Wednesday, 6 September 2017

The 2017 ISMB Retreat

The Institute of Structural and Molecular Biology (ISMB) at Birkbeck and UCL holds a conference each summer where all members of the Institute's constituent departments come together to discuss their research. In even years, this takes the form of the ISMB Symposium: two intense days of research talks from ISMB core members and invited research leaders. In odd years, the focus turns to the younger members of the Institute with the ISMB Retreat. This, usually held in Cambridge University's Robinson College, is a two-day residential event featuring plenaries from outside speakers, shorter talks by students and postdocs from the ISMB, an activity designed to get young researchers thinking about science-based careers outside an academic lab, and a poster session. The 2017 retreat, held on June 29 and 30, was the seventh in the series.

There is a full report of this retreat (written by me) with photos on the ISMB website. Do check it out! Here are just a few highlights that are relevant to students of structural biology.

The three keynote speakers were Lori Passmore (MRC Laboratory of Molecular Biology (LMB), Cambridge; Bill Rutherford (Imperial College, London); and Bart Vanhaesebroeck (UCL Cancer Institute). Structural biology was a feature of all three talks, although most prominently in Passmore's. She was recently appointed as a group leader at the LMB, where her group uses high resolution electron microscopy to study the process of poly-adenylation (link to PPS section 8): the addition of the 'poly-A tail' to newly synthesised messenger RNA molecules during the maturation process. Much of the work she discussed has still to be published, apart from a section on techniques development: a physicist in her group, Chris Russo, has worked with her to devise a novel, gold-based substrate for mounting specimens in the electron microscope that eliminates most specimen movement and thus increases resolution.

Bill Rutherford gave an engaging talk, unusually featuring hand-written slides, on the mechanism of action of photosystem II, one of the complex proteins involved in photosynthesis. The evolution of photosynthesis was responsible for the increase in oxygen in the Earth's atmosphere that made multicellular life possible, and Rutherford explained how the increase in oxygen had led to further changes in the structure and mechanism of the photosystem that had the overall effect of decreasing its efficiency. Bart Vanhaesebroeck's lecture, which ended the retreat, described the mechanism of the phosphoinositide-3-kinase family of proteins and their role in cancer. Inhibitors of these kinases (link to PPS section 5) might prove useful anti-cancer drugs, almost certainly as part of combination therapy.

The programme also included nine short talks by students and young postdocs. The excellent quality of these was highlighted by the judges of the best talk, who were unable to come to a consensus judgement. In the end, two equal prizes were given, to Jennifer Booker, who studies the structure of sodium channels (see e.g. this post from September 2016) in Bonnie Wallace's group at Birkbeck, and to Sapir Ofer, for a talk on her PhD studies of the structural and molecular biology of archaeal histones. Proteins in this family are responsible for packing DNA into the chromosomes of eukaryotic cells; bacteria pack DNA using an entirely different mechanism, but the mode of packing in archaea - single-celled organisms that have no nucleus and are defined as prokaryotes but are more closely related to eukaryotes than to bacteria - was unknown until recently.

The retreat always includes an activity to challenge ISMB students and postdocs to think about career opportunities beyond academic research, and this year's proved particularly popular. It was a Dragons' Den style competition in which the younger delegates were split into teams, assigned mentors and given an hour to create a fictitious life science company, develop a bid for funding and present this to a panel of 'dragons'. Three finalists were chosen to battle it out in front of the rest of the delegates. The competitors worked hard and all 'companies' produced ideas that held water to at least some extent, but there could be only one winner. That was a company named TerraNova, which presented an antibody-drug conjugate for primary progressive multiple sclerosis.

Tuesday, 18 July 2017

Highlights from the summer 2017 ISMB seminar programme

Regular readers of this blog will know that the Institute of Structural and Molecular Biology (ISMB) coordinates the research efforts in the Department of Biological Sciences at Birkbeck and two departments – Chemistry and Structural and Molecular Biology – at neighbouring University College London. Research in the participating departments is coordinated through six core research centres, and many grants, PhD studentships and experimental facilities are held in common.

Since 2010, too, these departments’ seminar programmes have been consolidated into termly series of ISMB seminars, giving the Institute’s researchers and students the chance to hear world-class scientists present their work. Most of each term’s seminars are centred round a theme, with recent themes including bioinformatics; the molecular basis of infectious disease; and, in the most recent series, ‘beyond signalling’. This post briefly describes two seminars in this series, both from researchers based in London and both closely concerned with disease mechanisms.

