Two-dimensional crystallization of the mouse serotonin 5-HT3A receptor

Ghérici Hassaïne, Henning Paul-Julius Stahlberg, Horst Vogel
Pergamon-Elsevier Science Ltd, 2017
Article

The mouse serotonin 5-HT3A receptor is a homo-pentameric ligand-gated ion channel (pLGIC) mediating fast excitatory neurotransmission in the central nervous system. The molecular mechanism of ion permeation of 5-HT3A receptors triggered by the neurotransmitter serotonin is not yet fully understood. The recent X-ray structure of the mouse serotonin 5-HT3A receptor in complex with a stabilizing nanobody revealed for the first time the entire structure of a mammalian pLGIC in detergent. Structural information of the receptor in a lipid bilayer however is still limited primarily due to the lack of 2D crystals of the receptor in a lipid bilayer. Here we present our results on the formation and improvement of diffracting 2D crystals of the mouse 5-HT3A by limited proteolysis and addition of conformational nanobodies. (C) 2016 Elsevier Ltd. All rights reserved.

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Ghérici Hassaïne, Henning Paul-Julius Stahlberg, Horst Vogel
Pergamon-Elsevier Science Ltd, 2017
Article

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https://infoscience.epfl.ch/record/224515?ln=fr

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La famille des récepteurs sérotoninergiques, aussi appelés récepteurs 5-hydroxytryptamine (abrégés en 5-HT), sont des récepteurs du système nerveux central et périphérique activés par la sérotonine.

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Rapid purification of active gamma-secretase, an intramembrane protease implicated in Alzheimer's disease

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Gamma-secretase is an unconventional aspartyl protease that processes many type 1 membrane proteins within the lipid bilayer. Because its cleavage of amyloid-beta precursor protein generates the amyloid-beta protein (Abeta) of Alzheimer's disease, partially inhibiting gamma-secretase is an attractive therapeutic strategy, but the structure of the protease remains poorly understood. We recently used electron microscopy and single particle image analysis on the purified enzyme to generate the first 3D reconstruction of gamma-secretase, but at low resolution (15 A). The limited amount of purified gamma-secretase that can be produced using currently available cell lines and procedures has prevented the achievement of a high resolution crystal structure by X-ray crystallography or 2D crystallization. We report here the generation and characterization of a new mammalian cell line (S-20) that overexpresses strikingly high levels of all four gamma-secretase components (presenilin, nicastrin, Aph-1 and Pen-2). We then used these cells to develop a rapid protocol for the high-grade purification of proteolytically active gamma-secretase. The cells and purification methods detailed here provide a key step towards crystallographic studies of this ubiquitous enzyme.

Wiley-Blackwell2008

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Thermal Unfolding of a Mammalian Pentameric Ligand-gated Ion Channel Proceeds at Consecutive, Distinct Steps

Cédric Deluz, Ghérici Hassaïne, Henning Paul-Julius Stahlberg, Menno Berend Tol, Horst Vogel

Pentameric ligand-gated ion channels (LGICs) play an important role in fast synaptic signal transduction. Binding of agonists to the beta-sheet-structured extracellular domain opens an ion channel in the transmembrane alpha-helical region of the LGIC. How the structurally distinct and distant domains are functionally coupled for such central transmembrane signaling processes remains an open question. To obtain detailed information about the stability of and the coupling between these different functional domains, we analyzed the thermal unfolding of a homopentameric LGIC, the 5-hydroxytryptamine receptor (ligand binding, secondary structure, accessibility of Trp and Cys residues, and aggregation), in plasma membranes as well as during detergent extraction, purification, and reconstitution into artificial lipid bilayers. We found a large loss in thermostability correlating with the loss of the lipid bilayer during membrane solubilization and purification. Thermal unfolding of the 5-hydroxytryptamine receptor occurred in consecutive steps at distinct protein locations. A loss of ligand binding was detected first, followed by formation of different transient low oligomeric states of receptor pentamers, followed by partial unfolding of helical parts of the protein, which finally lead to the formation receptor aggregates. Structural destabilization of the receptor in detergents could be partially reversed by reconstituting the receptor into lipid bilayers. Our results are important because they quantify the stability of LGICs during detergent extraction and purification and can be used to create stabilized receptor proteins for structural and functional studies.

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The Structure of the Mouse Serotonin 5-HT3 Receptor in Lipid Vesicles

Cédric Deluz, Luigino Grasso, Ghérici Hassaïne, Henning Paul-Julius Stahlberg, Horst Vogel

The function of membrane proteins is best understood if their structure in the lipid membrane is known. Here, we determined the structure of the mouse serotonin 5-HT3 receptor inserted in lipid bilayers to a resolution of 12 Å without stabilizing antibodies by cryo electron tomography and subtomogram averaging. The reconstruction reveals protein secondary structure elements in the transmembrane region, the extracellular pore, and the transmembrane channel pathway, showing an overall similarity to the available X-ray model of the truncated 5-HT3 receptor determined in the presence of a stabilizing nanobody. Structural analysis of the 5-HT3 receptor embedded in a lipid bilayer allowed the position of the membrane to be determined. Interactions between the densely packed receptors in lipids were visualized, revealing that the interactions were maintained by the short horizontal helices. In combination with methodological improvements, our approach enables the structural analysis of membrane proteins in response to voltage and ligand gating.

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