Computational prediction of 3D Structure of Bilitranslocase

Membrane Transporter: Drug Development Perspectives

Amrita Roy ChoudhuryNational Institute of Chemistry

Slovenia

29 November 2013

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

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Workflow

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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

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24-48 (TM1)75-94 (TM2)

220-238 (TM3)254-276 (TM4)

MLIHNWILTFSIFREHPSTVFQIFTKCILVSSSFLLFYTLLPHGLLEDLMRRVGDSLVDLIVICEDSQGQHLSSFCLFVATLQSPFSAGVSGLCKAILLPSKQIHVMIQSVDLSIGITNSLTNEQLCGFGFFLNVKTNLHCSRIPLITNLFLSARHMSLDLENSVGSYHPRMIWSVTWQWSNQVPAFGETSLGFGMFQEKGQRHQNYEFPCRCIGTCGRGSVQCAGLISLPIAIEFTYQLTSSPTCIVRPWRFPNIFPLIACILLLSMNSTLSLFSFSGGRSGYVLMLSSKYQDSFTSKTRNKRENSIFFLGLNTFTDFRHTINGPIS

PLMRSLTRSTVE

Algorithm 1 2 3 4 Predicted transmembrane regions

CPNN-PredαTM 24-48, 75-94, 220-238, 254-276TMpred 26-45, 75-102, 217-237, 256-278TopPred II 26-46, 72-92, 221-241, 257-277SOUSIPRED-TMR 27-46, 75-94, 256-277TMHMM 20-42, 256-278HMMTOP 20-43, 226-245, 257-277Phobius 20-41, 256-277SVMtm 27-41, 257-273DAS-TMfilter 27-42, 257-271MEMSAT 22-42, 257-275SCAMPI 21-41, 221-241, 256-276MemBrain 23-42, 74-82, 256-270Philius 19-41, 76-99, 255-279OCTOPUS 23-43, 254-274TOPCONS 21-41, 221-241, 259-279

Transmembrane region prediction

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Analysis of predicted transmembrane domains

6In discussion with Sabina Passamonti (University of Trieste)

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)

Stability assessment of transmembrane domains

In collaboration with Andrej Perdih, Tom Solmajer (KI) 8

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)

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)

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)

Transmembrane domain arrangements

Domain arrangement

All 3520 structures

100 top-scoring structures

ABCD 281 9ADBC 213 4ACDB 56 1ABDC 1330 44ACBD 862 27ADCB 778 15

12In collaboration with Max Bonomi, Andrej Sali (UCSF)

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NMR studies of the Bilitranslocase transmembrane domains – Igor Zhukov

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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