Paul Freemont holds a chair in protein crystallography at Imperial College, London. His group has solved the structure of several proteins linked to cancer, including a domain called the ‘RING finger’ that is found in the breast cancer susceptibility protein BRCA1. His talk to the ISMB, however, concerned a research interest that he shares with the Institute’s head, Gabriel Waksman: the membrane-bound protein complexes through which Gram negative bacteria secrete toxins and other molecules across the double bacterial cell wall and out of the cell. These bacteria have evolved at least six such ‘molecular machines’, with yet another found in mycobacteria such as M. tuberculosis. Waksman’s work in elucidating the structure of the Type IV system is covered extensively in section 11 of the PPS course (‘Structures of Membrane Proteins’).

Freemont’s seminar described the Type VI secretion system (T6SS), which was first identified as recently as 2006 in Vibrio cholerae (as its name suggests, the causative agent of cholera). The function of the T6SS is to eject proteins from the interior of the bacterial cell into an adjacent cell, which may be either bacterial or eukaryotic. Freemont’s lab mainly studies these systems in the bacterium Pseudomonas aeruginosa, an opportunistic pathogen that causes infections mainly in people who are already chronically ill, such as cystic fibrosis patients and those with severe burns.

This secretion system has been described as a ‘molecular syringe’. Its structure resembles that of the tail of a bacteriophage – a type of virus that affects bacteria – but it is inverted, with the tip of the tail pointing away from the bacterial cell wall and towards its target cells. In some species, the same secretion systems can target both eukaryotic cells and other bacteria. The system consists of a long sheathed tube, built up from many protein subunits, that is large enough to be easily viewed using electron tomography and that is tipped by a spike through which the protein to be delivered is ejected. Energy for cargo delivery is provided by the contraction of the tube, with a single contraction storing the energy equivalent of 1600 molecules of ATP. The whole structure is dynamic; it is assembled only when needed and disassembled after the cargo has been delivered, allowing the cycle to begin again.

Although the T6SS can sometimes act as a cell-to-cell ‘killing machine’, as in Vibrio cholerae, protein delivery to the target cell will often have rather more subtle effects, with Pseudomonas aeruginosa a case in point. This rod-shaped Gram negative pathogen uses three distinctly different type 6 systems, encoded on separate operons, to secrete effector proteins that interfere with the host immune system. Freemont and his group have solved the structures of several P. aeruginosa T6SS components using X-ray crystallography, throwing more light on their phage-like mechanism of action. Structures of an accessory protein (TagJ) and the ATPase that catalyses sheath disassembly (ClpV) were all published in the Journal of Biological Chemistry in 2014; some other component structures are yet to be published. TagJ is now known to interact both with ClpV, an AAA+ ATPase, and with components of the sheath, and this interaction allows the rapid disassembly that is required for the complete system to be reset. Each ATPase only interacts with the components from its own operon. Further structural studies, using high-resolution electron microscopy as well as X-ray crystallography, are expected to elucidate further details of these complex molecular machines and to suggest ways in which they might one day be targeted by the novel antimicrobial drugs that we so desperately need.

The second London-based seminar speaker, Miriam Dwek from the University of Westminster, had a somewhat unorthodox beginning to her research career at Oxford University’s first spin-off company, Oxford Glycosystems (now, after many mergers, part of pharma giant UCB). She has maintained her interests in glycobiology (the biology and biochemistry of sugars and polysaccharides) and its application to human disease – particularly breast cancer – into and throughout her academic research career.

Breast cancer is one of the most common cancer types, with 400,000 new cases occurring each year in Europe alone. Breast tumours can be divided into many subtypes with different genetic and biochemical profiles; although some are now easily treated with surgery, radiotherapy and/or drugs, others are often fatal (if perhaps after many years). Generally speaking, tumours are tractable when they are confined to breast tissue and the disease only becomes difficult to treat once it has spread. All cancer types metastasise in a particular pattern; breast cancers tend to spread first into nearby lymph nodes and then to the lungs, brain or bones.

Metastasis is a complex, multi-step process, and selecting the optimum treatment for each patient depends on detecting whether and how her tumour will metastasise as early as possible. Changes in the concentration of some biological molecules in body fluids have been associated with tumour growth and development, and these biomarkers can be used as easily-measurable surrogates of cancer development. One particularly well-known example is the prostate-specific antigen (PSA), a glycoprotein found in semen that is elevated in prostate cancer. No such clear-cut examples exist in breast cancer, but many subtler biochemical changes are known to occur. Dwek and her group have been exploring differences in protein glycosylation patterns between breast tumours and normal breast cells.

