Integral Membrane Proteins

Research

Approximately 30% of the genes in the human genome code for membrane proteins, molecules which are embedded in the lipid bilayers of cells and organelles. These proteins are essential for moving ions, nutrients, waste products, drugs and large molecules such as proteins and DNA across cell membranes. Membrane proteins such as G-protein coupled receptors (GPCRs) are also essential for signalling processes, which are critical for cells to communicate with their environment. Since membrane proteins are the gateways into cells they are often the site of action of drugs; In fact more than 50% of all known drugs interact specifically with membrane proteins.

Membrane proteins are unfortunately notoriously difficult to handle and study because they are designed to sit within the hydrophobic environment of the lipid bilayer. They tend to be unstable when extracted from their native environment. There are therefore fewer than 200 structures of membrane proteins known, less than 2% of all the known structures, and most of these are from bacterial proteins. Membrane proteins are therefore one of the most important remaining frontiers of structural biology research. At the SGC we are now applying our state-of-the-art high-throughput methods to overcome the bottlenecks in membrane protein research, so that we can reliably deliver pure membrane protein samples and structures of these fascinating and medically critical molecules.

Medical importance

Mutations in membrane proteins are involved in many common diseases, including heart disease, where malfunctioning ion channels are often implicated. Drugs targeted to calcium channels can control issues such as high blood pressure and angina. Membrane proteins are also involved in cancer, where errors in signalling pathways can lead to cells dividing out of control. Often specific membrane proteins are overproduced in cancer cells and are therefore targets for drug therapy. Diseases of the brain such as migraine, depression and Alzheimer's are all linked to problems with transporters and channels. Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene which encodes a chloride ion channel. Our understanding of these and many other diseases would benefit greatly from more structural and functional information on the proteins involved. We hope that by solving the structures of these proteins, understanding the underlying biochemistry and studying their interactions with substrates and inhibitors, we can provide more effective treatments for many of these medically highly important diseases.

Membrane proteins and the SGC

The Structural Genomics Consortium has extensive experience in using high throughput methods to solve protein structures, having deposited over 800 structures of soluble human proteins. We are now applying those methods to the challenging field of membrane protein structural biology. In phase I the SGC targeted bacterial membrane proteins and produced structures for the KirBac3.1 potassium channel, led by the former PI, Dr Declan Doyle (Gulbis et al., in preparation, Kuo et al., 2005), and the magnesium transporter, CorA, solved in collaboration with Dr Chris Koth (Lunin et al., 2006).

CorA - iSee datapack

KirBac3.1 - iSee datapack

Now in phase II the SGC is focussing primarily on the structures of human membrane proteins. We also work with mammalian homologues of target proteins when the human protein is intractable. In addition we have a program in Oxford and Toronto to work on soluble domains of selected proteins. Targets are chosen in collaboration with our pharmaceutical and academic partners according to their scientific interest and medical importance. These include GPCRs, metabolic enzymes, proteases, ion channels and transporters (click here for a description of these targets)

Technologies for studying IMPs

The SGC aims to develop generic methods that will enable the high throughput structure determination of human membrane proteins. We have selected the baculovirus/insect cell expression system which provides a lipid composition close to that of human cells and is a proven high throughput platform. To date this system has been used successfully for structural studies of 2 human and 7 other eukaryotic IMP proteins in the PDB. We are testing the multiconstruct approach which has proven highly successful for soluble proteins in the SGC. For each target protein we generate up to 12 constructs of varying length and different affinity tags, including the full length gene and a series of truncations to remove potentially disordered regions. A high throughput expression screen is used to identify targets that can be purified in milligram quantities for crystallisation. Each protein is initially purified in dodecyl maltoside (DDM) detergent and is subsequently screened for stability in a series of different detergents to identify the optimum conditions for stability and crystallisation.

Together with Frank von Delft at SGC we are developing improved methods for high throughput nanodrop crystallisation as well as the manipulation of fragile membrane protein crystals, and the efficient collection and analysis of diffraction data using intense synchrotron microfocus beamlines, available at resources such as Diamond Light Source Ltd, in Oxfordshire. Wherever possible crystallisation is performed with bound ligands and inhibitors to capture a single native conformation and provide key insights into function and drug design. We are also generating renewable antibody fragments against our purified proteins for use as affinity reagents and crystallisation aids.

References

• Carpenter, E.P., Beis, K., Cameron, A.D., Iwata, S., "Overcoming the challenges of membrane protein crystallography", Current Opinion in Structural Biology, 18 (5), (2008), 581-586.
• Lunin, V.V., Dobrovetsky, E., Khutoreskaya, G., Zhang, R., Joachimiak, A., Doyle, D.A., Bochkarev, A., Maguire , M.E. , Edwards, A.M., Koth, C.M., "Crystal structure of the CorA Mg2+ transporter", Nature, 440 (7085) , (2006), 833-837.
• Gulbis J.M., Kuo, A., Smith, B., Doyle, D.A., Edwards, A., Arrowsmith, C., & Sundstrom, M. In preparation.
• Kuo, A., Domene, C., Johnson, L. N., Doyle, D. A. and Venien-Bryan, C., "Two different conformational states of the KirBac3.1 potassium channel revealed by electron crystallography", Structure, 13 (10), (2005), 1463-1472.