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Solution NMR spectroscopy studies of an adrenergic receptor b1AR

Reference	BB/K01983X/1
Principal Investigator / Supervisor	Professor Daniel Nietlispach
Co-Investigators / Co-Supervisors
Institution	University of Cambridge
Department	Biochemistry
Funding type	Research
Value (£)	453,478
Status	Completed
Type	Research Grant
Start date	01/09/2013
End date	21/01/2017
Duration	41 months

Abstract

Although crystal structure determination of GPCRs has become increasingly available over the last years, there is a distinct lack of insight on the dynamic nature of these highly mobile signaling proteins. Downstream signal transduction events follow from extracellular ligand-induced interactions that dynamically change the functional state of the receptor. Information on this functional receptor plasticity is not accessible through crystal structure snapshots obtained in often non-native detergent environments. Considering predominantly structure based static arguments therefore is unlikely to reveal all the essential aspects of how GPCRs function. Hence, investigation through complementary techniques that can provide information on receptor dynamics is urgently required. NMR is well suited to provide such information, however, until now the success of extensive NMR studies on GPCRs has been obstructed by the unfavourable properties of these proteins; sample preparation being a major bottleneck for structural studies of GPCRs. We intend to pursue comprehensive solution NMR studies of a class A GPCR. Our study will concentrate on a conformationally thermostabilized mutant of turkey b1AR which has been extensively used in X-ray crystallography studies and which provides beneficial sample characteristics to enable extensive NMR studies. The receptor is stabilized predominantly in one conformation, which results in superior NMR spectral quality due to improved sample homogeneity. In addition, spin relaxation dispersion techniques can probe the presence of 'invisible' low-populated receptor states and will provide the missing information on the conformational dynamics of the receptor. Using uniform and selective isotope labelled b1AR samples we will utilise this approach to study what effect ligands of different efficacies have on the conformational dynamics of the receptor, which regions of the GPCR are affected and how the changes depend on the type of ligand bound.

Summary

Living organisms are made up of a very large quantity of cells. Each of these cells contains machinery that is essential to maintain and develop the life of a particular organism. These cells are surrounded by a waterproof lipid membrane, which encapsulates the mostly aqueous interior of a cell that includes also the essential molecular machinery. Every process of life both on a large as well as on a small scale involves continuous adaptations to a changing environment. Following such changes and responding to the demands that arise through the activities of the organisms the conditions within the individual cells need to be continuously adjusted. Every cell needs to be supplemented with nutrients for energy and building materials, waste products need to be removed and instructions need to be given for the multitude of different processes to act in a concerted manner. To facilitate these requirements across the impenetrable lipid membrane a large number of proteins are embedded into the cell membrane. These proteins connect the cell exterior with the inside of the cell and are called membrane proteins. A particular group of these proteins is responsible for relaying information in form of control signals across the membrane. The cells are using these proteins as sensors that relay a message from the exterior to the inside of a cell, where a cell is then able to understand what adjustments need to be made. The range of such control signals can be very diverse and there are therefore several hundreds of these sensors making this a particularly important group of membrane proteins. In fact it turns out to be the largest family of proteins in humans. Our work is looking in more details at these proteins, the so-called G protein coupled receptors GPCRs. We are trying to understand how exactly it is that these proteins work and in particular how different external control signals for a given sensor facilitate the different responses on the inside of the cell. Exposure of these proteins on the surface of the cells makes them easily accessible, which is crucial for them to work properly. It makes them also ideal targets for drugs in situations when our body malfunctions and needs drug therapeutic help. Therefore next to the academic interest in understanding how these proteins work there is a large interest from the pharmaceutical industry for the development of newer and better drugs from which our general well being will benefit. To be able to address such questions typically requires biologists and chemists to zoom in on a molecular level using a range of biophysical techniques, which allow us to see what is happening on an atomic scale. Our lab is using a technique called nuclear magnetic resonance (NMR), which allows us to study these GPCR proteins in a nearly native environment. For the technique to work the GPCR under study is removed from the cell membrane but is still kept surrounded by a very small portion of it. These proteins are extremely unstable and hence very tricky to study. We are concentrating on a particular member of the GPCR family, which has been modified and displays enhanced properties to assist our investigations. These sensor proteins are considered to be highly mobile and their dynamic nature strongly influences how they function. NMR is an excellent method that can describe which parts of these proteins are flexible. We are particularly interested in studying how the mobility in these proteins changes in the presence of different external signals so that we can correlate these variations with the given responses inside the cell. Most likely our results will allow us to make conclusions that are very general in nature as it is highly likely that other GPCRs will function following a similar way of action. So far GPCRs have been elusive to such studies and our work intends to generate this novel insight.

Impact Summary

Our work concentrates on understanding how G protein-coupled receptors (GPCRs) function. This remains one of the most important and challenging questions in biology, not only from a mechanistic perspective but also for general human health. GPCRs form the largest family of proteins in humans and regulate most aspects of normal physiology as membrane embedded signalling molecules. Obtaining insight into dynamics and structure of GPCRs is essential for the mechanistic understanding of their activation and forms a vital asset for future drug development. The function of these proteins is modulated by their dynamic nature and NMR is the only method, which is able to provide information on conformational dynamics at atomic resolution. We will use our extensive expertise in the study of membrane proteins by NMR to characterize the dynamic behaviour of GPCRs. This work will be immediately beneficial to academic/industrial labs, both nationally and internationally, as it will provide a proof of principle how NMR spectroscopy can reveal the plasticity of GPCRs and how signalling is altered by different ligands. Data will be generated through our work that correlates ligand properties and their efficacies with the conformational sampling of the receptor. This information will be highly complementary to existing structural data and benefit the wider signalling community and drug design. Our approach will be transferrable to other stabilized receptors. Using stabilising lipid environments we anticipate even wildtype proteins to become accessible eventually. Technical improvements through our work will also benefit the research community studying membrane proteins by NMR. Staff working on this project will immediately and directly benefit, as they will develop/acquire skills in protein work, NMR and data analysis leading to mechanistic understanding. This will improve their employment prospects in both academic and industrial environments. Training expert post-doctoral scientists in these skills will also be highly beneficial to future employers. By adding to the trained pool of researchers in the UK we will increase the economic competitiveness of the country by strengthening its position in the global academic and pharmaceutical market. Our previous work on large helical membrane proteins and the research pursued here make our lab one of the leading experts in the field in the UK and worldwide. Our expertise in the study of such proteins will provide a strong asset for the UK GPCR community who can benefit from our expertise and through collaborative efforts will be able to explore mechanistic features of other receptors. Our work will significantly add to the knowledge base of how GPCRs work and how downstream signalling responses can be influenced. It is becoming increasingly clear that next to G-proteins GPCRs interact also with a range of other proteins that result in non-G-protein related signalling processes. This harbours enormous potential for biotech and pharmaceutical industry to expand drug therapeutic interventions through the design of ligands that influence or bias specific signal transduction. In the medium term (2-10 years) pharmaceutical industry will be able to rely on NMR derived information to gauge the mode of action of these proteins. This information will be vital to guide the development of drugs. Effective remedies can only be developed from an understanding of the underlying molecular principles of the biology involved. Increased mechanistic understanding will lead to improved as well as new drug types, further expanding an already multi-billion pound market. This will increase revenue in the UK, secure employment in the future and generate wealth and prosperity. As GPCRs affect many areas of the human physiology, a multitude of novel therapeutic approaches will be able to counter the effects aberrant signalling has, helping large portions of the population. This will improve the general health of our society.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Pharmaceuticals, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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