Introduction 1 2 3 4 5 6 7 8 9 6 10 11 12 13 14 15 1 11 18 19 1 16 17 18 19 20 21 Fig. 1 19 19 21  18 19 21 19 21 in silico in silico 22 R 23 R S 24 2 in silico Fig. 2 R R S  Modelling considerations 25 27 28 27 28 27 29 30 31 32 33 34 35 in silico 36 34 35 11 18 11 19 18 18 11 To substantiate this hypothesis, the three dimensional architectures of the human and rat CYP11B enzymes were constructed using comparative modelling. For reasons of relevance only the CYP11B1 and CYP11B2 isoforms were investigated. We intend to show how knowledge of these various hydroxylation patterns of aldosterone precursors can result in working models for the substrate selective activity of the two isoforms. From here on, the human isoforms will be noted as hCYP11B1 and hCYP11B2, whereas the rat isoforms will be noted as rCYP11B1 and rCYP11B2. in silico 22 37 R 23 38 19 24 39 Methods Homology modelling 40 41 42 3 43 45 Fig. 3 Topology alignment of human and rat CYP11B isoforms to related cytochrome P450 enzymes of which a three dimensional structure has been elucidated. Indicated with a & are the Arg123 in alpha-helix B′ and Glu310 in alpha-helix I. Indicated with a * is the triple mutant L301P, E302D, A320V in alpha-helix I. Indicated with a ^ is the catalytic Thr318 in alpha-helix I. Indicated with a # is the conserved Glu459 in alpha-helix L  46 47 47 1 Table 1 Generic pair wise sequence identity (in percentages) between the human and rat CYP11B isoforms and cytochrome P450 enzymes for which a three dimensional structure has been elucidated Chains 101 102 107 108 119 55 51 2B4 2C5 2C8 2C9 2D6 3A4 h11B1 h11B2 r11B1 r11B2 r11B3  CYP101 – 17.3 30.2 37.7 39.6 38.5 23.5 21.2 23.1 30.8 23.1 11.5 19.6 26.4 24.5 24.5 26.4 26.4 2CPP CYP102 16.3 – 24.5 28.3 39.6 19.2 29.4 26.9 34.6 30.8 32.7 14.8 47.1 26.4 26.4 24.5 24.5 24.5 1BU7 CYP107 20.0 12.3 – 28.3 50.9 36.5 21.6 19.2 21.2 21.2 25.0 14.3 21.6 20.8 18.9 20.8 18.9 18.9 1JIN CYP108 23.2 15.8 22.1 – 35.8 28.8 29.4 25.0 26.9 28.8 25.0 17.9 29.4 26.4 26.4 28.3 28.3 28.3 1CPT CYP119 18.8 16.0 25.6 20.8 – 34.6 27.5 28.8 28.8 32.7 28.8 25.0 29.4 26.4 26.4 28.3 26.4 26.4 1F4U CYP55 21.2 11.2 28.5 24.7 24.3 – 19.6 17.3 19.2 21.2 23.1 18.5 19.6 18.9 17.0 17.0 17.0 17.0 1ROM CYP51 12.3 18.0 19.1 17.2 14.7 16.0 – 23.1 28.8 25.0 25.0 11.1 35.3 20.8 20.8 17.0 20.8 20.8 1EA1 CYP2B4 14.6 16.7 16.4 14.0 15.0 16.0 16.7 – 57.7 65.4 59.6 33.3 33.3 32.1 32.1 32.1 30.2 30.2 1SUO CYP2C5 16.8 17.8 16.6 14.8 16.9 15.0 14.9 51.0 – 69.2 78.8 44.4 33.3 32.1 32.1 28.3 32.1 32.1 1NR6 CYP2C8 15.8 17.6 16.4 15.5 15.8 15.8 13.4 53.8 73.6 – 69.2 44.4 31.4 30.2 30.2 30.2 30.2 30.2 1PQ2 CYP2C9 15.6 18.0 17.4 15.0 16.6 15.3 14.3 51.0 77.3 78.4 – 40.7 33.3 32.1 32.1 28.3 30.2 30.2 1OG2 CYP2D6 13.1 16.9 14.9 14.3 14.7 16.0 16.7 39.6 40.0 40.6 38.5 – 14.8 25.0 25.0 25.0 21.4 21.4 2F9Q CYP3A4 14.1 22.0 19.6 14.5 17.2 14.8 16.5 22.8 22.0 22.9 21.9 17.9 – 32.1 32.1 30.2 30.2 30.2 1W0E h11B1 16.0 16.3 14.6 15.5 16.1 12.0 14.9 17.4 17.6 15.8 17.3 14.9 17.1 – 98.1 81.1 86.8 84.9 – h11B2 15.3 16.9 13.6 14.8 15.8 12.0 14.5 17.4 17.6 16.2 17.7 15.5 17.5 93.6 – 83.0 88.7 86.8 – r11B1 12.3 15.4 13.2 13.8 15.3 11.0 13.4 17.4 16.7 16.6 17.7 14.4 17.3 63.6 63.6 – 88.7 86.8 – r11B2 15.3 15.8 14.4 14.5 16.6 13.0 14.7 15.9 16.2 15.8 17.1 13.5 16.8 68.2 68.8 82.6 – 98.1 – r11B3 15.4 15.8 14.4 14.5 16.6 13.0 14.7 15.9 16.2 15.8 17.1 13.8 16.8 68.6 69.2 83.0 97.3 – – Pseudomonas-Putida