Glycosylation is one of the most common post-translational modifications of amino acids (link is to PPS section 2). There are two basic types; one or (almost always) more monosaccharides can be bonded to the oxygen atoms of serine and threonine side chains (O-glycosylation) or to asparagine’s side chain nitrogen (N-glycosylation). The addition of the first residue to the amino acid and the subsequent extension of the chain are catalysed by enzymes in the transferase class. In O-glycosylation, in particular, the patterns of residues added to the glycan ‘branches’ differ between healthy breast epithelial cells and breast tumour cells, and this, in turn, can aid the process of cell adhesion (binding cells together), which is essential for tumour metastasis. Cadherins are glycoproteins that have important roles in cell adhesion, and Dwek’s group used glycoproteomics techniques to identify this as a potential biomarker of metastatic breast cancer. She considers that it is likely to be particularly useful for detecting metastasis in patients with estrogen receptor positive tumours and vascular invasion.

Other topics presented by leading researchers in this ISMB seminar series included nuclear receptors, collagen-binding proteins and protein targeting and translocation. The seminar programme will return in October, and I will doubtless be returning to it again in this blog.

Tuesday, 7 March 2017

Mapping the Evolution of Enzyme Function

The Institute of Structural and Molecular Biology, which combines the research endeavours of Birkbeck and University College London in these disciplines, runs a programme of weekly research seminars throughout the university terms. Each term’s seminars are linked by a theme, and the theme for the spring term of 2017 has been ‘Bioinformatics and Computational Biology’. Early in February, the Institute was delighted to welcome one of the UK’s foremost structural biologists, Professor Dame Janet Thornton, to give a talk in this series. Thornton was well known to many in the large audience, having spent the whole of the 1980s at Birkbeck, rising to be a professor in the School of Crystallography (now part of Biological Sciences). During the 1990s she held chairs at both Birkbeck and UCL and founded a biotech company, Inpharmatica, before leaving to direct the European Bioinformatics Institute (EBI) at Hinxton, near Cambridge. She has now stepped down from the directorship but maintains an active research group at the EBI.

The topic that Thornton chose to present was one that she had worked on throughout her long career: the structure, function and evolution of the enzymes. When she started studying proteins there were probably about 20 known structures. The PDB now holds well over 120,000 protein structures, and tens of thousands of these are of enzymes, so there is plenty of data to work with.

And enzymes are particularly easy to work with because their functions are so well characterised. Back in the 1960s an Enzyme Commission assigned a set of four numbers (‘EC numbers’) to each enzyme. There are six primary enzyme classes, each of which is divided into sub-classes and sub-sub-classes; the final number is a serial number that defines the enzyme’s substrate. So, for example, phosphoinositide phospholipase C is also known as EC; the 3 indicates that this enzyme is a hydrolase, the 1 that it acts on ester bonds and the 4 that it is a phosphoric diester hydrolase. The other top-level classes are the oxidoreductases (1); the transferases (2); the lyases (4); the isomerases (5); and the ligases (6). EC numbers define enzyme function rigorously, so referencing them in computer programs is straightforward.

Thornton and her group chose to focus on those enzymes that have a well-characterised catalytic function that is mainly involved in small-molecule metabolism. All enzymes with these characteristics were grouped into homologous superfamilies (that is, families of proteins with a clear evolutionary ancestor) and the members of each superfamily were annotated with EC numbers as a proxy for their function. For example, the superfamily of enzymes that are clearly related to phosphoinositide phospholipase C by structure and function includes not only enzymes classified as but also sphingomyelin phosphodiesterases D ( and phosphatidylinositol diacylglycerol-lyases ( The two phosphodiesterases have the same chemistry (as specified by the first three EC numbers) but act on substrates with very different shapes, while the chemistry of the enzymes differs significantly from the others.

In this example, comparing the structures of enzymes with the EC numbers and showed that active site residues involved in their reaction mechanism and the bound metal ion in each one that is necessary for catalysis superimpose very well, but the rest of the active site varied significantly to allow substrates with distinctly different sizes and shapes to bind. In contrast, the lyase has a similar-shaped active site to but no bound metal and different catalytic residues. In this case is likely that a single amino acid change, removing an aspartic acid residue and therefore a negative charge, has removed the ability of the enzyme to bind a metal ion and thus changed the reaction that the enzyme catalyses.

Enough data was available to group the enzymes in this superfamily, and in another 275, into phylogenetic trees to map out the evolutionary route taken within each superfamily and catalogue all possible evolutionary changes of function. Some of these are much more complex than the one outlined above. For example, the analysis showed that five classes of flavin-dependent mono-oxygenases with different chemistry were evolutionarily related. Here, the change in chemistry seems to have arisen not from a simple substitution of one amino acid for another but a change in the multi-domain architecture of the protein.

