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Structure and dynamics of G protein-coupled receptors by NMR spectroscopy

Final Report Summary - NMRGPCR (Structure and dynamics of G protein-coupled receptors by NMR spectroscopy)

The biochemical processes of living cells involve a numerous series of reactions that work with exceptional specificity and efficiency. The tight control of this intricate reaction network stems from the architecture of the proteins that drive the chemical reactions and mediate protein–protein interactions. Indeed, the structure of these proteins will determine both their function and interaction partners. A detailed understanding of the proximity and orientation of pivotal functional groups can reveal the molecular mechanistic basis for the activity of a protein. Together with X-ray crystallography and electron microscopy, NMR spectroscopy plays an important role in solving three-dimensional structures of proteins at atomic resolution. In the challenging field of membrane proteins, G-protein coupled receptors (GPCRs) form a remarkable modular system that allows transmission of a wide variety of signals over the cell membrane, between cells and over long distances in the body. The signal can be a change in concentration of peptides, hormones, lipids, neurotransmitters, ions, odorants, tastants, etc., or an influx of photons to the eye. GPCRs convey these signals to the inside of the cell and elicit a series of reactions involving other proteins, nucleotides and metal ions, which eventually deliver a message and an appropriate cellular and physiological response. Many physiological processes in mammals depend on those GPCRs, which are also the targets for a large portion of all pharmaceuticals drugs. About a thousand human genes code for these receptors, and they are involved in sensing a wide range of extracellular stimuli. Examples include the adrenergic receptors, dopamine receptors, histamine receptors, the light receptor rhodopsin, and the many odor and taste receptors.

NMR spectroscopy is the only experimental method, which yields both structural and dynamical information on biomolecules at atomic resolution with minimal invasiveness and at close to natural conditions. As such it can provide unique information to understand the connection between primary structure, tertiary structure, dynamics and function. It was the goal of this work to apply and further develop these strengths of NMR technology with the aim to reveal general principles of GPCRs structure-function relations.

I benefited from the extensive knowledge from the host laboratory in GPCR structural biology, which has developed a successful mutagenesis strategy to produced thermostabilized recombinant receptors. My work focused on three different receptors: the human arginine vasopressin receptor type II (h_AVPR2), the human β-1 adrenergic receptor (h_ADRB1) and the turkey β-1 adrenergic receptor (t_ADRB1). With stabilized GPCRs I explored several alternative strategies for incorporation of magnetically active isotopes using the prokaryotic expression systems Escherichia coli (E. coli). Expression of heterologous proteins in E. coli is frequently associated with incorrect folding and accumulation of the recombinant protein in cytoplasmic aggregates named inclusion bodies (IBs). Targeting of GPCRs to IBs combines many advantages. IBs are mechanically stable and can be easily isolated from other cell constituents by centrifugation, they are not toxic to the cell, and they are resistant to proteolytic degradation. Use of α5 integrin (α5I) fragment as a targeting partner greatly facilitated the expression of h_AVPR2 and h_ADRB1 and their productions in IBs were massive. This strategy implied, however, that the receptors thus expressed, must be subsequently folded to their native state, which constitutes a difficult challenge. The folding efficiency depends on the competition between protein aggregation and 3D structure formation, as well as on the ability of the receiving surfactant to stabilize the native 3D state of the folded receptor. Different refolding environments were tested such as classical detergents, lipid-detergent mixtures, bicelles, lipid vesicles and original surfactants such as APols. Successful folding was achieved for both receptors mentioned from IBs but in such low amount that no expression of isotope labeled protein was considered.

GPCRs are found in plasma membranes, so the most obvious strategy to express these recombinant receptors is to target them in the inner membrane of E. coli. This approach was performed on the human and the turkey β-1 adrenergic receptors (h_ADRB1, t_ADRB1). An efficient way to insert the seven transmembranes could be achieved by fusing the two GPCRs to different helper partners. First, thioredoxin A (TrxA), fused on the N-terminus of both β-1 adrenergic receptors, was tested for its ability to promote disulfide bond formation crucial for ligand binding and consequently signaling. A significant amount of protein was expressed and purified. However the receptor could only be kept in solution mostly in presence of one group of lipids so called the Fos-Cholines that some researchers have classified into the “harsh” category of surfactants. The lack of stability in milder detergents was attributed to the absence or incorrect formation of the two disulfide bonds. Many efforts have been done to amend this problem using different redox systems without any major successes. In addition to catalyzing disulfide bond formation like TrxA, the DsbA fusion tag was tested on h_ADRB1 as it provides also a signal sequence for translocation into the periplasmic space. Unfortunately no expression was detectable for this construct despite an extensive screening of conditions such as E. coli strains, culture media, pH, temperatures, IPTG concentrations, ligand additions, and induction times. Finally the combination of the maltose-binding protein (MBP) used as an N-terminal fusion partner, with TrxA added on the C-terminus of t_ADRB1 was investigated. The membrane expression of the receptor was highly successful yielding a large amount of fusion protein. Despite a significant amount of receptor lost during the purification steps such as immobilized metal ion affinity chromatography (IMAC), dialysis and sepharose-alprenolol affinity chromatography, a reasonable amount of pure receptor was produced. The quality of the receptor made was assessed through its ability to bind or not a set of selective and non-selective ligands for β-1 adrenergic receptors. The thermostability could also be determined using a thiol-specific fluorochrome dye (CPM) for stability profiling that could be compared with material produced in eukaryotic cells. After confirmation that the construct was suitable for NMR studies a 15N-labelled t_ADRB1 was produced. Unfortunately, the funding for this fellowship stopped after the 24 months period and no further investigations were possible.