Dr Liz Carpenter
|Research Area:||Protein Science and Structural Biology|
|Technology Exchange:||Crystallography, Drug discovery and Protein interaction|
|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 25% 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.
The three dimensional structures of these large macromolecules are the key to understanding their function and designing drugs to inhibit them. In the Integral Membrane Protein group at the SGC we use the technique of X-ray crystallography to solve membrane protein structures. We then study the structures in complex with inhibitors and drugs, using this information to improve and extend the available treatments for disease. The IMP group at the SGC studies proteins from a variety membrane protein families, including ion channels which are critical for heart and nerve function, transporters which move solutes, nutrients and waste products across membranes and ABC transporters which move waste products and drugs into and out of cells. In the past two years we have established a working high-throughput system for the producing human membrane proteins for structural studies. This system has allowed us to obtain crystals for three human membrane proteins and pure protein from more than 30 proteins and we are developing these projects for structure function studies. In less than two years we obtained our first structure, the first structure of a human ABC transporter, the mitochondrial ABCB10 protein which is thought to be involved in protecting the heart during oxidative stress.
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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
Structural Studies of Human ABC Transporters
The aim of this project is to use X-ray crystallography, biochemical and biophysical studies to understand how a human mitochondrial ABC transporter, ABCB10, contributes to the formation of red blood cells and how it helps mitochondria to cope with oxidative stress. Oxidative stress within mitochondria can lead to damage to mitochondrial DNA, one of the primary causes of mitochondrial failure during aging in humans. Understanding how ABCB10 functions in cells could therefore contribute to the ...
Structural studies of human ion channels
Ion channels are critical for the function of almost every aspect of the human body, playing essential roles in the brain, nervous system and the heart. Defects in these channels lead to a host of medical conditions such as heart disease, cystic fibrosis and depression. We aim to study these complex, membrane embedded proteins using X-ray crystallography to solve their three dimensional structures. to date there are few structures of mammalian ion channels and only two human channels. Many ...