Analysis of a drug bound to a G protein-coupled receptor

Introduction

G protein-coupled receptors (GPCRs) are the largest family of integral membrane proteins inthehumangenomeareimportantdrugtargets.

Within the human genome there are coded over 1000 GPCRs and compounds that act at these targets comprise more than 50% of the current market for human drugs. Understanding how GPCRs function and bind to drugs is of importance to researchers in the search for new drugs. A better understanding will facilitate design and help to avoid compounds with undesirable side effects.

This practical exercise will provide a general overview of the GPCR protein and will explore the design of a compound within the active site using a GPCR structure with an agonist.

Aim

To investigate GPCR protein structure and the amino acids involved in the binding of an ^{agonist using the }2^{-adrenergic receptor as an example. To design novel ligands to improve} potency and selectivity.

The Report:Please answer the questions in the exercise in your report. No Aims, Methods, etc. are required. All images should include a figure legend.

Background

GPCRstructure

NH₂

Extracellular loops

Intracellular loops

SchematicdiagramofaGPCR

• GPCRs are proteins that transfer signals from outside the cell into the cell interior.
• GPCRs are embedded in the cell membrane with regions (loops) exposed to inside and outside of the cell.
• The protein passes through the membrane 7 times (hence 7TM). There is an eighth helix that is located inside the cell (and does not pass through the membrane).
• The transmembranesectionof all GPCRs are very similar but the extracellular and intracellular loops can vary greatly.

GPCRfunction

GPCRs which interact with a huge variety of different neurotransmitters or hormones which can be classified in the following manner:

• Monoamines(e.g. adrenaline, dopamine, 5HT)
• Nucleotides
• Lipids
• Neuropeptides (e.g. leu and met-enkephalin), peptide hormones and protein hormones
• Glycoprotein hormones
• Glutamate
• Calcium ions
• The binding sites for their various ligands differs between the classes.
• The binding site for monoamines, nucleotides and lipids is deep within the structure between the transmembrane helices.

• Upon binding of the chemical messenger to the GPCR the G protein then interacts with the receptor-ligand complex. The G protein then carries the ‘message’ to the next target in the signalling cascade.

Crystal structure of an opioid bound to the mu opioid receptor

Information about the crystal structure is available from the Protein Data Bank (PDB) http://www.rcsb.org/.

Question 1.

GotothePDBdatabasesiteandsearchforentry4DKL.This is the structure of the mu opioid receptor bound to the ligand, β-funaltrexamine, which is an analogue of buprenorphine.

Three lipid molecules and one polyethylene glycol molecule are also present in the crystalstructure.

The original paper that discusses the crystal structure is available here: https://www.nature.com/articles/nature10954.pdf (or on Moodle). The supplementary information from the paper will also help answer this question (on Moodle also).

Find the structure of the β-funaltrexamine. Using a diagram, show how the ligand has made a covalent bond with the receptor. The formation of the covalent bond permanently attaches the ligand to the receptor, which assists with protein crystallisation for X-ray crystallography.

Loadingthecrystalstructureintopymol

Obtain the 4DKL protein crystal structure of the mu opioid receptor to the irreversible morphine-like antagonist β-funaltrexamine (β-FNA).

In the PyMOL> command box type:

fetch 4DKL

oruse the command FileGet PDB (if it exists).

Pymol mouse commands:

Left mouse: rotate or select Right mouse: scale

Middle mouse (wheel): click to centre structure Pymol display matrix:

Structures can be turned on and off by clicking on the grey section

A = action S = show H= hide

L= label

C= colour

Theproteinstructure

Show the structure in cartoon format (other representations are possible too) AllHideeverything

4DKLShowcartoon

Show the small (organic) molecules in the crystal structure AllShoworganicsticks

Note that the protein is made up of three domains. One of these is the 7-transmembrane (7TM) region, the focus of our interest, while the remaining sections were used to help examine the agonist state of the receptor.

The crystal structure contains genetically engineered domains that are not part of the GPCR. These were used to help crystallise the protein.

