G-protein-coupled receptors (GPCRs) are integral membrane proteins involved in signal transduction, and constitute major drug targets for disease therapy. Sequence analysis of the human genome suggests that there are up to 1000 different GPCRs, each interacting specifically with ligands ranging from odorant molecules to neurotransmitters to hormones. However, despite their striking clinical relevance, high-resolution structural information on GPCRs is only available for the visual pigment rhodopsin, which can be purified in large amounts from natural tissue. Structure determination of any other GPCR requires a suitable heterologous expression system and a robust large-scale purification scheme to obtain milligram quantities of functional receptor protein for 3D crystallization studies. We focus on the three-dimensional structure determination of GPCRs by x-ray crystallography. This includes heterologous expression, large-scale purification of receptors in functional form, pharmacological characterization, receptor crystallization, and use of antibody fragments for co-crystallization experiments. We explore currently also the structure of a neuropeptide ligand bound to its GPCR. In the past year, I accomplished the following towards these goals: (1) Large-scale fermentation, automated large-scale receptor purification and 3D crystallization experiments: Progress towards structure determination of integral membrane proteins is given by obtaining milligram quantities of functional receptors on a regular basis, i.e. every week. With Dr. Joseph Shiloach and his co-workers (Biotechnology Unit NIDDK), we have obtained E. coli cells from 200-liter fermentors with receptor material of high quality. We have used a large-scale, automated purification scheme for the GPCR neurotensin receptor to produce on a weekly basis 5 mg of functional receptor protein suitable for 3D crystallization set-ups. We have explored so far more than 1600 crystallization conditions, and trends regarding receptor stability and crystallisability are becoming now clear. (2) Identification of additional GPCRs suitable for crystallization: Although GPCRs seem to be similar, there are fundamental differences regarding expression levels and biochemical behavior, with profound consequences for successful crystal formation. Currently, we have identified 2 promising GPCRs: a formyl peptide receptor and a chemokine receptor. (3) Co-crystallization of bacterially expressed Fv antibody fragments with integral membrane proteins has successfully led to high-resolution 3D structures of a bacterial cytochrome oxidase and the yeast cytochrome bc1 complex. In addition, the high-resolution structures of bacterial potassium channels in complex with monoclonal Fab antibody fragments have been reported by MacKinnon's group. Co-crystallization with antibody fragments may also be beneficial for the structure determination of GPCRs, allowing better crystal contacts and constraining receptor flexibility. We have pursued the generation of antibody fragments along 3 lines: generation of antibody fragments by phage display against lipid-reconstituted receptors, and against detergent-solubilized membrane proteins, and by 'classic' means. (3.1) Functional reconstitution of receptors into lipid vesicles: These proteoliposomes are currently being used to select for antibody fragments by the phage display system in collaboration with a group in Germany. (3.2) I have visited Andrew Bradbury's laboratory in Los Alamos, to use his phage display library for selection of antibodies against membrane proteins in detergent solution. Experiments were done with a bacterial membrane protein to explore and solve potential difficulties arising from the presence of mild detergents. The technology is now established in the laboratory and I will apply this to GPCRs. (3.3) Generation of monoclonal antibodies in mice: This project is commissioned out to commercial companies, using a bacterial membrane protein as a test candidate, to explore and solve potential difficulties associated with the generation of conformation-specific antibodies. Once this project is completed and evaluated, we will address GPCRs if feasible. (4) In collaboration with Marc Baldus at the Max-Planck-Institute for Biophysical Chemistry, Gottingen, Germany, we have completed the structure determination of isotopically labeled neurotensin, bound to its receptor, by solid state NMR techniques. Our ability to produce receptors on a regular basis, and to efficiently reconstitute receptors into lipid has allowed us to achieve this goal. I will continue this collaboration to obtain structural information about which residues of the ligand interact with which residues of the neurotensin receptor. (5) NIH Intramural Aids Targeted Antiviral Program (IATAP) FY 2003/04: Together with Susan Buchanan, I was awarded IATAP funding on 'Structure determination of chemokine HIV-1 co-receptors' (FY03: $ 100,000). (6) The Membrane Protein Interest Group (MPIG), which I chair, provides a forum for discussion relating to all aspects of membrane proteins (functional and structural). The MPIG was well received on campus and has been very active (see www.nih.gov/sigs/mpig/).
