S. Debaveye’s research while affiliated with KU Leuven and other places

The effect of ligands on the biochemical behavior of Alpo6. (A) SEC-profile derived from Alpo6-eGFP purified in the absence of ligand (blue). (B) SEC-profile derived from Alpo6-eGFP purified in the presence of glycine (green). (C) SEC-profile derived from Alpo6-eGFP purified in the presence of taurine (pink). (D) SEC-profile derived from Alpo6-eGFP purified in the presence of GABA (orange). (E) SEC-profile from Alpo6-wt purified in the presence of glycine. (F) Histogram displaying the relative fluorescence of the oligomeric peak height derived from FSEC-TS experiments on Alpo6-eGFP. Alpo6-eGFP incubated at 4°C (black) and at 65°C (blue) in the absence of ligands. Alpo6-eGFP incubated at 65°C in the presence of glycine (green), taurine (pink) and GABA (orange). (TIF)

Multiple sequence alignment. Multiple sequence alignment including full-length sequences of the putative cation-selective channels Alpo1-4 (blue), anion-selective channels Alpo5-7 (yellow), CLRs with known structures (pink) and human CLR subunits with high sequence identity to the identified homologues. The degree of amino acid conservation is displayed in shades of blue. Secondary structure elements, retrieved from the m5-HT3A R crystal structure (Hassaine G, Deluz C, Grasso L, Wyss R, Tol MB, Hovius R, et al. X-ray structure of the mouse serotonin 5-HT3 receptor. Nature. 2014;512: 276–281), are indicated above the alignment, α-helices and β-strands are colored in red and green, respectively. (TIF)

The identification of Nb9 as crystallization chaperone for Alpo1. (A) FSEC profiles from Alpo1-eGFP (red) and Alpo1-eGFP in complex with Nb9 (green). (B) Histogram displaying the relative fluorescence of the oligomeric peak height of Alpo1-eGFP incubated at 4°C (red), Alpo1-eGFP incubated at 65°C (orange) and Alpo1-eGFP in complex with Nb9 incubated at 65°C (green). (TIF)

Cys-loop receptors are membrane spanning ligand-gated ion channels involved in fast excitatory and inhibitory neurotransmission. Three-dimensional structures of these ion channels, determined by X-ray crystallography or electron microscopy, have revealed valuable information regarding the molecular mechanisms underlying ligand recognition, channel gating and ion conductance. To extend and validate the current insights, we here present promising candidates for further structural studies. We report the biochemical and functional characterization of Cys-loop receptor homologues identified in the proteome of Alvinella pompejana, an extremophilic, polychaete annelid found in hydrothermal vents at the bottom of the Pacific Ocean. Seven homologues were selected, named Alpo1-7. Five of them, Alpo2-6, were unidentified prior to this study. Two-electrode voltage clamp experiments revealed that wild type Alpo5 and Alpo6, both sharing remarkably high sequence identity with human glycine receptor α subunits, are anion-selective channels that can be activated by glycine, GABA and taurine. Furthermore, upon expression in insect cells fluorescence size-exclusion chromatography experiments indicated that four homologues, Alpo1, Alpo4, Alpo6 and Alpo7, can be extracted out of the membrane by a wide variety of detergents while maintaining their oligomeric state. Finally, large-scale purification efforts of Alpo1, Alpo4 and Alpo6 resulted in milligram amounts of biochemically stable and monodisperse protein. Overall, our results establish the evolutionary conservation of glycine receptors in annelids and pave the way for future structural studies. EW and MN are co-first authors

Overview of the detergents and their concentrations used during solubilization and (fluorescence) size-exclusion chromatography ((F)SEC). (TIF)

Significance In this study we take advantage of a recently described chimera of the α7 nicotinic acetylcholine receptor (nAChR) and acetylcholine binding protein (AChBP), termed α7-AChBP. To date, more than 70 crystal structures have been determined for AChBP in complex with ligands that occupy the orthosteric binding site. Here, we use an innovative screening strategy to discover molecular fragments that occupy allosteric binding sites. In combination with X-ray crystallography we determine a molecular blueprint of three different allosteric sites in α7-AChBP. Using electrophysiological recordings on the human α7 nAChR we demonstrate that each of the three sites is involved in allosteric modulation of the receptor. Our study contributes to understanding the sites of allosteric binding in ion channels.

