Dr Liz Carpenter
|Research Area:||Protein Science and Structural Biology|
|Technology Exchange:||Crystallography, Drug discovery and Protein interaction|
|Scientific Themes:||Protein Science & Structural Biology and Physiology, Cellular & Molecular Biology|
|Keywords:||Membrane proteins, X-ray crystallography, Protein structure, ion channels, high throughput methods and drug design|
Membrane proteins are the gateways to the cell. All cells and organelles are surrounded by an oily, impermeable lipid bilayer and many small molecules can only cross this barrier by passing through protein molecules embedded in the bilayer. Many nutrients, ions, waste products and even DNA and proteins enter and leave cells only via proteins which are tightly controlled, thus maintaining the integrity of the cell. Communication between cells is also mediated by these proteins often by binding signaling molecules outside cells and amplifying the signal by triggering chemical reactions inside the cell. These diverse functions are fulfilled by a huge variety of membrane proteins, in fact approximately 15% of all the genes in the human genome code for these proteins. Given their location on the surfaces of cells, it is not surprising that membrane proteins are often found to be the targets for drugs, such as the calcium channel blockers used to treat heart disease and potassium channel blockers which are used in diabetes treatment. Indeed membrane proteins are involved in the development of many diseases, including heart disease, cancer, cystic fibrosis, Alzheimer’s, Parkinson’s and other neurological diseases, kidney disease and epilepsy.
My group at the structural Genomics Consortium in Oxford aims to solve the three dimensional structures of human membrane proteins using X-ray crystallography. We purify proteins, pursuad them to form crystals, and then expose them to a beam of X-rays. The resulting diffraction patterns can then be used to understand the positions of all the atoms in the protein. We then study the structures in complex with inhibitors and drugs, using this information to improve and extend the available treatments for disease. There are less than 50 structures of human membrane proteins known and we therefore seak to develop methods to make this process more efficient. In the past four years we have established a working high-throughput system for the producing human membrane proteins for structural studies.
The IMP group at the SGC studies proteins from a variety membrane protein families, including ion channels enzymes and ABC transporters. To date we have solved structures of of proteins in three different areas:
1. We solved the first structure of a human ABC transporter, ABCB10, a mitochondrial protein which is important for heme production and for resistance of mitochondria to oxidative stress.
2. Premature ageing syndromes can be caused by a failure in the processing of the lamin proteins, which form a network of fibres underlying the nuclear membrane within cells. We have solved the structure of a zinc metalloprotease, ZMPSTE24, which is responsible for two steps in this processing. This structure has allowed us to see how mutations in the ZMPSTE24 protein can lead to premature ageing diseases, which provide a model for normal ageing.
3. Recently we have solved and deposited the structure of a human ion channel, TREK-2, one of the family of K2P proteins which are responsible for the background leak current that helps to maintain the membrane potential and also are susceptible to a range of physiological and pharmacological stimuli.
There are no collaborations listed for this principal investigator.
