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.

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.

New protein structures presented at the 2013 BCA Winter Meeting

The work of crystallographers in the UK is supported through the British Cryatallographic Association, which has about 700 members based in academia and industry. It is organised into four groups representing different disciplines within crystallography, including one for structural biologists called, not surprisingly, the Biological Structures Group. Every December, this group organises a one day conference to present some of the most recent developments in structural biology. I have blogged these meetings before, and searching this blog for "winter meeting" will find a few of those posts.

The 2013 meeting was billed as both a "final" event in the centenary year of the Braggs' landmark discoveries and part of the build-up to the International Year of Crystallography, but these were not the only anniversaries highlighted there. 2013 also marked the sixtieth anniversary of the publication of the structure of DNA. The 2013 Winter Meeting was held in King's College London, which played a very important part in that discovery: Maurice Wilkins and Rosalind Franklin, who obtained the X-ray diffraction patterns that led to the discovery of the double helix, were based there. (Wilkins shared the Nobel Prize for this discovery with Watson and Crick; Franklin died in 1958, four years before that prize was awarded.) And the first precise physical model of the double helix is still on display in the college.

Maurice Wilkins' original DNA model

The first researcher to speak at the meeting was Birkbeck's own Professor Bonnie Wallace. Her work on the structures of voltage gated sodium channels has been described on this blog before, most recently in April 2013. These proteins are responsible for the transport of ions in and out of cells, an essential signalling mechanism in all multi-cellular organisms. Their structures, however, are among the most intractable of all membrane proteins (PPS section 11, to be released in May, covers this fast moving field). Wallace has used a combination of X-ray crystallography, spectroscopy and molecular dynamics to explore the structure and mechanism of sodium channels in bacteria. The bacterial sodium channel is simpler than the mammalian equivalent, consisting of a tetramer in which helices from each monomer line the pore. The Wallace group's most recent strucure (PDB 3ZJZ) shows the position of the C-terminal domain of these channels for the first time. This domain consists of a coiled coil formed by one helix from each monomer that is linked to the rest of the protein by a flexible region. Moving the coiled coil up and down causes a conformational change that allows the channel to open and close.

The technique of rational or structure-based drug design, which involve modelling the interactions between a library of potential ligands and a protein binding site, has proved particularly successful in the design of anti-viral drugs. Several inhibitors of HIV protease and of influenza virus neuraminidase that were designed in this way have become very successful drugs. David Stuart from the University of Oxford and the Diamond synchrotron gave a talk illustrating how structure-based in silico techniques are now being applied to design drugs against another virus family: the Picornaviruses. Members of this large family are responsible for a diverse range of diseases, ranging in humans from polio to the common cold. The foot-and-mouth virus, which affects livestock and which devastated parts of the UK countryside in 2001, is also a member of this family.

One of the viruses studied in Stuart's goup is a human picornavirus that causes similar symptoms to the foot-and-mouth virus and that represents a serious threat to public health in East Asia. The disease is known as hand foot and mouth virus, and the virus as CAV16: like all picornaviruses, it consiss of a single strand of RNA enclosed within an icosahedral (20-sided) protein capsid. The intact virus particles are very fragile and diffraction patterns must be captured before the particles disintegrate in the X-ray beam. Stuart and his Chinese collaborators have used one of the microfocus beamlines at Diamond to take snapshots of the virus structure at several points during its life cycle. One of these is of an "uncoating intermediate" that shows one of the viral proteins (VP1) emerging from the capsid so that it can be embedded in the membrane of a host cell Ren et al., 2013). Stuart and his co-workers are now designing compounds to bind to these intermediate structures and prevent the virus from entering its human host cells.

