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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 - PowerPoint PPT Presentation
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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
2
Workflow
3
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
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
5
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)
13
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
14
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