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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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Take a look at our website!

http://go.to/gpcr

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Learning points

• Programming in Python

• What-IF

• GPCRs

• Website building (e.g. CGI)