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Amrita Roy Choudhury National Institute of Chemistry Slovenia 29 November 2013

Computational prediction of 3D Structure of Bilitranslocase Membrane Transporter: Drug Development Perspectives. Amrita Roy Choudhury National Institute of Chemistry Slovenia 29 November 2013. Introduction – Bilitranslocase. Plasma membrane organic anion transporter protein

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Amrita Roy Choudhury National Institute of Chemistry Slovenia 29 November 2013

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  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

  3. Sequence analysis Workflow 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

  5. Transmembrane region prediction 24-48 (TM1) 75-94 (TM2) 220-238 (TM3) 254-276 (TM4) MLIHNWILTFSIFREHPSTVFQIFTKCILVSSSFLLFYTLLPHGLLEDLMRRVGDSLVDLIVICEDSQGQHLSSFCLFVATLQSPFSAGVSGLCKAILLPSKQIHVMIQSVDLSIGITNSLTNEQLCGFGFFLNVKTNLHCSRIPLITNLFLSARHMSLDLENSVGSYHPRMIWSVTWQWSNQVPAFGETSLGFGMFQEKGQRHQNYEFPCRCIGTCGRGSVQCAGLISLPIAIEFTYQLTSSPTCIVRPWRFPNIFPLIACILLLSMNSTLSLFSFSGGRSGYVLMLSSKYQDSFTSKTRNKRENSIFFLGLNTFTDFRHTINGPISPLMRSLTRSTVE

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

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

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

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

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

  12. Transmembrane domain arrangements In collaboration with Max Bonomi, Andrej Sali (UCSF)

  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

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