All cells, whether prokaryotic or eukaryotic, contain long molecules of DNA that must be packaged in order to fit into the confined space available. Fortunately for developers of anti-bacterial drugs (and users of antibiotics) bacterial cells package DNA using a different mechanism from mammalian ones. In bacteria, enzymes called topoisomerases bind to, cut and re-join double-stranded DNA so that it can be unwound or untangled ahead of replication. Ivan Laponogov, a postdoctoral research assistant at King's College, described recent work in his group on the structure of one of these enzymes. Bacterial topoisomerase II ia a target for an important class of antibiotics, the fluoroquinolones, but resistance to these drugs is increasing.

These enzymes are powered by ATP and act as "clamps", capturing one double-helical strand of DNA and passing it through a break in another to remove supercoils and knots in the nucleic acid structure. The structure presented at this meeting was the first of a complete topoisomerase dimer bound to DNA in the "open clamp" position. This structure was solved with and without a fluoroquinolone drug (levofloxacin) bound. The structure with drug bound showed that molecule intercalating between DNA bases at the point where the nucleic acid would be cleaved, preventing that cleavage. The structure without the drug showed the DNA in a different position; the position of a functionally important magnesium ion also changed between the structures.

Many essential cellular processes involve a post-translational modification in which poly-(ADP ribose) or PAR is added to amino acid side chains, and the processing of this molecule involves a wide variety of enzymes. Inhibitors of one of these, poly-(ATP ribose) polymerase or PARP, have recently been developed as drugs against cancer. David Leys from the University of Manchester described his work on the structure of another enzyme in the PAR life cycle: poly-ADP-ribose glycohydrolase (PARG), which catalyses the removal of PAR from proteins.

Mammalian PARG enzymes have three domains, a N-terminal regulatory region and two C-terminal domains forming the catalytic region; the equivalent bacterial enzymes lack the N-terminus. Leys and his groups first solved structures of a bacterial PARG bound to ADP-ribose (PDB 3SIG) and to a known inhibitor with a similar structure. They found that a C-terminal helix in the protein was clamped around the terminal ribose of PAR, enabling the release of a single ADP-ribose from the polymer. This basic mechanism is similar in the mammalian enzyme. More recently, the Leys group has solved the structure of PARG bound to an intact PAR substrate (PDB 4L2H); modelling studies based on this structure suggest that the enzyme acts predominantly as an exo-glycohydrolase, that is, it catalyses the removal of one residue at the end of the polymer chain. Understanding the structure and mechanism of these enzymes should enable us to develop small-molecule inhibitors of PARG, and these may one day rival the PARP inhibitors as anti-cancer drugs.

A hundred years on from the "invention" of crystallography and sixty years on from the structure of DNA, these elegant, fascinating and complex structures presented at one meeting give a snapshot of recent progress in structural biology. Furthermore, each of these structures has already provided insights into human disease that may yet lead to the development of useful drugs.

Science Week 2013: Structures of Sodium Channels

Since 2010, Birkbeck College has held a week of lectures, most often in the spring, to highlight some of the research carried out in the School of Science. This year’s speakers included Professor Bonnie Wallace from the Department of Biological Sciences, who presented a fascinating and accessible lecture on the structures of sodium channels, and what these new structures are already teaching us human health, and particularly about some rare neurological diseases.

Professor Nicholas Keep, Dean of the Faculty of Science (and director of the MSc in Structural Molecular Biology and the second-year option TSMB) introduced Professor Wallace. She has been at Birkbeck for about twenty years and now directs the department’s impressive research work on the structural biology of membrane ion channels. You will learn a lot about membrane proteins in general in section 11 of PPS; they are ubiquitous, are responsible for the transport of both chemicals and signals into and out of cells, and form some of the most important drug targets. They are also, as Wallace made very clear in her talk, some of the most challenging of all proteins for structural biologists to work with.

All cell membranes are semi-permeable, which means that some substances can pass across them easily while others are excluded. Ions, which are charged, are generally excluded by the hydrophobic (“water hating”) membranes. This could be something of a problem, as ion transport into and out of cells is an essential physiological process. Ion channels are evolution’s solution to this problem: proteins embedded in membranes that allow ions to selectively enter and leave cells.

