Structural Secrets of an Ancient Viral Plague

Research in Biological Sciences at Birkbeck, and several related departments at neighbouring University College London, is combined into the Institute of Structural Molecular Biology. The Institute holds a regular seminar programme - every Wednesday lunchtime during termtime - in which it invites excellent scientists, many with links to the colleges, to present their research. A few weeks ago, the seminar speaker was an electron microscopist, Sarah Butcher, who is based at the University of Helsinki in Finland. Her group has been investigating the structure of a virus that causes a very well-known disease: measles.

Measles has been known of for millennia. The disease (although of course not its cause) was first described in ancient Egypt. It is one of the most infectious viruses known, but people who encounter measles (if at all) as an unpleasant childhood affliction are often surprised to learn that it is a killer. About 164,000 people lost their lives as a result of measles infection in 2008, most from lingering immunosuppression rather than the acute infection. Most deaths occur in Africa and south Asia; a smaller epidemics have recently arisen in the UK when the MMR vaccination lost popularity over the MMR autism scare.

The measles virus is a paramyxovirus; an enveloped virus with a single strand of RNA as its genome, and closely related to the viruses that cause mumps, respiratory syncytial virus (RSV) infection and para-influenza in infants and children. It has two surface proteins and iis thought to attach directly to the membranes of the cells it infects via one of these.

Until recently, structural studies of the measles virus have been fairly limited. Many groups have studied it using an electron microscopy technique called negative staining, but that can only see the virus' surface. Structures of one intact measles virus protein and domains of three others have been deposited in the Protein Data Bank; the haemagglutinin (e.g. PDB code 2RKC); two separate domains of the phosphoprotein (1OKS and 2K9D) and a structure of fragments of two proteins simply called P and N bound together (1T60).

Sarah Butcher and her group used a technique called cryo-electron microscopy, which allows the interior of viruses to be visualised, to study the measles virus. Their results led them to focus on the matrix protein, which is thought to be important for the assemby of the virus (the protein coloured cyan in the images below). All previous models had placed the matrix protein covering the inner part of the viral membrane. What the Butcher group saw, however, was completely different. They could see a protein surrounding parts of nucleocapsid - the viral RNA and its associated, protective protein - and further analysis identified this as the matrix protein. The matrix binds tightly to parts of the nucleocapsid to make rod-like structures, and these fold into anti-parallel units that are somewhat remniscent of antiparallel beta sheets in proteins. This model suggests that the process of virus replication will be more complex and yield more potential drug targets than has previously been thought.

Two models for the organisation of proteins and RNA in the measles virus. Top: the old model, with the matrix protein (cyan) surrounding the virus coat. Bottom: the Butcher group model, with the matrix protein surrounding parts of the nucleocapsid. Figure credit: Proc. Nat. Acad. Sci. USA (2011)

Structures of proteins from other viruses, particularly HIV and influenza, will be covered quite extensively later in the PPS course. We don't study the technique used in this study, cryo-electron microscopy, in PPS but it is covered in one of the options for the second year of the PPS course, Techniques in Structural Molecular Biolog

Welcome to new students!

A warm welcome to the Principles of Protein Structure blog to all students who have just started studying Birkbeck's Principles of Protein Structure course!

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. Throughout the taught course (but more often in the later part of the course) I will post reports of recent developments. 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 overall title of the programme for Autumn 2011 is "“Proteins of the Future: Mechanism, Evolution and Design” which is closely connected to the content of the PPS course. Other posts may be reports from conferences or summaries of recently published papers in protein structure, protein bioinformatics and allied areas.

Do, if you get a chance, look through some of the earlier posts on the blog 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!

And the best of luck for the 2011-12 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

Waking Up to Structural Biology

Professor Nicholas Keep (known to generations of Birkbeck students as Nick) was appointed to a chair in biomolecular structure in 2009. It was June 2011, however, before he gave his inaugural lecture at the college. In this lecture he gave an overview of the techniques he uses as an experimental structural biologist and some of the discoveries he has made through them.

Nick’s career so far has been a glittering one. He took both his degrees at Cambridge and did postdoctoral work at UCL and back in Cambridge at the prestigious Laboratory of Molecular Biology (LMB) before being appointed as a lecturer at Birkbeck in the mid-90s. Since then he has risen steadily through the ranks and is now not only Professor but Executive Dean, with academic and financial oversight of the whole Faculty of Science. It is perhaps not surprising that it took him two years to fit in his inaugural lecture.