TRPV3 is a thermosensitive ion channel primarily expressed in epithelial tissues of the skin, nose and tongue. The channel has been implicated in environmental thermosensation, hyperalgesia in inflamed tissues, skin sensitization and hair growth. While TRP channel research has vastly increased our understanding of the physiological mechanisms of nociception and thermosensation, the molecular mechanics of these ion channels are still largely elusive. In order to better comprehend the functional properties and the mechanism of action in TRP channels, high-resolution 3-D structures are indispensible, as they will yield the necessary insights into architectural intimacies at the atomic level. However, structural studies of membrane proteins are currently hampered by difficulties in protein purification and establishing suitable crystallization conditions. In this report, we present a novel protocol for the purification of membrane proteins, which takes advantage of a C-terminal green fluorescent protein (GFP) fusion. Using this protocol, we purified human TRPV3. We show that the purified protein is a fully functional ion channel with properties akin to the native channel using planar patch clamp on reconstituted channels and intrinsic tryptophan fluorescence spectroscopy. Using intrinsic tryptophan fluorescence spectroscopy, we reveal clear distinctions in the molecular interaction of different ligands with the channel. Altogether, this study provides powerful tools to broaden our understanding of ligand interaction with TRPV channels and the availability of purified human TRPV3 opens up perspectives for further structural and functional studies. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.

Pentameric ligand-gated ion channels (pLGIC) catalyze the selective transfer of ions across the cell membrane in response to a specific neurotransmitter. A variety of chemically diverse molecules, including the Alzheimer's drug memantine, block ion conduction at vertebrate pLGICs by plugging the channel pore. We show that memantine has similar potency in ELIC, a prokaryotic pLGIC, when it contains an F16'S pore mutation. X-ray crystal structures, using both memantine and its derivative, Br-memantine, reveal that the ligand is localized at the extracellular entryway of the channel pore, and the pore is in a more closed conformation than wild-type ELIC in both the presence and absence of memantine. However, using voltage clamp fluorometry we observe fluorescence changes in opposite directions during channel activation and pore block, revealing an additional conformational transition not apparent from the crystal structures. These results have important implications for drugs such as memantine, which block channel pores.

Pentameric ligand-gated ion channels (pLGICs), such as nicotinic acetylcholine, glycine, γ-aminobutyric acid GABAA/C receptors, and the Gloeobacter violaceus ligand-gated ion channel (GLIC), are receptors that contain multiple allosteric binding sites for a variety of therapeutics, including general anesthetics. Here, we report the x-ray crystal structure of the Erwinia chrysanthemi ligand-gated ion channel (ELIC) in complex with a derivative of chloroform, which reveals important features of anesthetic recognition, involving multiple binding at three different sites. One site is located in the channel pore and equates with a noncompetitive inhibitor site found in many pLGICs. A second transmembrane site is novel and is located in the lower part of the transmembrane domain, at an interface formed between adjacent subunits. A third site is also novel and is located in the extracellular domain in a hydrophobic pocket between the β7–β10 strands. Together, these results extend our understanding of pLGIC modulation and reveal several specific binding interactions that may contribute to modulator recognition, further substantiating a multisite model of allosteric modulation in this family of ion channels.

In the present study, we report for the first time, the molecular, biochemical and electrophysiological characterization of the components present in the soluble venom from Mesobuthus gibbosus (Brullé, 1832). According to the epidemiological and clinical situation of scorpion envenomation cases M. gibbosus scorpion is one of the most important health-threatening species of Turkey. Despite the medical importance reported for M. gibbosus, there is no additional information on toxin peptides and venom components to clarify the toxic effect of the M. gibbosus sting. Biochemical characterization of the venom was performed using different protocols and techniques following a bioassay-guided strategy (HPLC, mass spectrometry and Edman degradation sequencing). Venom fractions were tested in electrophysiological assays on a panel of six K(+) channels (K(v)1.1-1.6) by using the two-electrode voltage clamp technique. Three new α-KTx peptides were found and called MegKTx1, MegKTx2 and MegKTx3 (Mesobuthus gibbosus, K(+) channel toxin number 1 to 3). A cDNA library from the telson was constructed and specific screening of transcripts was performed. Biochemical and molecular characterization of MegKTx peptides and transcripts shows a relation with toxins of three different α-KTx subfamilies (α-KTx3.x, α-KTx9.x and α-KTx16.x).