The group constructed an ‘EC exchange matrix’ from this data to show how many times each top-level EC class had changed into each other class during evolution. While most changes in chemistry left the top-level class – the basic type of the reaction – unchanged, every possible change had occurred at least once in evolutionary history. In fact, 11% of the changes catalogued were changes to top-level class. The diagram below illustrates this data in a series of six circles, one for each ‘original’ enzyme class, with the width of each strip indicating the number of transitions from one class to another: for example, the thick red strip going from the ‘top’ to the ‘bottom’ of the top left-hand circle illustrates that a lot of transitions from oxidoreductases (class 1) to transferases (class 2) have been observed.

An overview of functional evolution in enzymes. © Nicholas Furnham & Sergio Martinez Cuesta, EBI

They then looked in much more detail at the changes observed in the catalytic site of each superfamily during evolution, and found that active sites differ in ‘plasticity’. At one extreme there is the TIM barrel ‘superfold’, which is a scaffold that holds amino acids with different chemistry in similar positions to catalyse many different reaction types. At the other extreme, there are seven superfamilies in which the catalytic residues are 100% conserved. It is interesting to try to correlate sequence similarity with ‘functional similarity’, but this runs into the problem of how to define functional identity. With enzymes, any measure of functional similarity will include a contribution from the chemical similarity of the substrates and this is difficult to gauge, particularly as most of the best computational tools were written for commercial drug discovery and are therefore not in the public domain. Preliminary results suggest that there is some correlation, but it is much weaker than that between sequence and structural similarity.

Thornton summed up her lecture by re-stating that evolutionary changes to enzyme substrate specificity are much commoner than those to basic chemistry. Evolution has, however, given rise to an explosion in enzyme function. The EC system has catalogued a total of 2,994 unique enzyme functions, but only 379 different structures (CATH superfamilies) are known to have enzymatic activity. Most enzyme functions will therefore have evolved from another function, with each catalytic activity arising independently only a few times throughout evolutionary history. The evolutionary relationships within enzyme superfamilies are complex and there are many ways in which their function can diverge.

Much of the work Thornton presented has been described in a 2012 paper in PLoS Computational Biology; its lead author, Nick Furnham from the Thornton group at the EBI, is now a group leader at one of Birkbeck’s neighbouring colleges, the London School of Hygiene and Tropical Medicine. PPS students will learn much more about the structure, function and mechanisms of enzymes in section 10 of the course, ‘Protein Interactions and Function’.

The most recent paper from the Thornton group on this topic is:
Furnham N, Dawson NL, Rahman SA, Thornton JM, Orengo CA. Large-Scale Analysis Exploring Evolution of Catalytic Machineries and Mechanisms in Enzyme Superfamilies. Journal of Molecular Biology 428 (2016) p.253-267

Tuesday, 24 January 2017

Seeing the Wood for the Trees in Structural Biology

The British Crystallographic Association (BCA) was set up in 1982 to support UK scientists working in crystallography and other structure-based sciences. It has five specialist groups (four discipline-based, and one for young crystallographers): the Biological Structures Group for structural biologists holds its main annual conference each December, generally just before the Christmas break. Several of these one-day Winter Meetings have been previously described in this blog. The 2016 meeting, however, was particularly relevant for anyone connected to Birkbeck: not only was it held in the college, but it celebrated the work of one of the college’s most distinguished structural biologists, Steve Wood. The meeting title was, of course, a pun on his name.

Wood worked with Professor Sir Tom Blundell at Birkbeck in the 1990s to solve the structure of an important small human protein, serum amyloid P component (SAP or pentraxin; PDB 1SAC). This protein forms pentamers that bind to amyloid fibres and it is thought to be involved in the protection of those fibres from breakdown by proteases. Pentraxin-binding compounds that interfere with this process might be useful as treatments for amyloidosis and other diseases associated with protein aggregation, perhaps including Alzheimer’s disease.

Blundell, a former head of Birkbeck’s Crystallography Department and now emeritus professor of Biochemistry at the University of Cambridge, kicked off the meeting in fine style. He had known Wood since they were, respectively, a young lecturer and a PhD student at the University of Sussex in the 1970s, and they have published over 60 papers together. His talk surveyed the structural biology of multi-protein signalling systems over the last 40 years. The earliest such system to be discovered involved the control of blood sugar levels through insulin and glucagon binding to their receptors. The general principles developed through structural studies of this relatively simple system have been applied to other, more complex ones including the interaction between the breast cancer susceptibility protein BRCA2 and a recombinase enzyme that controls one type of DNA repair. Mutations that interfere with this binding lead to greatly enhanced susceptibility to some cancer types. Blundell’s group at Cambridge set up a database, CREDO, to catalogue the interactions involved in all macromolecular complexes in the PDB. Many protein-protein interactions are now actual or potential drug targets. Some promising drugs for solid tumours act by inhibiting the interactions between cyclins and cyclin dependent kinases (CDKs) that drive cells through the cell cycle. Astex Pharmaceuticals, the drug discovery company set up by Blundell and some of his Cambridge colleagues in 1999, has one such CDK inhibitor – ribociclib – that has completed Phase III clinical trials for advanced breast cancer.