Bacillus Megaterium Saccharopolyspora-Erythreaea Pseudomonas Archaeon Sulfolobus Solfataricus Fusarium-Oxysporum Mycobacterium Tuberculosis Oryctolagus Cuniculus Homo Sapiens 42 30 34 35 48 33 49 11 18 50 51 52 53 54 11 18 3 43 45 3 55 2 36 11 18 19 11 18 19 56 Ligand docking 57 default 1 5 57 binding R 2 N binding 58 Docking of steroids was performed in the presence of an iron-bound oxygen atom and their conformations were afterwards checked to investigate alternate orientations of the steroid in the active site cavity. The protein structures used for docking the substrates were the unequilibrated structures, whereas for docking the inhibitors, the hCYP11B1 and hCYP11B2 models were used after they were equilibrated with the ligand 18-hydroxycorticosterone. All docking runs were performed in the absence of water molecules. For each inhibitor, the best ranked pose was used as input for the molecular dynamics study. Molecular dynamics of inhibitors 59 55 60 3 3 3 3 3 3 Cellular assay for measuring inhibitor in vitro activity 61 62 62 64 2 Results and discussion Model quality n−1 n α,n n n α,n n n+1 65 2 Table 2 Validation results for the lowest energy models of CYP11B1 and CYP11B2 and the crystal structures which were used for the template, part I  Ramachandran Plot (core regions) (%) Ramachandran Plot (favourable regions) (%) a b Template PDB, resolution CYP101 (2CPP, 1.63 Å) 92.1 100.0 96.0 197 CYP2C5 (1NR6, 2.10 Å) 87.8 99.2 93.6 195 Model hCYP11B1 78.8 94.7 84.1 126 hCYP11B2 78.7 94.7 87.5 125 hCYP11B2-TripMut 80.6 96.5 81.1 117 rCYP11B1 79.7 96.5 80.2 113 rCYP11B2 82.4 96.5 80.1 114 a b For both hCYP11B1 and hCYP11B2 around 95% of the residues are positioned in the favoured and core regions of the Ramachandran Plot, indicating that for hybrid models, the structures are of acceptable quality. Due to the high quantity of alpha-helices and beta-sheets, the majority of residues is positioned in the expected regions. The residues which are situated in disallowed and unfavoured regions of the plot, are located in loop regions outside the active site. In total, 9 residues in the hCYP11B1 model are situated in the disallowed regions and 15 residues in the unfavoured regions. For the hCYP11B2 model, 10 residues are situated in disallowed regions and 14 residues in the unfavoured regions. The causes for these disparities are several insertions or deletions introduced in the models for which the structural minimisation was not sufficiently adequate to correct the backbone dihedrals. In particular, these regions are a relatively large insertion between alpha-helix D and beta-sheet 3-1, and an insertion between helix G and H. 66 67 2 3 68 Table 3 Validation results for the lowest energy models of CYP11B1 and CYP11B2 and the crystal structures which were used for the template, part II MOE-protein report Observed CYP101 (2CPP) Observed CYP2C5 (1NR6) Observed CYP11B1 model Observed CYP11B2 model a Parameter Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D. Trans-Omega 176.6 2.7 178.9 0.9 172.0 7.7 172.0 7.7 180.0 5.8 C-alpha chirality 32.8 3.6 34.3 1.7 30.8 11.1 30.8 10.8 33.8 4.2 Chi1-gauche minus −63.0 17.4 −63.3 14.8 −62.3 21.7 −62.6 22.1 −66.7 15.0 Chi1-gauche plus 55.4 20.7 56.3 16.4 51.5 26.7 53.0 28.3 64.1 15.7 Chi1-trans 185.3 13.3 184.2 12.9 186.3 21.9 186.8 20.3 183.6 16.8 Helix phi −65.2 11.9 −67.4 15.5 −60.7 19.7 −61.1 19.8 −65.3 11.9 Helix psi −41.2 