TCD Researchers Play Important Role in International Research on Human Transmembrane Protein
Feb 22, 2011
International collaborative research led by the Stanford University School of Medicine, involving Trinity College Dublin researchers recently published in Nature shows how an important human transmembrane protein functions at a molecular level. Trinity’s team played an important role in the research by implementing novel, high throughput methods and instrumentation to crystallise the receptor which revealed its structure and function. The findings are significant in that the particular human transmembrane protein known as β2-adrenergic receptor, a G protein-coupled receptor (GPCR), is the focus of a series of drugs for the treatment of asthma. This new research on its structure and function has the potential of leading to the development of improved drug therapies.
There are over 750 human GPCRs distributed throughout the body with representatives in almost every cell type. They function in myriad ways enabling us to interact with our environment, and with one another through the sense of sight and smell. They also play key roles in heart and lung function, in how we respond to hormones and neurotransmitters and by extension how they influence mood and behaviour, and are involved in immunity and inflammation. It is apparent therefore why a full suite of properly functioning GPCRs is integral to human health and wellbeing. About a third of drugs on the market today target GPCRs.
The research sets out to understand how one of these GPCRs, the β2-adrenergic receptor, works at a molecular level. The receptor is long enough to comfortably span the five nanometre width of the cell’s outer protective membrane. In this way, one end of the receptor can sense what is happening outside the cell and transmit the information it collects there to the cell’s interior for appropriate action. The fight-or-flight hormone adrenaline mediates its activity by way of the β2-adrenergic receptor. When the receptor binds adrenaline shot into the blood by the adrenal gland it snaps into action by binding with its cognate G-protein located inside the cell. This interaction triggers a series of reactions within and between cells that are part of the body’s response to adrenaline. These include vasodilation and constriction, smooth muscle relaxation, hearth muscle contraction and mobilisation of energy reserves in the liver and muscle. The β2-adrenergic receptor is an important pharmaceutical paradigm for the larger family of GPCRs some of which are targeted by beta blockers.
Trinity’s Professor of Membrane Structural and Functional Biology, Martin Caffrey in the Schools of Medicine and Biochemistry & Immunology explained: “New and improved drugs are always in demand. But to design them in a rational way we need to know how the receptor, in this case the β2-adrenergic receptor, is put together in a structural sense and how this structure enables it to function as a receptor and a communicator of information.”
“By structure we refer to the arrangement in three-dimensions of the receptor’s constituent atoms, amino acids and the ligands it binds. We would also like to know how this structure changes when adrenaline nudges the receptor and how this facilitates downstream signalling.”
“The only way to get such detailed information for a complicated membrane protein like the β2-adrenergic receptor is to use what is called macromolecular crystallography. This is a method that requires a well ordered crystal of the receptor. The crystals must then be irradiated with and scatter X-ray photons in a way that can be used to decipher the receptor’s structure.”
“One of the big challenges in this protracted and involved process is to coax the protein into the regular and ordered lattice of a crystal. This is particularly difficult in the case of GPCRs because they continually flit about structurally in the plane of the membrane and, at any one moment, can be seen to exist in a number of different conformations or shapes. The particular conformation assumed, in turn, dictates the receptor’s biological activity. To get a collection of receptor molecules to crystallise, ideally they should all assume the one stance or conformation.”
Professor of Molecular and Cellular Physiology at Stanford University, Brian Kobilka, who led this research project, devised a strategy for locking the receptors into what amounts to a single conformation by covalently or irreversibly splicing an adrenaline look-alike molecule into the binding site of the receptor. So stabilised and rendered uniform the receptor was successfully crystallised and its structure solved.
The Trinity College Dublin group contributing to the work include research scientists Mr Joseph Lyons and Dr David Aragăo and Professor Martin Caffrey who are members of the Membrane Structural and Functional Biology (MF&SB) group affiliated with the Schools of Medicine and Biochemistry & Immunology.
The Trinity team’s contribution to the work was to implement novel, high-throughput methods and instrumentation to crystallise the stabilised β2-adrenergic receptor. They employed custom-designed robots that dispense nano-litre volumes of a highly viscous, protein-laden lipidic liquid crystal or mesophase into home-built, multi-well glass sandwich plates for crystallisation screening. The mesophase mimics the lipid bilayer membrane in which the receptor resides in the cell. When treated appropriately it undergoes a transition to a second mesophase in which receptor molecules preferentially cluster, arrange themselves regularly in two- and then three-dimensional arrays, and eventually form crystals. The crystals typically are very small, just a tenth to a hundredth of a millimetre in size and are extremely fragile. They were harvested carefully from the toothpaste-textured mesophase, cryo-cooled in liquid nitrogen and then shipped in special Dewars from the laboratory at TCD to a synchrotron X-ray facility, the Advanced Photon Source, at the Argonne National Laboratories on the outskirts of Chicago. The synchrotron is a high-energy, particle accelerating machine about a mile in circumference that produces an extraordinarily intense beam of X-rays perfectly matched in size to that of the crystal. There the team used state-of-the-art technologies to centre the crystal in the laser-like X-ray beam and to collect diffraction data which, upon processing and modelling by researchers at Stanford, generated the structure reported in Nature.
The project was led by Brian Kobilka, Professor of Molecular and Cellular Physiology at Stanford University School of Medicine. Professor Kobilka assembled an interdisciplinary team from across the globe that included molecular biologists, protein biochemists and biophysicists, crystallographers, synthetic organic chemists and molecular modellers. In all, 18 individuals from seven institutions in the USA and Europe contributed to the work. Each played a complementary role in providing insights into the molecular workings of these medically and pharmaceutically important membrane protein receptors.
Trinity’s Membrane Structural and Functional Biology group is funded through Science Foundation Ireland, the USA National Institutes of Health and the European Union Framework Seven Programme.
Trinity in the News: