Identification number of the residue near the docking site

Yizhou YuImperial College London, Department of Life Sciences, London, United Kingdom

Molecular modelling of propofol binding to GABAA receptors

reveal a novel gating mechanism

BACKGROUND1. Many general anesthetics enhance the actions of the neurotransmitter

γ-aminobutyric acid (GABA) at the GABA type A receptor.1,2

2. No agreement as where anesthetics bind on the receptor.3

3. GABAAR signalling mechanisms revealed by structural pharmacology in

2019.4

1. Measure the movement

across conformations

Propofol binds to pre-formed pockets

in the receptor to stabilise

conformations. By comparing different

structures of the GABA receptors, I

determine where the regions with the

most movement are. Propofol, or similar

anaesthetics, would bind to those mobile

regions to stabilise the receptor pore.

2. Dock propofol at M286 6

and H2677

Propofol was docked near sites with thehigh movement and identified usingphotolabeling as propofol binding sites.Vina outputs the best docking mode,which includes the docking conformation(how the ligand is positioned) andlocation on the receptor, as well as therelated energies involved.

Identify state-dependent regions on the receptor, which would

account for the ability of anesthetics to affect channel opening by

binding differentially to the open and closed states using AutoDock

Vina (Vina).5

Aim: Identify anaesthetic binding sites using an in

silico docking program

DISCUSSION• Etomidate and propofol may bind to locations in the receptor with high

movement, near the residues 200s and 270s respectively (Fig 1).

• Propofol docks near both previously photolabelled residues (M268 and

HIS267), resolving controversies (Fig 2).

• Specifically, propofol docking on the resting state holds the prospect of opening

the receptor pore. Further calculations are required to validate this hypothesis

(Fig 3.).

• I created a suite of programs that prepares the receptor and ligand files for

docking using Vina on a Linux cluster and subsequently visualises the output

files on PyMol.

References1.Franks, N. P. (2008) General anaesthesia: From molecular targets to neuronal pathways of sleep and arousal. Nature Reviews Neuroscience. 9 , 5, 370.

2. Nelson, L. E., Guo, T. Z., Lu, J., Saper, C. B., Franks, N. P. & Maze, M. (2002) The sedative component of anesthesia is mediated by GABA(A) receptors in an endogenous sleep pathway.

Nature Neuroscience. 5 , 10, 979.

3. Franks, N. P. (2015) Structural comparisons of ligand-gated ion channels in open, closed, and desensitized states identify a novel propofol-binding site on mammalian γ-aminobutyric acid

type A receptors. Anesthesiology: The Journal of the American Society of Anesthesiologists. 122 , 4, 787-794.

4. Laverty, D., Desai, R., Uchański, T., Masiulis, S., Stec, W. J., Malinauskas, T., Zivanov, J., Pardon, E., Steyaert, J. & Miller, K. W. (2019) Cryo-EM structure of the human α1β3γ2 GABA A

receptor in a lipid bilayer. Nature. 565 , 7740, 516.

5. Forli, S., Huey, R., Pique, M. E., Sanner, M. F., Goodsell, D. S. & Olson, A. J. (2016) Computational protein–ligand docking and virtual drug screening with the AutoDock suite. Nature

Protocols. 11 , 5, 905.

6. Jayakar, S. S., Zhou, X., Chiara, D. C., Dostalova, Z., Savechenkov, P. Y., Bruzik, K. S., Dailey, W. P., Miller, K. W., Eckenhoff, R. G. & Cohen, J. B. (2014) Multiple propofol-binding sites in

a γ-aminobutyric acid type A receptor (GABAAR) identified using a photoreactive propofol analog. Journal of Biological Chemistry. 289 , 40, 27456-27468.

7. Yip, G. M., Chen, Z., Edge, C. J., Smith, E. H., Dickinson, R., Hohenester, E., Townsend, R. R., Fuchs, K., Sieghart, W. & Evers, A. S. (2013) A propofol binding site on mammalian GABA A

receptors identified by photolabeling. Nature Chemical Biology. 9 , 11, 715.

8. Pravda, L., Sehnal, D., Toušek, D., Navrátilová, V., Bazgier, V., Berka, K., Svobodová Vařeková, R., Koča, J. & Otyepka, M. (2018) MOLEonline: A web-based tool for analyzing channels,

tunnels and pores (2018 update). Nucleic Acids Research. 46 , W1, W373.

Acknowledgements: I thank Prof Nick P. Franks for day-to-day guidance, as well as Dr Suhail Islam and Mr Florian Guitton for providing computational resources.

Allows flexible residues?

Run on a Linux cluster?

Easy to use?

Customisable?

Code developmentME

TH

OD

Repository: code and

detailed report

a) Resting vs pre-active b) Resting vs desensitised

En

erg

y in

kca

l/m

ol

⍺1 subunit

𝛽2 subunit ⍺1 subunit

𝛽2 subunit

𝛾2 subunit

3. Measure the size of the

receptor poresThe pore size of the dockedconformations were measuredusing MOLEonline8 to investigatethe effect of propofol binding atthese sites. Residues lining thereceptor pore were alsovisualised.

Figure 1. Comparison of the RMSD values of the

closed & resting state (6HUG) against the

desensitised (6HUO) conformation in a) and pre-

active (6HUJ) conformation in b) confirms

previously-identified propofol-binding sites. The

distances between the α carbon were computed and

shown in 2 colour-coded views each, with one at a

180° rotation of the other. The inter-chain RMSD

values are graphed on the right of the molecular

structures, with chain B in pink and chain E in cyan.

These are the 𝛽e a2 subunits which are reputed to

mediate channel closure and propofol binding. In both

figures, the MET 286 residue is labelled in green and the

HIS 267 residue in red.

Figure 2. Molecular docking of propofol near the MET 286 and HIS 267 sites indicate

that both sites identified by photolabeling are propofol-binding sites. The energy

values of the HIS 267 and MET 286 sites are not significantly different (Welch Two Sample

t-test, t = -0.58, p-value = 0.57). The horizontal reference line is the experimentally-derived

threshold for propofol binding, suggesting both sites can bind propofol.Figure 3. GLU 270 was both lining the receptor pore and also

selected to adopt a different protein conformation by Vina. Upon

docking, the carboxyl group of this residue flexes and seems to open

the receptor pore. The original residue is labelled in cyan, and the

flexed residue is in green with its oxygen projected inwards. I therefore

propose that GLU 270 acts as a gate for the GABA receptor, which

can allow or prevent ion influx in the closed state. Propofol binding

may directly open this gate to allow chloride influx.