Wednesday, 22 December 2010
Before the main business could begin, however, the delegates heard two interesting short talks. The first was by Martin Welsh, coordinator of life sciences research at Diamond, the UK's synchrotron facility (located in the Oxfordshire countryside). This provides the most intense radiation sources for X-ray crystallography (and other analytical techniques) in the country, and although it has been open since 2007 it is still complete. The UK scientific community is still deciding the range of problems to be addressed by the last set of beam lines, which in turn will determine how they are constructed. He called for the structural biology community to become fully involved in this process, as it risks losing out to the physical scientists.
The second introductory talk was by the BCA's President, Professor Elspeth Garman from the University of Oxford: first structural biologist to hold this position for many years. She explained how the relationship between technological developments in X-ray crystallography and the biological insights that it provides is as strong now as it was when Dorothy Hodgkin (Nobel Laureate in Chemistry 1964) was solving the structures of penicillin, vitamin B12 and (in 1969) the peptide hormone, insulin.
Highlights of structural biology research were then presented in three main sessions. The first of these featured three keynote lectures by distinguished structural biologists. The only common feature of the biological systems described was the fact that metal ions formed a crucial part of their mechanism of action.
First, Chris Schofield from the University of Oxford described his group's work elucidating how mammals sense and respond to changes in the concentration of oxygen. This work can be said to date back to the nineteenth century, when climbers, and people living at high altitude where oxygen concentrations are lower, were first observed to have increased numbers of red blood cells. It is now known, in part thanks to Schofield's work, that cells respond to low oxygen levels through a transcription factor called Hypoxia Inducible Factor 1 (HIF-1), which in turn regulates the production of another protein, erythropoietin, which is involved in the production of red blood cells. HIF-1 is upregulated if oxygen concentrations are low and broken down by the proteasome when concentrations are high. Schofield has used crystallography to determine the structural basis of the "switch" between the stable (low oxygen) and unstable (high oxygen) forms of this enzyme. The stable form contains two key proline residues (link is to section 2) and these are oxidised to hydroxyproline to give the unstable, high oxygen form. The enzyme that catalyses this oxidation reaction, HIF prolyl hydrolase (PHD) binds an iron ion in its active site unusually tightly, which causes it to react unusually slowly; it is this slow reaction that enables it to react to long-term changes in oxygen concentration.
Ben Bax, from GlaxoSmithKline (GSK) in Stevenage (and formerly a Birkbeck postdoc), then described how X-ray crystallography is being used to design novel inhibitors of bacterial proteins called DNA topoisomerases. These are enzymes that catalyse the winding and unwinding of DNA molecules, a process that is different enough in bacteria for inhibitors of these enzymes to be specific and effective antibacterial drugs. Bacteria have two forms of this enzyme, topoisomerase II (or DNA gyrase) and topoisomerase IV. Both these, confusingly, are known as type II topoisomerases; these are the target of the fluoroquinolone antibiotics, which have been in clinical use since 1962 and had sales of over $7Bn in 2009. Bax's group at GSK has solved the structure of a complex between the antibiotic moxifloxacin and topoisomerase IV from Acinetobacter baumannii, revealing that the enzyme-inhibitor interaction involves a magnesium ion that is not involved in the catalytic reaction (PDB entry 2XKK). They have also solved the structure of DNA gyrase from Staphylococcus aureus in complex with a novel bacterial topoisomerase inhibitor (PDB entry 2XCS), which sheds new insight into the enzyme's mechanism and the role of manganese ions in it.
The final talk in this session was given by Wyatt Yue, from the Structural Genomics Consortium in Oxford, and concerned the structure and function of an enzyme, methylmalonyl-CoA mutase (but known, thankfully enough, as MUT) which is involved in the metabolism of vitamin B12 (also known as cobalamin). This vitamin is essential for life; bacteria can synthesise it, but mammals need to take it in through their diets. Its structure was solved by Dorothy Hodgkin in 1954. It can bind cobalt in any of its oxidation states: Co(I), Co(II) and Co(III). Although only two enzymes including MUT require vitamin B12 as a co-factor, its deficiency can cause severe disease. Yue and his group have solved the structure of MUT bound to vitamin B12 (PDB entry 2XIJ) and that of an associated protein, a GTP-binding protein known as MMAA (PDB entry 2WWW). This protein is believed to act as a "gatekeeper", necessary for binding MUT to the appropriate, adenosyl form of vitamin B12. Two copies of the same MMAA mutation, which abolishes its interaction with MUT, have recently been identified in a patient with the metabolic disorder methylmalonic aciduria.
