1. a project of david lutje hulsik and tim hulsen may 7th, 2001 2
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A project of David Lutje Hulsik and Tim Hulsen
May 7th, 2001
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What are GPCRs?
• Membrane-bound receptors
• A very large number of different domains both to bind their ligand and to activate G proteins.
• 6 different families
• Transducing messages as photons, organic odorants, nucleotides, nucleosides, peptides, lipids and proteins.
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• Seven transmembrane regions
GPCR Structure
• Conserved residues and motifs (i.e. NPXXY)
• Hydrophobic/ hydrophilic domains
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GPCR-G protein coupling
• Agonist binding to receptor becomes stronger upon G protein coupling
• GDP is released
• G protein takes up GTP
• G protein binds to activated receptor
• GTP uptake triggers release of G protein from receptor
• Receptor gets activated by agonist
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Research goals
• To determine whether predictions made about the structure of GPCRs are correct
• To see which methods give the best results
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• Residue numbering: Schwartz / Baldwin
(e.g. V.16)
Ballesteros-Weinstein
(e.g. 6.50)
etc.
Major research difficulties
• Bacteriorhodopsin as template
• Available high-resolution structural information
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Bacteriorhodopsin
• Photosynthetic bacteria
• Conformation
• Helical arrangement
• G proteins not involved
• Proton pumping
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Studies on GPCRs
• Mutation studies
• Photoaffinity
• Protease studies
• Cystein scanning
• NMR
• Spinlabel
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Methods
• Collecting data
• Structure validation with WHAT-IF
• Making alignment with the use of several articles which compare a GPCR with bacteriorhodopsin
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Collecting Data
• Articles Oldfashioned library work Online libraries (PubMed)
• Online databases GPCRDB
• Websites Different GPCR-groups
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Making Alignments
• Structural alignment rhodopsin/bacteriorhodopsin
---------- -WIWLALGTA LMGLGTLYFL VK-------- BRh ----PWQFSM LAAYMFLLIM LGFPINFLTL YVTVQ----- Rh
• Comparing with alignments made by other GPCR-experts
---------- -WIWLALGTA LMGLGTLYFL VK-------- BRh ----PWQFSM LAAYMFLLIM LGFPINFLTL YVTVQ----- Rh---------P EWIWLALGTA LMGLGTLYFL VKGM------ BRh Vriend ---------Q FSMLAAYMFL LIMLGFPINF LTLY------ Rh Vriend
Difference: +3
• Helix 3 means trouble Differences were larger then +10.
Complete alignment
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Structure validation with WHAT-IF I
• Structure predictions by Baldwin et al.
Electron density maps493 GPCR (a.a.)sequences
Helical orientationInteracting residues
Helical orientation as predicted was correct
Only a few residues interact::17 G P VII:18
:18 N A II:11 D II:14
:21 V A II:11 Y VII:21
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Structure validation with WHAT-IF II
• Structure predictions by Thirstrup et al.
Construction of zinc binding site-opioid receptor
Helical orientationHelix-helix interactions
Measured distances between zinc ion and residues too large;
even with the use of the ‘tors’ command
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Structure validation with WHAT-IF III
• Structure predictions by Greenhalgh et al.Spin label; electron paramagnetic resonance spectroscopy
Mapping residue positions(relative to aqueous boundaries)
Residue ContactGreenhalgh et al.
Contact WhatIF Difference
Arg-82 Extracellular about 5 Tyr-79 6.1 1.1
Asp-85 Extracellular about 9 Tyr-79 10.8 1.8
Asp-96 Intracellular within 7 Val-101 8.8 1.8
Differences are within range; spin-labeling could be a reasonably safe way to predictthe structure of membrane proteins
Distances for bacteriorhodopsin (in Å):
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Conclusions
• Predicting a structure with such low level of homology is very hard
• Most predictions are in the right direction, but still need some refinement
• Availability of real data (e.g. electron density maps) improves structure prediction
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Learning points
• Programming in Python
• What-IF
• GPCRs
• Website building (e.g. CGI)