16.5 −37.6 17.9 −42.5 25.1 −41.9 25.2 −39.4 11.3 Chi1-pooled S.D.  15.5  13.8  22.5  21.6  15.7 Proline phi −65.8 11.3 −61.9 9.5 −65.3 19.6 −67.6 20.5 −65.4 11.2 Dihedral outliers 0 4 15 17   Bond angle outliers 0 0 3 4   Bond length outliers 0 0 0 1   Results were generated with the MOE module: protein Eval. The thresholds were chosen to be 5 for the Z-Score and 70 for the vanderWaals contacts a [ 68 ] 69 70 Protein-substrate interactions 3 4 3 11 18 19 4 Fig. 4 18 20 21 3  Table 4 Hydroxylation distance table (iron atom–carbon atom) after minimisation with MOE (distances in Angstrom)  hCYP11B2 hCYP11B2-TripMut HCYP11B1 rCYP11B1 rCYP11B2 C11 C18 C19 C11 C18 C19 C11 C18 C19 C11 C18 C19 C11 C18 C19 DOC 4.72 4.30 5.61 4.37 4.65 5.32 4.30 4.56 5.48 4.30 4.75 4.83 4.70 4.24 5.54 18OH-DOC 4.33 4.30 a 4.31 4.51 b 4.31 4.60 b 4.30 4.68 b 4.32 4.31 a B 5.39 4.06 5.46 5.37 4.40 5.22 5.43 4.39 5.28 5.33 4.49 4.94 5.28 4.21 5.20 18OH-B 4.86 4.21 a 5.42 4.64 c 5.38 4.62 d 5.47 4.62 d 5.29 4.35 a 18 20 18 18 11 11 18 20 21 21 4 34 3 ( 4 ) 35 5 Fig. 5 R S S R R  71 36 4 36 11 18 18 3 19 18 11 19 21 11 18 19 21 18 11 11 18 18 20 11 18 11 11 18 11 11 18 18 18 20 18 18 4 18 11 18 18 18 18 18 18 gem- 18 gem- 72 Protein-inhibitor interactions R R S 73 74 R S S R 5 S R R R R 5 S S 75 R 5 Table 5 Correlation of docking and molecular dynamics results to in vitro data for both human CYP11B1 and CYP11B2 models  IC50 (nM) Goldscore ΔG Goldscore (kcal/mol) ΔG Chemscore (kcal/mol) non-bonded  hCYP11B1 Metyrapone 46.4 ± 10.4 57.33 −8.43 −8.73 −48.4 ± 3.7 R 0.5 ± 0.2 66.21 −9.38 −9.25 −56.0 ± 2.4 R 118.6 ± 8.9 54.01 −8.07 −8.14 −38.4 ± 2.5 S 39.5 ± 4.4 56.67 −8.36 −8.77 −56.3 ± 3.4 hCYP11B2 Metyrapone 207.8 ± 4.5 49.99 −7.64 −7.95 −36.2 ± 7.5 R 1.7 ± 0.9 65.21 −9.28 −9.21 −54.4 ± 2.9 R 6.0 ± 1.9 63.20 −9.06 −9.38 −55.9 ± 3.3 S 171.2 ± 51.7 53.81 −8.05 −8.12 −44.3 ± 1.8 non-bonded 3 R R Molecular dynamics 6 Fig. 6 R R S  During the first 500 ps, the RMSD increased and the protein still adapted towards its optimal conformation. After this point in time hardly any change in the three dimensional structures of the proteins was observed. The largest fluctuations of the protein were found in the flexible regions with peak values located in the structures around alpha-helix D (not shown). In the random coil following alpha-helix D we introduced a large insertion of seven amino acids, which elongates the alpha-helix by one turn before it connects to the following beta-sheet. Inside the water box, this region is found to protrude into the water without any stabilising protein interactions and unfolds due to interaction with water. In all the simulations we observed an opening of the active site and the continuous flow of water molecules in and out of the active site cavity. Several water molecules retained key positions, such as the water molecules that make up the channel towards the conserved Glu459 (not shown). 7 R R 8 R S R S Fig. 7 R R  Fig. 8 S R R S  R S R S 5 76 R S R Conclusion 11 18 19 3 21 R R S R S The constructed models are useful tools in trying to understand some of the molecular mechanisms involved in ligand binding and substrate conversion for the CYP11B family. As such, these models might also be appropriate tools for more detailed protein-inhibitor modelling studies as well as for ligand design or database screening, following further model optimisation and model tuning.