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Cross-linking and molecular modelling to determine the structure and dynamics of the intracellular regions of ATP gated P2X receptor ion channels

Reference	BB/M000990/1
Principal Investigator / Supervisor	Professor Richard Evans
Co-Investigators / Co-Supervisors	Dr Ralf Schmid
Institution	University of Leicester
Department	Cell Physiology and Pharmacology
Funding type	Research
Value (£)	345,527
Status	Completed
Type	Research Grant
Start date	30/11/2014
End date	29/11/2017
Duration	36 months

Abstract

P2X receptors for ATP are ligand gated ion channels and at least one receptor subtype is expressed by almost every cell type. It is clear that the intracellular amino (N) and carboxyl (C) termini play central roles in the gating/regulation of P2X receptors. The X-ray structures of the apo and ATP bound zebrafish P2X4 receptors provided a major advance in molecular understanding. However, the intracellular regions were truncated to aid crystallization and so there is no information on these regions. Our pilot studies on native and cysteine mutant human P2X1 receptors have shown that lysine and cysteine reactive cross-linking reagents can be used to provide distance constraints to generate molecular models of the intracellular N and C termini. To test and develop these models we will use a range of cross-linking compounds, mass spectrometry (CXMS), cysteine mutants, and gel shift assays to identify additional molecular distances in the N and C termini. Agonist binding induces marked conformational changes at the intracellular ends of the transmembrane segments. Therefore to map the corresponding movements at the intracellular termini we will determine cross-linking in the apo state (in the presence of apyrase to break down endogenous ATP) as well as following irreversible agonist binding with photo-activatable 2-azido ATP. The studies will be done in an iterative cycle of data collection, modelling and further testing of predictions to refine and validate models of the structures of the N and C termini of the P2X1 receptor. The total conservation of motifs in both the N and C termini of mammalian P2X receptor subunits suggests that their orientation may also be conserved. We will use information from our models to make/test predictions on distances in the human P2X2 receptor. These studies will provide the first 3D models of the interactions of the N and C termini and will address the molecular basis of their contributions to channel properties.

Summary

Cells within the body communicate with one-another through the release of chemicals recognised by specific cell surface receptors. One example of such a chemical is ATP that binds to P2X receptors. In humans there are at least 13 different types of P2X receptor that vary in their properties, for example in terms of sensitivity to ATP. P2X receptors play an important role in a range of normal bodily functions e.g. in control of blood pressure, blood clotting and taste sensation, as well being potential drug targets for the treatment of pain and neurodegenerative diseases e.g. Alzheimer's disease. P2X receptors are membrane proteins with parts on the outside of the cell that recognize the ATP molecule, a region that passes through the cell wall (that is associated with the channel turning on) and a region that is inside the cell (intracellular). The intracellular region is made up of separate parts that play an important role in the controlling how long the receptor is active for. Recent studies have shown the 3D structure of a P2X receptor at rest and following ATP binding and this has provided a major advance in our understanding of how this novel family of receptors works. However to obtain these structures the intracellular parts of the receptor were removed, and therefore we have no information on the structure of the intracellular regions, or the mechanism of how they regulate the receptor. This information is essential to understand the fundamental mechanisms associated with the activity of this distinct family of receptors. The intracellular regions of the receptor are made up of a combination of different building blocks called amino acids, and some of these have unique properties that can be used to measure molecular distances in the receptor. One such chemically unique amino acid is lysine and several of these are present in the intracellular regions of the P2X1 receptor. We used a drug, DSS, to determine the distance between lysine residues in the intracellular region. The DSS molecule can be thought of as a stick with a lysine reactive group/ball at either end. When we treated the P2X1 receptor with DSS it will bind to lysine residues and two lysine residues can be bound together (cross-linked) if the distance between them is equivalent to that of the two balls at the end of the stick. We can then extract the P2X1 receptor and use a technique called mass spectrometry to identify which lysine residues have been cross-linked by the DSS. This provides us with a distance constraint between the lysine residues that can be used in computer simulations to provide a model of the 3D structure of the intracellular parts of the receptor. In this study we will use a range of chemical cross linkers (that vary in the length of the stick and the chemical reactivity of the groups at either end) as molecular rulers to determine how close defined residues in the intracellular parts of the receptor are. These distances will then be used to develop 3D models of the receptor that give rise to predictions that can be tested to validate and refine the model. We will test the effects of the cross-linker in the absence and presence of ATP to determine whether there is a movement in the relative positions of the intracellular regions that is associated with turning on of the receptor. We will also compare the pattern of cross-linking between the P2X1 receptor that turns off quickly in response to ATP and the P2X2 receptor that shows a sustained response as long as ATP is present. In this way we will be able to determine the 3D shape of the intracellular parts of P2X receptors and if these change when they are turned on and off. This will provide a fundamental insight into how these receptors work at the molecular level, and give insight into variations between receptors, and why genetic mutations affect receptor properties that can lead to imbalances in signalling and disease.