ABCB10 is one of the three ATP-binding cassette (ABC) transporters found in the inner membrane of mitochondria. In mammals ABCB10 is essential for erythropoiesis, and for protection of mitochondria against oxidative stress. ABCB10 is therefore a potential therapeutic target for diseases in which increased mitochondrial reactive oxygen species production and oxidative stress play a major role. The crystal structure of apo-ABCB10 shows a classic exporter fold ABC transporter structure, in an open-inwards conformation, ready to bind the substrate or nucleotide from the inner mitochondrial matrix or membrane. Unexpectedly, however, ABCB10 adopts an open-inwards conformation when complexed with nonhydrolysable ATP analogs, in contrast to other transporter structures which adopt an open-outwards conformation in complex with ATP. The three complexes of ABCB10/ATP analogs reported here showed varying degrees of opening of the transport substrate binding site, indicating that in this conformation there is some flexibility between the two halves of the protein. These structures suggest that the observed plasticity, together with a portal between two helices in the transmembrane region of ABCB10, assist transport substrate entry into the substrate binding cavity. These structures indicate that ABC transporters may exist in an open-inwards conformation when nucleotide is bound. We discuss ways in which this observation can be aligned with the current views on mechanisms of ABC transporters. Hide abstract
Mutations in the nuclear membrane zinc metalloprotease ZMPSTE24 lead to diseases of lamin processing (laminopathies), such as the premature aging disease progeria and metabolic disorders. ZMPSTE24 processes prelamin A, a component of the nuclear lamina intermediate filaments, by cleaving it at two sites. Failure of this processing results in accumulation of farnesylated, membrane-associated prelamin A. The 3.4 angstrom crystal structure of human ZMPSTE24 has a seven transmembrane α-helical barrel structure, surrounding a large, water-filled, intramembrane chamber, capped by a zinc metalloprotease domain with the catalytic site facing into the chamber. The 3.8 angstrom structure of a complex with a CSIM tetrapeptide showed that the mode of binding of the substrate resembles that of an insect metalloprotease inhibitor in thermolysin. Laminopathy-associated mutations predicted to reduce ZMPSTE24 activity map to the zinc metalloprotease peptide-binding site and to the bottom of the chamber. Hide abstract
The mechanism by which nucleotide-binding domains (NBDs) of ABC transporters power the transport of substrates across cell membranes is currently unclear. Here we report the crystal structure of an NBD, FbpC, from the Neisseria gonorrhoeae ferric iron uptake transporter with an unusual and substantial domain swap in the C-terminal regulatory domain. This entanglement suggests that FbpC is unable to open to the same extent as the homologous protein MalK. Using molecular dynamics we demonstrate that this is not the case: both NBDs open rapidly once ATP is removed. We conclude from this result that the closed structures of FbpC and MalK have higher free energies than their respective open states. This result has important implications for our understanding of the mechanism of power generation in ABC transporters, because the unwinding of this free energy ensures that the opening of these two NBDs is also powered. Hide abstract
Outer membrane proteins are structurally distinct from those that reside in the inner membrane and play important roles in bacterial pathogenicity and human metabolism. X-ray crystallography studies on >40 different outer membrane proteins have revealed that the transmembrane portion of these proteins can be constructed from either beta-sheets or less commonly from alpha-helices. The most common architecture is the beta-barrel, which can be formed from either a single anti-parallel sheet, fused at both ends to form a barrel or from multiple peptide chains. Outer membrane proteins exhibit considerable rigidity and stability, making their study through x-ray crystallography particularly tractable. As the number of structures of outer membrane proteins increases a more rational approach to their crystallization can be made. Herein we analyse the crystallization data from 53 outer membrane proteins and compare the results to those obtained for inner membrane proteins. A targeted sparse matrix screen for outer membrane protein crystallization is presented based on the present analysis. Hide abstract
The nucleobase-cation-symport-1 (NCS1) transporters are essential components of salvage pathways for nucleobases and related metabolites. Here, we report the 2.85-angstrom resolution structure of the NCS1 benzyl-hydantoin transporter, Mhp1, from Microbacterium liquefaciens. Mhp1 contains 12 transmembrane helices, 10 of which are arranged in two inverted repeats of five helices. The structures of the outward-facing open and substrate-bound occluded conformations were solved, showing how the outward-facing cavity closes upon binding of substrate. Comparisons with the leucine transporter LeuT(Aa) and the galactose transporter vSGLT reveal that the outward- and inward-facing cavities are symmetrically arranged on opposite sides of the membrane. The reciprocal opening and closing of these cavities is synchronized by the inverted repeat helices 3 and 8, providing the structural basis of the alternating access model for membrane transport. Hide abstract
Membrane protein structural biology is still a largely unconquered area, given that approximately 25% of all proteins are membrane proteins and yet less than 150 unique structures are available. Membrane proteins have proven to be difficult to study owing to their partially hydrophobic surfaces, flexibility and lack of stability. The field is now taking advantage of the high-throughput revolution in structural biology and methods are emerging for effective expression, solubilisation, purification and crystallisation of membrane proteins. These technical advances will lead to a rapid increase in the rate at which membrane protein structures are solved in the near future. Hide abstract