flexibility observed in loop regions. When the refined MD model of PsuDJ1-1 dimer was overlaid on the hDJ1 template an RMSD of 1.834 Å was achieved – an indication of the strong conservation of structure between the two molecules (Supplementary Fig. 2b). The residues comprising each secondary structure region for the hDJ-1 template and the PsuDJ-1.1 models are indicated in Supplementary Table 4.
Supplementary Table 3. Analysis of the template and resultant PsuDJ-1.1 homology model (HM) and PsuDJ-1.1 HM after 30 ns of molecular dynamics simulation (MD) with the protein structure validation tools ERRAT, Profiles-3D Verify Score and Procheck.
hDJ-1 (PDB: 2R1U) / PsuDJ-1.1 HM / PsuDJ-1.1 HM after MDERRAT (%) / 92.676 / 72.77 / 95.015
Profiles-3D Verify Score(%)* / 100.00% / 99.73% / 100.00%
Procheck / 94.9% core
4.4% allowed
0.0% generously allowed
0.6% disallowed / 92.3% core
4.6% allowed
2.8% generously allowed
0.3% disallowed / 89.3 % core
9.2 % allowed
0.0 % generously allowed
1.5 % disallowed
*Percentage of the residues with an averaged Profiles-3D Verify Score > 0.2.
Supplementary Table 4. Comparison of the secondary structure regions observed in the hDJ-1 crystal structure (PDB: 2R1U) and the modelled PsuDJ-1.1 structures.
1 / 16-28 / 15-27 / 15-27
2 / 58-64 / 57-61 / 57-61
3 / 76-83 / 75-81 / 74-79
4 / 86-97 / 83-95 / 83-95
5 / 109-114 / 106-112 / 105-112
6 / 127-133 / 124-131 / 127-132
7 / 158-173 / 154-169 / 154-168
8 / 175-185 / 171-181 / 171-179
B Strand / hDJ-1 (PDB: 2R1U) / PsuDJ-1.1 HM / PsuDJ-1.1 HM after MD
1 / 5-10 / 4-8 / 4-9
2 / 32-37 / 31-36 / 31-37
3 / 44-45 / 42-44 / 42-44
4 / 51-52 / 49-51 / 50-52
5 / 56-57 / 54-55 / 55-56
6 / 69-72 / 67-70 / 67-70
7 / 101-105 / 98-102 / 99-101
8 / 122-123 / 118-120 / 119-120
9 / 140-141 / 135-137 / 136-137
10 / 146-148 / 142-143 / 142-144
11 / 152-155 / 148-151 / 148-151
Supplementary Fig. 2. a) The initial PsuDJ-1.1 homology model (in red) overlaid on a representative snapshot of of the PsuDJ-1.1 dimer (in blue) after 30 ns of molecular dynamics simulation. The root mean square deviation (RMSD) between the two was 1.776Å. b). The hDJ1 structure (in yellow) overlaid on a representative snapshot of the PsuDJ-1.1 dimer (in blue) after 30 ns of molecular dynamics simulation with an RMSD of 1.834 Å. Images were generated using Pymol (DeLano 2002).
Supplementary Fig. 3. ERRAT plots for a) the hDJ-1 2R1U template; b) the PsuDJ-1.1 HM and c) the PsuDJ-1.1 MD simulated structure. Two lines are drawn on the error axis to indicate the confidence with which it is possible to reject regions that exceed that error value. For PsuDJ-1.1 HM (panel b) the low quality regions are located in the b-1 strand and adjacent loop region, the b-6 strand and a-3 helix region and the b-9 strand region.
References
Bowie JU, Lüthy R, Eisenberg D (1991) A method to identify protein sequences that fold into a known three-dimensional structure. Science 253:164-170
Colovos C, Yeates T (1993) Verification of protein structures: patterns of nonbonded atomic interactions. Protein Sci 2:1511-1519
DeLano WL (2002) The PyMOL Molecular Graphics System. In. DeLano Scientific LLC, San Carlos, CA, USA. http://www.pymol.org
Laskowski R, Rullmannn J, MacArthur M, Kaptein R, Thornton J (1996) AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J Biomol NMR 8:477-486
1