Garry Taylor, who gave the next talk, joined Blundell’s group as a postdoc soon after its move to Birkbeck in the mid-70s, where he established a long, productive collaboration with Wood and with Jim Pitts, who now directs the PPS course. Taylor is now a professor at the University of St Andrews in Scotland where he studies the structure and mechanism of sialidases. These enzymes hydrolyse (break) the bond between a terminal sialic acid residue and the remainder of a polysaccharide or glycoprotein; both bacterial and viral sialidases are involved in the pathology of infectious disease. All sialidases share a catalytic domain with a characteristic beta propeller fold, but the bacterial enzymes have a separate carbohydrate-binding domain (CBD). This binds tightly to the sialic acid substrate of all sialidases, including that of influenza virus neuraminidase (which will be covered in detail in section 10 of the PPS course). Taylor and his group were awarded a grant to explore the idea that this domain, alone, might bind tightly enough to sialic acids on the surface of influenza virus host cells to prevent both virus entry and the release of progeny virions. They have now developed multi-valent CBDs that can protect mice from challenge with a lethal dose of influenza virus. Taylor suggested that, if these molecules are as successful in protecting against influenza in human trials, they might also be useful prophylactics for other respiratory pathogens that bind to cells via sialic acid receptors.

Jonas Emsley, one of Wood’s many PhD students at Birkbeck, is now at the University of Nottingham where his group studies the structures and mechanisms of proteins involved in blood coagulation. His talk focused on the activation and assembly of proteases in the contact system, in which the presence of ‘foreign’ surfaces such as bacteria triggers several physiological processes including blood clotting. Inappropriate activation of this system has been linked to heart disease and stroke, and mice that lack either of the coagulation factors Factor XI and Factor XII are protected to some extent from thrombosis. Factor XI, which is activated by Factor XII, contains four repeats of a domain with six conserved cysteine residues that can be drawn in the shape of an apple, hence its name of ‘apple domain’. The protein circulates as a dimer with the monomer-monomer interactions mediated by one apple domain and the catalytic domains sitting on top of the eight apple domains like a cup on a saucer. There is a pocket on the surface of each apple domain, and the pocket on the second such domain binds a conserved tripeptide, DFP, that is found in many of its substrates. Small-molecule inhibitors of this interaction might be useful anticoagulants.

Structure of factor XI apple domain with bound peptide substrate showing the conserved DFP motif. Image (c) Jonas Emsley

Other speakers included Birkbeck’s Helen Saibil, whose ground-breaking high resolution electron microscopy of protein complexes has been covered many times in this blog (see e.g. posts from April 2015 and July 2013) and Neil McDonald, now based at the Francis Crick Institute in London, who described some largely unpublished work on the structure and mechanism of RET receptor tyrosine kinases. Appropriately, however, the final talk was devoted to Wood’s structure: SAP. It was given by Simon Kolstoe who joined the Wood group in Southampton as a PhD student in 1999, moved with him back to UCL and is now at the University of Portsmouth. He first presented a ‘potted history’ of structural studies of this protein, describing how a competitive inhibitor of SAP-amyloid binding was developed as a potential treatment for amyloidosis at the turn of the millennium. This compound, CPHPC, was found to deplete SAP levels in serum but, unfortunately, clinical amyloid levels were unchanged. A high-resolution structure of this compound binding to SAP was published in 2014 (PDB 4AVV). Kolstoe and his co-workers have now turned their attention to SAP binding to DNA, which might also be clinically relevant.

The meeting ended with the usual votes of thanks, with the award of a poster prize to Jingxu Guo from University College London, and with a gift to Wood: a molecular model of a SAP-drug complex, presented by Tony Savill of Molecular Dimensions Ltd.

Image of two molecules of SAP coordinated with five molecules of CPHPC. Image (c) Simon Kolstoe, PDB 4AVV

Monday, 10 October 2016

Welcome to PPS students 2016-17!

This post is extremely like those I have written at this time of year for the past few years. This is because what I have to say now is very, very similar...