There were two further sessions at the conference, which I don't have room to blog about: one covered metal ion transport and catalysis, and the other metals in proteins that function as transporters, receptors and chaperones. There is some more information on the conference website.
Thursday, 25 November 2010
Doublecortin is a MAP that stabilises the most commonly found type of microtubule, which is composed from thirteen protofilaments. Mutations in both proteins that destabilise the interaction between them can cause devastating neurological diseases. A recent paper by Carolyn Moores, an electron microscopist working at Birkbeck, has highlighted details of the interaction between these proteins.
You can read a summary of this paper here (PDF format). This is one of a series of summaries of high impact papers by Birkbeck scientists that I have written for the website of the Institute of Structural and Molecular Biology, which links researchers in these disciplines at Birkbeck with those at UCL.
If you have time once you have read the summary you may explore the structures of these proteins further by downloading the atomic coordinate files that were "docked" into the electron density in this paper from the PDB. The PDB entry used for the alpha-beta tubulin dimer structure is 1JFF and the one used for the doublecortin domain that is bound to tubulin is 1MJD.
Friday, 5 November 2010
At the end of October Birkbeck College hosted its first Science Week, featuring lectures from distinguished researchers in all science departments. Two talks by members of the Department of Biological Sciences highlighted the beauty and importance of protein structure: the topic of the PPS course. Both lecturers came originally from North America; both moved to Birkbeck about twenty years ago; and both have achieved very significant honours as researchers in structural biology since.
Proteins, DNA and the Components of Life
Professor Bonnie Wallace’s research focuses on the structural analysis of proteins and peptides bound to membranes, and particularly on those involved in ion transport and cell signalling. Her talk in Science Week was entitled “Proteins, DNA and the Components of Life”. She began by describing proteins and nucleic acids as large molecules – “macromolecules” – that are essential components of all living cells. They are both polymers of smaller units; as you should know by now, proteins are polymers of amino acids and nucleic acids similarly are polymers of nucleotides. Amino acids combine as “beads on a string” in many different ways to form the vast array of known proteins; the functional – structural, metabolic or transport among many others – of each is determined by its three-dimensional structure. The techniques used for determining protein (and DNA) structure, including X-ray diffraction, which have developed from fairly crude beginnings about fifty years ago, now allow even large structures to be determined quickly. The section of Professor Wallace’s talk that introduced basic protein structure was illustrated with a physical model of a protein from the 1970s that was then compared with modern molecular graphics representations of protein structures. “Cartoon”, “ball and stick” and “space-filling” protein models, as well as stereo views that can give the illusion of three-dimensionality, are all useful for different purposes.
DNA molecules also are linear polymers, and these – particularly in eukaryotic cells – are extremely long and need to be packed very tightly if they are to fit into the tiny cell nuclei. The packing mechanism involves interactions between the DNA molecule and proteins called histones, to form a structure resembling “beads on a string” that condenses further to form fibres and eventually chromosomes.
The final part of Professor Wallace’s talk concerned the evolutionary differences between DNA in different individuals and in different species, celebrating the tenth anniversary of the publication of the first draft of the human genome. Genomics has provided a useful correction to human arrogance by revealing that many organisms have larger, if not necessarily more complex, genomes than ours. More importantly, differences between bacterial and human cells, between cancer cells and normal ones, and between dysfunctional (mutated) and normal versions of disease-linked proteins, are enabling us to design and develop drugs against a wide range of diseases. Important drugs for infectious diseases including influenza and HIV have been designed using “structure-based” techniques. Professor Wallace ended her talk by describing some of her group’s work on the structure of a protein that is embedded in a cell membrane and provides a channel for sodium ions to pass into cells. Anti-epileptic drugs such as lamotrigine work by binding to and blocking these channels, which will be discussed in detail in the section on Membrane Proteins towards the end of the PPS course.
Model for the structure of a human sodium channel
The Arms Race between Man and Pathogen
Professor Helen Saibil studies proteins using electron microscopy, which can determine the structure of very large proteins and of protein complexes embedded in cell membranes. Her talk, intriguingly titled “The Arms Race between Man and Pathogen”, focused on two pieces of her group’s research into proteins that punch holes, or pores, in membranes. These independent studies led to the discovery of an unexpected similarity between a group of bacterial proteins that cause damage during infection and one of human (and other mammalian) proteins that help protect against viral infection and blood cancers.