Much of Wallace’ work over the last ten years has focused on the structures of voltage gated sodium channels. These open to allow sodium ions to enter cells, and close to prevent them from doing so, in response to changes in potential across the membrane, and they are found throughout nature. Small molecules can bind to these channels, holding them either open or closed; some of these are severely toxic, but others are important drugs for cardiac arrhythmias, epilepsy, and pain.

Human voltage gated sodium channels are composed of a single protein chain, divided into four similar domains. Each of these domains has six transmembrane helices, four of which (labelled S1-S4) act as a voltage sensor while the other two (S5 and S6) fold together to form an eight-helix pore. This protein has so far proved impossible to crystallise, and the breakthrough involved a bacterial protein. Similar proteins are found in the membranes of some species of bacteria, enabling them to live in “extreme” environments that are rich in salt. Their structures are similar to those of the human protein, but in this case the channel is built up from a complex of four identical proteins, each of which is homologous to a single domain of the human channel.

Although this simpler bacterial protein proved easier to work with than the human protein, it was still not at all easy. It took over ten years for Professor Wallace and her group to isolate the gene, clone and purify the protein, obtain crystals and finally solve the structure of the pore. The structure was finally solved using the powerful X-rays generated at Diamond, the UK’s only synchrotron radiation source located near Harwell in Oxfordshire.

These channels exist in three different structural forms: “open”, “closed” and “inactivated”. Many years before the detailed structures were solved Wallace and her group had used a biophysical technique, circular dichroism (CD) spectroscopy, to examine the conformational changes that occurred when mammalian and bacterial channels switched from one state to the other. As always, however, the full atomic-crystal structures yielded very much more information.

The first of these structures to be solved was a slightly strange one: the pore was held in the “closed” conformation that prevents sodium ions from entering the cell, although the voltage sensor was in the structure associated with the “active” state (PDB code 3RVY). The “top” part of this structure, towards the extracellular membrane surface, has a hydrophobic surface, and the pore in this part of the membrane acts as a selectivity filter to allow sodium ions in while keeping others, including potassium and calcium ions, out. Wallace and her group were the first to solve the structure of a fully open channel and showed that the upper portion of the channel containing the selectivity filter was virtually unchanged. The conformational change associated with opening and closing the channel occurs at the internal or cytoplasmic side of the protein (PDB 4F4L). When the pore closes, a small turning motion of the “bottom” part of the helical bundle causes the diameter of the pore to shrink, in a motion rather like the closure of a camera lens; the resulting channel is too small for sodium ions to pass through, so any inside the pore become trapped there.

Two subunits of the bacterial sodium channel pore in the “open” conformation, shown as a ribbon structure

All voltage gated sodium channels have a domain at the C-terminal end of the molecule that is necessary for channel activity but that was not visible in any of the crystal structures. Wallace and her group looked at this part of the molecule in the bacterial protein using a particularly powerful form of CD spectroscopy called synchrotron radiation CD spectroscopy that she had pioneered, and showed that each subunit had an extremely flexible protein chain separating the pore from a C-terminal helix. Using this information, the group have proposed a novel mechanism for channel opening in which the conformational change in the pore is enabled by these helices oscillating up and down.

Two subunits of the bacterial sodium channel pore in the “open” conformation, shown as a ribbon structure

The final part of Wallace’ talk was devoted to the role of sodium channels in health and disease, and as a drug target. A few unfortunate individuals have mutations in a type of channel that is involved in the response to painful stimuli. If this channel is jammed open, patients experience a constant, burning pain termed erythromelalgia, most commonly in their hands and feet. Wallace showed that an equivalent mutation from phenylalanine to valine at the base of one of the bacterial protein subunits caused the channel to open just enough for ions to pass through. There are also people in whom these channels are jammed in the closed position, and they feel no pain, even if they walk on hot coals. It may one day be possible for drugs based on our knowledge of these structures to be designed to ease both these conditions.