I would like to offer a warm welcome to the Principles of Protein Structure blog to all students who have just started studying Birkbeck's Principles of Protein Structure (PPS) course, and a welcome back to any who have taken a break in studies and intend to complete the course this year.

I run this blog to link the material that you will be studying in the course to new research developments in the areas of protein structure and function and related aspects of biotechnology and medicine. I might, example, report on talks given in the ISMB seminar series run jointly by the Department of Biological Sciences at Birkbeck and research departments in neighbouring University College London. The programme for Autumn 2016 focuses on the molecular and structural biology of infectious disease; there is material on similar topics in section 10 of the PPS course, 'Protein Interactions and Function'. Other posts may be reports from conferences or summaries of recently published papers in protein structure, protein bioinformatics and allied areas. Look out for an account of a conference on structural assemblies at Birkbeck in December that will honour the 50-year career of one of our emeritus professors, Steve Wood.

Some earlier posts on this blog were written by "guest blogger" Jill Faircloth, who took the MSc in Structural Molecular Biology a few years ago and is now working as a freelance science communicator. She introduces herself in this post written in March 2012, in which she also describes how she found the later part of the PPS course and her thoughts on the two choices available for the second year of the MSc.

Do, if you get a chance, look through some blog posts from earlier years to see the kind of topics that we will be discussing. However, don't be discouraged if at this stage of the course you find the science presented there difficult to understand. I can assure you that it will get easier!

I particularly recommend that you look at a couple of posts from December 2013 and July 2014 about the history of structural science, particularly X-ray crystallography. Crystallography was the first method to be developed for solving the structure of biological macromolecules, and it is still the most important. The year 2014 was designated by the United Nations as the International Year of Crystallography, marking the year between the centenaries of the publication of the first papers on X-ray diffraction and the award of the 1915 Nobel Prize for Physics to the father-and-son team of William and Lawrence Bragg who made the principal discoveries.

So - the best of luck for the 2016-17 PPS course and for your studies at Birkbeck! We hope that many of you will go on to complete our MSc in Structural Molecular Biology.

Best wishes,

Dr Clare Sansom
Senior Associate Lecturer, Biological Sciences, Birkbeck and Tutor, Principles of Protein Structure

Monday, 19 September 2016

Shining light on the 3D structures of membrane proteins

A symposium was held at Birkbeck on August 10, 2016 to honour one of the college’s most distinguished structural biologists, Professor Bonnie Wallace. This was organised by a postdoctoral member of her group, Lee Whitmore, and her long-term colleague and collaborator Dr Bob Janes from Queen Mary, University of London to celebrate her 65th birthday. It featured speakers from five continents, all of whom had collaborated with her or worked in her lab at some point in their careers.

Wallace joined what was then the Department of Crystallography at Birkbeck in 1990 after holding several research positions in her native USA. Her research on the structure and function of membrane proteins has won her several prestigious awards, including the Biochemical Society’s triennial AstraZeneca award in 2010. This is given for outstanding research in a UK or Irish laboratory that leads to the development of a new method or reagent. She has made significant contributions to both the development of circular dichroism spectroscopy as a tool for investigating the structures of proteins (including membrane proteins) at less than atomic resolution, and to studies of membrane protein structures using crystallography and electron microscopy. Her studies of voltage-gated sodium channel structures have led to some important insights about their functions in health and disease.

The symposium was divided into three sessions, with the first devoted to circular dichroism spectroscopy and the second two to membrane proteins. A general report of the day has been published on the Biological Sciences website; here, to fit in with the remit of the PPS course, I concentrate on the sections on membrane proteins.

The first talk was on electron microscopy, given, appropriately enough, by one of the pioneers of the field: Richard Henderson from the MRC Laboratory of Molecular Biology in Cambridge. Throughout most of the 1970s and 1980s he and his collaborator, Nigel Unwin, worked on the development of electron microscopy techniques for the study of protein structures. Most of their work involved the proton pump, bacteriorhodopsin, which is found in very high concentrations in the purple membranes of Halobacteria. At the beginning, this work was very time-consuming: it took them a year to locate the C-terminus of the protein, and another to determine the binding site of its ligand, retinal.

The first near atomic resolution structures of this protein were obtained in the mid-1990s. At about that time, too, he switched the focus of his interest from the structures of ‘2D crystals’ of bacteriorhodopsin to those of ‘blob-like’ single particles: isolated protein chains or, more often, membrane-embedded protein complexes. The list of biologically and medically important complexes to have been solved using this technique is now growing rapidly, and includes rotary ATP synthase (see the previous post on this blog); the next complex in the electron transport chain, known as respiratory complex I; and gamma secretase, which is a potential drug target for Alzheimer’s disease.