Impact Summary

The primary impact of our work will be to provide the first validated 3D models of the intracellular regions of a P2X receptor. The work has direct relevance to the Academic community, in particular those with an interest in ion channels. The crystallization of apo and ATP bound zfP2X4 receptors has had a major impact on our functional understanding. However, it has been suggested that other states are likely to account for key experimental data. The intracellular amino and carboxyl termini of P2X receptors play a major role in the regulation of channel properties and many membrane proteins, including P2X receptors, have been truncated to optimize crystallization for structural studies. Our pilot cross-linking and modelling data suggest that the intracellular regions, and/or the membrane environment may play an important role in stabilizing a native state of the receptor. P2X receptor properties are essentially the same whether they are expressed endogenously or recombinantly. Thus our work using P2X receptors and mutants expressed in HEK293 cells will provide structural information in essentially a native environment. Our combined iterative approach of using cross-linking, mass spectroscopy/gel shift assays to develop and test molecular models of the organization/structure of the intracellular regions of the P2X receptor may provide an approach that can be used on the intracellular regions of other ion channels and proteins. In addition by collecting data from P2X receptors at rest, and following activation, for both desensitizing P2X1 and non-desensitizing P2X2 receptors, we will be able to study the molecular mechanisms of receptor desensitization and explain previous mutagenesis work. This will provide fundamental information on mechanisms of ion channel gating that may also be applied to the ASIC/ENaC/DEG family of trimeric ligand gated channels, that share some architectural features with P2X receptors, and were also truncated to aid crystallization. Structural information is key to understanding the function of proteins, but in particular membrane proteins and receptors, or parts of them, are often not accessible to high resolution structure determination via X-ray or NMR methods. In silico methods such as ab initio protein structure prediction or protein-protein docking are not yet reliably generating structural models that are useful for the interpretation of biological data. One way to address this problem is using low resolution experimental data to drive molecular modelling. For example using residual dipolar coupling (RDC) NMR data in protein-protein docking has substantially improved the quality of protein-protein docking models. In this project we use distance constraints derived from cross-linking to improve structure prediction and derive models that enable us to interpret biological data. We would envisage that analogous approaches could be applied to other receptors and membrane proteins where structural information is limited. The impact of our work will be initially through the publication of our research findings that will be of interest to the academic community. In the medium to long term similar approaches may be used for other ion channels/receptors/membrane proteins. The post-doctoral researcher will benefit from continued scientific training, including the interpretation of molecular modelling and bioinformatics data. This will be important for their career development and be applicable to both academic and industrial biotechnology positions. In addition there will be continuing ongoing professional development of presentations skills (oral, written and numerical analysis). The project will also provide training opportunities for MSc students in Bioinformatics doing 3 months projects on P2X receptor modelling. This will give them direct practical experience as well as training them in presentation skills and time management.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	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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