Rotate the structure and identify the 7TM region. To help clearly visualize the 7TM domain we will remove the other domains.

In the PyMOL> command box type:

select xxx, resi 1000-1200 select yyy, resname so4

select zzz, resname 1pe [ don’t forget the comma ]

Delete the bits we don’t want

xxxActionremove atoms yyyActionremove atoms zzzActionremove atoms

Colour the protein chains as a rainbow (N-terminus blue, C-terminus red) AllColourspectrumrainbow

We now have the structure with the key 7TM domain (and some substructures that interact with the protein). Take some time to inspect the 7TM structure. Can you identify helices 1- 8?

Question 2.

Maketwodiagrams: [ use print screen or use Draw/Ray in top right corner ]

1. Onefromtheviewoflookingacrossthemembrane.
2. Onelookingdownontopofthe7TMdomain(fromtheoutsideofthecell)

Using your diagrams. a) briefly describe the structure of the protein b) identify the boundligandandthe3boundlipidmolecules.

Analysingtheboundlipid

In the crystal structure, there is a cholesterol molecule (residue name CLR, residue number 614) that we will explore further.

Displaythelipidbindingsite

The first step will involve hiding everything.

4DKL  Hide everything

To display the glycerol ester and surrounding amino acids type the following into the command box:

In the PyMOL> command box type:

select clr, resi 614 expand 6.0

Show the atoms

clrShowsticks Colour the atoms by element

AllColourby element where C is green.

Colour the lipid a different colour Click on the lipid to select it.

SeleColourby element where C is aqua.

Label residues

clrlabelresidues Centre the structure

clrActioncenter

Question 3.

Explainwhyyouthinkamoleculeisfoundinthatparticularposition?Whattypeofinteractionsdoescholesterolmakewiththereceptor?Forthereportdescribethetypesofinteractionsandlisttherelevantresidues.

Theligandbindingsite

Displaytheligandbindingsite

To look at this more closely we need to display the ligand (β-funaltrexamine, BF0) and residues close to this compound. (Note last character is zero).

First hide everything

All  Hide  everything

All  Label clear [ might be necessary ] Display β-FNA and surrounding amino acids define a selection

In the PyMOL> command box type:

select bf0, resn bf0 expand 6

Label residue names

bf0Labelresidues bf0showsticks

Colour the ligand a different colour from the other residues Select and centre the ligand

seleColourby element(colour scheme) sele->Action->center

MeasuringdistancesinPymol.

Choose:

Wizard (menu)Measurementdistance

Click on atoms to measure distance. Choose Done when finished. [ if you do not have a Wizard menu:

In the PyMOL> command box type:

wizard distance

]

Identifytheligand-receptorinteractions

Identify the colvalent linkage that has been created between the ligand and protein.

An aspartic acid residue (Asp, one letter code D) makes strong interactions with the ligand.

Question 4.

Whatisthenumberofthisresidue?Whattypeofinteraction(s)doestheligandmakewiththisresidue? (Note that hydrogen atoms are not shown in the crystal structure). How does thisresiduecontributetothemuopioidpharmacophore?(seelectureNotes).

Question 5.

O

Pethidine

a)Drawadiagramthatcomparesthestructureofpethidinetoβ-FNA.b)Basedonthestructureofβ-FNA,identifythelikelyresiduesthatinteractwithpethidine.

DrugDesign

Finally, we wish to design a new compound using β-FNA as our template. The compound β- FNA is derived from morphine, being substituted at the 6 position and having the double bond in the C ring reduced.

HO

A

B

^O E ^H

C _N

^HO 6

One way to improve affinity (and possibly selectivity) of our template for the receptor is to design a compound which makes an additional interaction with the receptor. To do this we need to identify a residue adjacent to the ligand that might be able to provide an additional interaction.

Question 6.

Design ligands modifying the 6-position of β-FNA and that make new interactions with the receptor. Explain why you think they would improve ligand binding.