Molecular simulation and modelling techniques have developed alongside those of structural biology and for almost as long. Mark Sansom, a professor of structural bioinformatics at the University of Oxford, described simulations of membrane proteins. He started his talk describing a program to visualise and analyse the pores through the centres of membrane proteins that was written by Oliver Smart (now at the EBI) when he was a postdoc in Wallace’s group. This program, HOLE, is relatively simple but is still widely used. Sansom’s current work uses molecular dynamics to model the membrane bilayer with numbers of embedded proteins, focusing particularly on interactions between those proteins and the lipids of the membrane.

Not surprisingly, there were several talks about the ion channels that have been a focus of so much of Wallace’s more recent research: voltage-gated sodium channels. Hugh Hemmings from Weill Cornell Medicine, New York, USA described how these channels have become useful targets for anaesthetic drugs. General anaesthesia is a drug-induced coma characterised by unconsciousness, immobility and amnesia; an effective anaesthetic will achieve all these and a wide variety of molecules have been employed to greater or lesser effect since the nineteenth century. Many of these target proteins involved in the release of neurotransmitters by pre-synaptic nerves, including ion channels; sodium channels were first proposed as anaesthetic targets in the late 1970s but fell out of favour for several decades. Interest in this mechanism of anaesthesia has revived with the use of the bacterial proteins – a focus of Wallace’s structural studies – as a model system. Hemmings’ current studies focus on the mechanism through which volatile anaesthetics such as isoflurane inhibit the passage of sodium ions through these channels.

Crystal structure of the NavMs voltage-gated sodium channel pore depicted in ribbon motif and viewed from the membrane normal direction. each of the four monomers in the tetrameric structure is depicted in a different colour (from Naylor et al., 2016 - Wallace lab paper). The transmembrane sodium pathway run through the middle of the structure, from top to bottom.

Lin Field of Rothamsted Research, Harpenden, UK, described research leading to a very different application of sodium channel blockers: as insecticides. Insects cause an immense amount of crop damage worldwide, but non-specific insecticides might be toxic either to humans or to beneficial insects such as bees. The mechanism of the pyrethroid class of insecticides was unknown when the first members of this class were patented, but they are now known to bind to voltage-gated sodium channels and prevent their closure. Structural studies of these proteins have shown how mutations that are known to lead to pyrethroid resistance can prevent the molecules from binding, and why these compounds have very little effect on the very similar mammalian channels. Researchers hope that these studies are taking us nearer to the development of ideal, ‘designer insecticides’ that are only harmful to pest species.

Further talks were given by Wallace’s first Ph.D. student at Birkbeck, Declan Doyle, who is now at the University of Southampton; by Per Bullough from the University of Sheffield; and by Dame Carol Robinson, the first woman to be appointed as a full professor of chemistry at the University of Oxford. The symposium ended with a summary and vote of thanks from Janes, who stressed that it did not mark Wallace’s retirement: she still loves science and has many questions to answer. I hope that I will be blogging innovative research from the Wallace lab for many years to come.

Wallace’s research has been described in this blog on several previous occasions – see in particular this post from April 2013 and this one from November 2010. The use of cryo-electron microscopy to determine atomic resolution structures of proteins is covered in depth in our Techniques in Structural Molecular Biology course, which is one of the options for the second year of the Structural Molecular biology MSc.

Thursday, 4 August 2016

ATP synthase: a new drug target for tuberculosis

The London Structural Biology Club (LSBC) is a network for students and researchers working in all aspects of structural molecular biology and based in London and the south-east of England. Once a term, members get together for an afternoon of research talks and discussion followed by refreshments (generally featuring pizza and beer). These meetings are often held at Birkbeck, and we have featured them on the PPS blog before (see this post from 2008 and this one from 2012).

The LSBC meeting for the summer term of 2016 was also held at Birkbeck, and hosted and chaired by Alfonso de Simone, a lecturer in NMR spectroscopy at Imperial College London. One of the four talks was given by Thomas Meier, also from Imperial College. Meier, who has worked at ETH Zurich, Switzerland, and the Max Planck Institute of Biophysics in Frankfurt, Germany, was appointed to a chair of structural biology at Imperial just over a year ago. His research concerns the structure and function of a tiny, complex ‘molecular machine’, ATP synthase (the link is to our page on that enzyme in section 10 of the PPS course). This enzyme is a ‘rotary ATP synthase’, catalysing the conversion of the electrochemical energy of ion transfer across the cell membrane into chemical energy stored in ATP. Meier and his group have solved the complete structure of this multi-subunit enzyme complex using a combination of X-ray crystallography and electron microscopy, and shed light on its role as a target for a novel class of drugs against tuberculosis.

