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.

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.