Download X-ray and Cryo-EM Structures for Novel Human Membrane Protein

X-ray and Cryo-EM Structures for Novel Human Membrane Protein Targets in Diabetes and
Obesity
Supervisor:
Professor Liz Carpenter
Department:
Structural Genomics Consortium, Nuffield Department of Medicine
Background
Many genes have been identified as associated with diabetes and obesity through large scale
sequencing efforts. A large number of these genes are integral membrane proteins and this class of
proteins are regarded as challenging for drug design because they are difficult to produce, assay and
obtain structural information. The field of human integral membrane proteins (IMPs) structure and
function is however developing rapidly and these targets represent an untapped resource for novel
diabetes and obesity targets. Prof. Liz Carpenter’s Integral Membrane Proteins Group at the SGC in
Oxford works with a broad range of IMPs, including ion channels, solute carriers, ABC transporters
and membrane enzymes. There are potential diabetes targets in all these families. In the past 6 years
this group has solved structures of seven human membrane proteins, with examples from each of
these families (Quigley et al., Science, 2013; Shintre et al., PNAS, 2013; Dong et al., Science, 2015).
We have also developed assays appropriate to each protein, either in house, or through
collaborations. Therefore at the SGC, we are uniquely well-placed to produce IMP diabetes targets,
solve their structures (by X-ray, serial femtosecond crystallography or cryo-electron microscopy),
develop assays and identify probe molecules to study their roles in healthy and patient-derived cells.
Novo Nordisk’s programme to study secreted proteins, through protein production, antibodies and
peptides as therapeutics, aligns well with our work on plasma membrane proteins, as these proteins
are accessible at the surface of cells, and are thus amenable to similar treatments to secreted
proteins. Production of integral membrane proteins in proteoliposomes, nanodiscs or detergents for
therapeutic purposes would be feasible and would extend the number of diabetes targets that can
be studied at the protein level.
Aims
We aim to:
1. Study novel diabetes and obesity integral membrane protein targets, both targets that are
already identified as important for diabetes, and new targets, as further sequencing of
genomes leads to the identification of new proteins that are relevant to diabetes.
2. Develop methods for producing of these proteins in a range of formats
3. Work with Novo Nordisk to produce antibodies against these proteins
4. Solve 3D structures of these targets, with antibodies and in complex with small molecule
binders.
5. Develop assays for function, stability, small molecule and antibody studies.
6. Design specific probe molecules to activate and inhibit these proteins in cells.
Description of work
1. Identify up to 20 human integral membrane proteins that are of interest to Novo Nordisk.
Our current target list includes 14 membrane proteins that are associated with diabetes and
obesity. We would work with Novo Nordisk scientists to select current targets and identify
new targets that are of value for diabetes and obesity.
2. A number of these targets have already been screened for expression in insect cells. The
fellow would develop purification protocols for these in parallel with testing new targets for
expression, using the SGC’s high-throughput methods. We have screened over 400 IMPs to
date and successfully produced 10% of them, so it is essential for the fellow to start with a
range of targets.
3. The fellow would develop methods to insert the proteins into proteoliposomes, nanodiscs
or lipodiscs. They would then work with Novo Nordisk scientists to determine if these
proteins are sufficiently stable to be used as therapeutic agents. Mutagenesis to stabilize the
proteins would be an option for proteins of lower stability.
4. Protein in proteoliposomes would be used for antibody production in Novo Nordisk’s labs
either by the fellow themselves working in NN labs, or by NN antibody production experts.
5. For the targets that can be produced, the fellow would then use a range of structural
biology techniques available in our lab, include X-ray crystallography, serial femtosecond
crystallography and electron microscopy to solve the structures of at least one of their
targets.
6. For well-behaved targets the fellow would develop assays to assess the function of the
protein. We use generic thermostabilization assays to test binding of small molecules to
membrane proteins on a medium-throughput scale and then test the effect of compounds
using transport assays, electrophysiology assays or enzymatic assays, depending on the type
of protein. These assays will be done either in house, with NovoNordisk scientists or through
our extensive network of collaborators. These assays can be used to test the effects of
stabilization methods, antibodies and peptides on purified protein and protein in
membranes. Small molecules can be used to probe the function of proteins in healthy and
disease d cells and in animal models.
Expected outcomes
1. As integral membrane protein targets are challenging to study, we would expect that within
3 years the fellow would assess the tractability of up to 20 IMP and then focus on five
targets that are most likely to give a structure and valuable data for a therapeutic.
2. They would to produce at least three proteins, including developing protocols for producing
the protein in proteoliposomes, nanodiscs and lipodiscs.
3. They would use thermostability assays to test stability of preps and to determine whether
small molecules, lipids or binding proteins would stabilize the protein.
4. The fellow would aim to solve the structure of at least one of the proteins, using whichever
technique is most appropriate for the protein.
This work will open up new target areas in the integral membrane proteins field as a collaboration
between Novo Nordisk and the SGC. All the research performed in the SGC labs will be published
without restriction, once the structures are solved.
Recent relevant publications
1. Dong YY, Pike, AC, et al. (2015) K2P channel gating mechanisms revealed by structures of
TREK-2 and a complex with Prozac. Science 347: 1256-1259.
2. Stansfeld PJ, Goose JE, et al. (2015) MemProtMD: Automated Insertion of Membrane
Protein Structures into Explicit Lipid Membranes. Structure 23, 1350-1361.
3. Quigley A, Dong YY, et al. (2013) The structural basis of ZMPSTE24 dependent laminopathies.
Science, 339(6127), 1604-1607.
4. Shintre CA, Pike AC et al. (2013) The first human ABC exporter structure reveals the initial
steps in the transport cycle. Proc Natl Acad Sci USA, 110, 24: 9710-9715.