ATP synthase is a highly dynamic enzyme complex, which makes it particularly difficult to study structurally. The complete enzyme comprises two motor sub-complexes; most of one, termed Fo, is embedded in the membrane and the other, F1, is in the matrix. They are tightly coupled with each other and linked by subunits forming an outer stalk and an inner, central stalk. From a functional point of view, the ATP synthase consists of a rotor and stator part, both sharing subunits from the F1 and the Fo sectors. The motors are driven either by the proton (or sometimes Na+) motive force to form ATP, or in opposite direction by the hydrolysis of ATP to pump ions across the membrane. Similar enzymes are found in the inner membranes of bacteria and mitochondria and in the thylakoid membranes of chloroplasts.

John Walker of the MRC Laboratory of Molecular Biology in Cambridge was awarded a share of the 1997 Nobel Prize in Chemistry for determining the structure of the bovine F1 motor using X-ray crystallography (e.g. PDB 1E79). This subcomplex consists of five different types of subunits; three pairs of similar alpha and beta subunits are arranged alternately around the rotor (gamma / delta / epsilon subunits), which harbours an asymmetric coiled coil domain in the gamma subunit. The nucleotides ADP and ATP can bind to the interfaces between the beta and alpha subunits; the central gamma subunit rotates in 120o steps, causing conformational changes that in turn change the affinity of the three catalytic sites for the nucleotides. Rotation in one direction, driven by the energy of ion transfer across the membrane, leads to synthesis of ATP from ADP and phosphorus (Pi); rotation in the other direction will hydrolyse ATP back into ADP and Pi, thus releasing the energy required to pump ions.

The membrane-embedded Fo motor consists of the rotor part, a ring of identical c-subunits termed the c-ring and the stator part, a single-chain a-subunit that grants access and release pathways for ions. Each c-ring subunit is a helical hairpin with its N and C termini on the cytoplasmic side; the number of subunits is constant within a species but varies between species, as far as we know today, between 8 and 15 c-subunits. Meier’s group has solved the structures of a number of c-rings from different species of bacteria, helping to elucidate the rotor’s mechanism of action; essentially, protons (or in some cases Na+ ions) can reach one of the c-ring subunits by an ion pathway mediated by the stator a-subunit, where they lock to a free ion binding site on the c-ring, rotate with the c-ring for almost a complete 360o turn to reach the second release pathway that leads to the other side of the membrane. The ion translocation causes rotation of the Fo ring and with it the complete central stalk that protrudes the F1 headpiece.

Artist's impression of an ATP synthase molecule embedded in a membrane. Image © Laura Preiss, Max Planck Institute of Biophysics, Frankfurt, Germany

Until recently, ATP synthase has not been thought of as a drug target, principally because the structures and mechanisms of the bacterial and human enzymes are so similar. Now, however, it has emerged that it is the target for bedaquiline, the first novel drug to be approved for treating tuberculosis (TB) for over 40 years. And new drugs for TB are needed very badly: in 2015, over 9 million people contracted this disease and about 1.5 million died from it. Tens of thousands of TB cases each year are multidrug resistant (MDR) or even of the extremely drug resistant (XDR) variant for which no other clinically approved antibiotic is available anymore. Bedaquiline, however, is effective against both, MDR- and XDR-TB strains.

Functional analysis has shown that bedaquiline, which is a diarylquinoline, acts by binding to and halting the rotation of the Fo rotor. Meier and his co-workers have now solved the structure of the drug bound to the c-ring from a similar, non-pathogenic bacterial species, Mycobacterium phlei. This structure has nine c-subunits and shares over 80% sequence identity with the M. tuberculosis c-subunit variant (100% match at and around the drug binding surface). The crystal structure (PDB 4V1F; Preiss et al. (2015)) shows the drug occupying the proton-binding site on each of the nine subunits and thus preventing proton transfer. This stalls the rotation of the Fo motor, preventing rotation and thus the synthesis of ATP in F1. Small differences between the structures of the proton-binding sites account for the exquisite specificity of bedaquiline for the Fo rings of mycobacteria and thus for its efficacy and safety as an anti-tubercular drug.

Meier ended his talk by explaining that TB-causing bacteria will eventually – perhaps sooner rather than later – develop resistance to bedaquiline, just as they have to every previous drug that has entered the clinic. There is therefore a pressing need to develop further drugs that act at the same target, and his group’s structural studies are proving useful in the search for bedaquiline analogues.

The London Structural Biology Club has a public Facebook group, which can be found here.