1. Computational prediction of 3D Structure of Bilitranslocase Membrane Transporter: Drug Development Perspectives Amrita Roy Choudhury National Institute of Chemistry Slovenia 29 November 2013

2. Introduction – Bilitranslocase  Plasma membrane organic anion transporter protein  340 residues long  Distribution – hepatic cells, gastric, intestinal and renal epithelium, vascular endothelium, brain cells  No sequence homolog  Presence of motif conserved in phycocyanins  Function – transport of organic anions like bilirubin, anthocyanins, flavonoids, nicotinic acid  Potential candidate for drug target 2

3. Workflow 3 Sequence analysis Transmembrane region prediction Stability assessment of predicted transmembrane domain Transmembrane domain arrangement analysis NMR studies of transmembrane domains Towards possible functional mechanism

4. Sequence analysis  Grand Average of Hydropathicity – 0.255 (marginally hydrophobic)  Conserved motif 1 (bilirubin- binding motif) BTL residues 62-80 V-[ISA]-[CAT]-[AE]-D-S-Q-G-[RQ]-[FH]-L- S-S-[TF]-[EC]-L-[QF]-V-A  Conserved motif 2 BTL residues 220-228 G-[SK]-[VAD]-[QK]-C-[ASV]-[GR]-[LD]-I 4

5. 24-48 (TM1) 75-94 (TM2) 220-238 (TM3) 254-276 (TM4) MLIHNWILTFSIFREHPSTVFQIFTKCILVSSSFLLFYTLLPHGLLEDLMRRVGDSLVDLIVICE DSQGQHLSSFCLFVATLQSPFSAGVSGLCKAILLPSKQIHVMIQSVDLSIGITNSLTNEQLCGFG FFLNVKTNLHCSRIPLITNLFLSARHMSLDLENSVGSYHPRMIWSVTWQWSNQVPAFGETSLGFG MFQEKGQRHQNYEFPCRCIGTCGRGSVQCAGLISLPIAIEFTYQLTSSPTCIVRPWRFPNIFPLI ACILLLSMNSTLSLFSFSGGRSGYVLMLSSKYQDSFTSKTRNKRENSIFFLGLNTFTDFRHTING PISPLMRSLTRSTVE Algorithm1234 Predicted transmembrane regions CPNN-PredαTM24-48, 75-94, 220-238, 254-276 TMpred26-45, 75-102, 217-237, 256-278 TopPred II26-46, 72-92, 221-241, 257-277 SOUSI PRED-TMR27-46, 75-94, 256-277 TMHMM20-42, 256-278 HMMTOP20-43, 226-245, 257-277 Phobius20-41, 256-277 SVMtm27-41, 257-273 DAS-TMfilter27-42, 257-271 MEMSAT22-42, 257-275 SCAMPI21-41, 221-241, 256-276 MemBrain23-42, 74-82, 256-270 Philius19-41, 76-99, 255-279 OCTOPUS23-43, 254-274 TOPCONS21-41, 221-241, 259-279 Transmembrane region prediction 5

6. Analysis of predicted transmembrane domains 6In discussion with Sabina Passamonti (University of Trieste)

7. Stability assessment of transmembrane domains  20 ns molecular dynamics (MD) simulations using CHARMM  Alpha helical conformation  Fully solvated DPPC membrane  Analyze trajectories  Analyze RMSD and backbone torsion angles 7In collaboration with Andrej Perdih, Tom Solmajer (KI)

8. Stability assessment of transmembrane domains In collaboration with Andrej Perdih, Tom Solmajer (KI)8

9. Stability assessment of transmembrane domains Average RMSD  TM1 – 1.23  TM2 – 0.59  TM3 – 0.52  TM4 – 0.65 9In collaboration with Andrej Perdih, Tom Solmajer (KI)

10. Transmembrane helix-helix interaction 1.Based on complete transmembrane domain (SaliLab) 2.Based on residue contact (TMhit)  Predicted transmembrane helix-helix interactions  TM2-TM3  TM1-TM4 10In collaboration with Max Bonomi, Andrej Sali (UCSF)

11. Transmembrane domain arrangements  Monte Carlo (MC) simulation  Constraints – DOPE, excluded volume, packing, distance, diameter, tilt, depth, interaction  2 million conformations  3520 clusters  Score the representative all-atom models for each cluster  Analyze distribution 11In collaboration with Max Bonomi, Andrej Sali (UCSF)

12. Transmembrane domain arrangements Domain arrangement All 3520 structures 100 top-scoring structures ABCD2819 ADBC2134 ACDB561 ABDC133044 ACBD86227 ADCB77815 12In collaboration with Max Bonomi, Andrej Sali (UCSF)

13. 13 NMR studies of the Bilitranslocase transmembrane domains – Igor Zhukov

14. Discussion – towards functional mechanism of BTL  TM2 and TM3 play significant role in transport channel formation, ligand binding and mediation  Conserved Ser (73, 74, 229) and Cys (75, 224) are solvent-accessible  Probable allosteric nature  Probable bi-directional transport system 14