Pore-forming protein toxins are “weapons” that bacteria can use in order to break holes in the cells of their human or other animal hosts. They are used by a wide range of bacteria, including some very dangerous ones; one example is Streptococcus pneumoniae, which, as its name implies, causes pneumonia. Unusually, these proteins can exist in two forms, one that is water-soluble and another that is membrane-embedded. The change between these forms requires a considerable rearrangement of the protein’s shape, with one hydrophobic (“water-hating”) segment that is located deep in the core of the soluble protein uncurling to form a “beta-hairpin” structure that embeds in the membrane.
In 2005, Professor Saibil’s group used electron microscopy to determine the structures of two forms of the pore forming toxin from S. pneumoniae, pneumolysin, that together illustrate its mechanism of action. Initially, the protein associates into ring-shaped “pre-pores” of about forty monomers that sit on the membrane surface; the pore itself is formed when part of each monomer changes shape and burrows deep into the membrane. They were able to fit the more detailed X-ray crystal structure of a related protein as a soluble monomer into the shape of the pre-pore protein, and show the type of rearrangement that would be necessary for part of that protein to “collapse” into the membrane.
Another protein that has been studied by Professor Saibil and her colleagues recently is perforin, which is secreted by some lymphocytes (white blood cells that form an important part of the immune system) and which also punches holes in membranes: this time, in the membranes of infected or cancerous cells. This allows the lymphocyte to deliver toxins to the affected cell that trigger the process of apoptosis, or programmed cell death. People with a defective form of this protein suffer from severe viral infections and have a greatly increased risk of blood cancers.
There is no detectable similarity between the sequence of perforin and that of pneumolysin. However, when the first structure of a human perforin-like molecule was discovered in 2007, researchers were surprised to discover that its fold was similar to that of the bacterial protein. It became clear that human and bacterial pore-forming proteins must have evolved from a common ancestor. Protein structures are more tightly conserved than gene and protein sequences, and the structures of distantly related proteins are often much more clearly similar than their sequences.
Hoping to understand more about the mechanism of this protein, Professor Saibil’s group then used electron microscopy to solve the structure of perforin assemblies in contact with membranes. They observed pore structures that were similar, but not identical, to those of pneumolysin. In particular, the perforin structures indicated a much smaller conformational change between non-bound and membrane bound forms than the one seen with pneumolysin. Also, the perforin molecule seemed to be arranged “inside out” in the pore structure as compared to that of pneumolysin, an observation that the group was able to verify with biochemical tests. This finding was very unexpected, but turned out to be consistent with a twist between two parts of the pneumolysin molecule that does not occur in perforin. Both molecules have “arms” that extend down into the membrane to form the sides of the pore, but perforin’s arms are much longer and do not require as big a conformational change to reach down into the membrane.
This story of two related proteins, one on each side of the battle between man and pathogen, features many aspects of protein structure research that are covered during the PPS course. These include the beta-hairpin structure, the interatomic forces that determine the way proteins fold, and the structures of membrane proteins. If, however, you have been inspired to learn more about the way electron microscopy is used to study protein structure, you should aim to take our second-year MSc module, Techniques in Structural Molecular Biology.
Tuesday, 5 October 2010
I have run this blog in some recent years to help students understand how what they study relates to current research in protein structure and in areas related to it such as drug design and development. This year, I will be posting accounts of lectures, conferences and new papers in the areas covered by the course, and link them to the scientific literature more widely. Comments are welcomed and if any of you would like to have a go at blogging I can set you up as an author.
We have a weekly seminar programme shared between Biological Sciences at Birkbeck and the other departments in the Institute of Structural and Molecular Biology, and later in the course I will occasionally post reports of these seminars when they seem particularly relevant to the course material. There is a theme for the seminars each term, and this term (Autumn 2010) this is "Cellular and Structural Biology of Infection".
Before then, however, I will be blogging from Birkbeck Science Week at the end of October. There will be two lectures then that are very relevant for this course, both given by senior professors in the Department of Biological Sciences: Prof. Helen Saibil on the structures of bacterial protein toxins and Prof. Bonnie Wallace on proteins and DNA. Do check back on this blog afterwards as I hope that will give you an idea of both the beauty and the elegance of our subject. Or - if you are within reach of London on either of those days - do come and hear what they have to say! Science Week lectures are free and open to all, although registration is required.
Enjoy the blog - and do let us know what you think of it!