He began his lecture with a whistle-stop tour of the history of structural biology, beginning at Birkbeck and with the first Professor of Crystallography here, J.D. Bernal (see June 2008 post). Before he even arrived in London, however, Bernal had published (with Dorothy Hodgkin) the first ever diffraction pattern to be obtained from a protein crystal. Very much later, Nick as an undergraduate Cambridge student was inspired by two Nobel laureates, Max Perutz (who published the first-ever three-dimensional protein structure, that of myoglobin, in 1958) and Tim Hunt, to specialise in this aspect of molecular science. His PhD research was on the structure of methylmalonyl-coA mutase , an enzyme that binds to vitamin B12. This provides another link back to Dorothy Hodgkin, whose Nobel Prize in 1964 was awarded partly for determining the structure of this important vitamin.

He then spent a few minutes going through the essential principles of protein crystallography. This involves purifying and crystallising a protein and then exposing the crystals to a parallel beam of X-rays. These X-rays are deflected from the atoms in the crystal in a regular way to produce a diffraction pattern, and this can be interpreted to give first a map showing the density of electrons in the molecule, and then a model of the positions in space of all atoms in the molecule (missing out, in most cases, the lightest atom, hydrogen). In an ideal case, this whole process can now take a month or so, but this is rare: many structures still take years to solve. Birkbeck now has excellent facilities for purifying proteins and growing crystals. We can collect the X-ray data on site but often go to more powerful machines – synchrotrons – located in the UK and beyond. Britain’s first synchrotron is still in use, but in Jordan; it was replaced about five years ago by a state-of-the-art facility, Diamond , near Harwell in Oxfordshire. Birkbeck’s scientists also use synchrotrons elsewhere in Europe; Nick’s favourite is in Grenoble.

Nick then went on to describe some of the research projects that he has led or contributed to at Birkbeck. Working with Lin Field and Jing-Jiang Zhou at Rothamsted Research in Hertfordshire, he solved the structure of an insect protein that binds odorant molecules, which has led to some useful insights into the mechanism behind insects’ extremely sensitive sense of smell. Much of his work, however, is and has been in proteins that are involved in one way or another with human disease. Duchenne Muscular Dystrophy is a progressive, muscle-wasting genetic disorder that almost always affects boys and that is caused by mutations in a large muscle protein called dystrophin. Nick and his colleagues have solved the structures of a part of this protein that binds to another muscle protein, actin, and of related proteins.

It was only when he reached some of his final examples that the pun in his title, “Waking up to Structural Biology”, became clear. The bacterium Mycobacterium tuberculosis (see also April 2011 post ) infects the lungs of about a third of the world’s population. In most people, however, it remains in a wholly benign, dormant condition. In about 5-10% of cases, however, the dormant bacteria will “wake up” when an infected individual is under stress (for example, by exposure to another infection) and overt tuberculosis (TB) develops. Bacteria in the dormant stage are untreatable by any current TB drugs. Nick and his group first studied the structure and function of a protein known as resuscitation promoting factor that is involved in this “waking up” process,. He observed similarities between its sequence and that of a very well-known protein, lysozyme , which breaks down bacterial cell walls, and later, when the structure was solved, it was seen to have a lysozyme-like fold. This led to his identification of a glutamate residue as key to this enzyme’s activity. He is now looking at other proteins involved in M. tuberculosis resuscitation including a small heat shock protein (a protein that helps keep other protein structures stable under stresses such as raised temperatures), Acr1. This is the most abundant protein in dormant TB. He ended with a glimpse of a new unpublished TB protein structure.

Nick concluded a fascinating lecture by thanking his lengthy list of co-authors and particularly his research group, stressing the collaborative nature of science, and Gabriel Waksman, head of the Department of Biological Sciences, closed the proceedings by praising his achievements in teaching and administration as well as research.

Both second-year options in the MSc Structural Molecular Biology programme are concerned with the techniques used to study the structures of biomolecules. Techniques in Structural Molecular Biology (TSMB) is a general course covering crystallography, NMR, electron microscopy and some of the molecular biology and bioinformatics techniques associated with them, whereas Protein Crystallography (PX) is, as its name implies, a more specialist course.

The Structural Biology of Pilus Biosynthesis: Or, How Bacteria Man the Pumps

Science Week at Birkbeck College was celebrated for the second time in early May 2011 with presentations from each of the college’s three science departments. The two lectures from researchers in Biological Sciences were linked by the common theme of nano-machines in biology. Just as a car engine, for example, is built up from many interacting parts, so some proteins work together in large complexes to do particular jobs within cells. Professor Helen Saibil, whose ground-breaking work in electron microscopy has featured earlier in this blog (see November 2010 post) presented some of her research into chaperones, protein machines that carry out “quality control” work enabling other proteins to form into and stay in the precise three-dimensional shapes they need to function.

The other speaker from Biological Sciences was Professor Gabriel Waksman, a distinguished structural biologist who combines a very successful research career with running both Birkbeck’s Biological Sciences department and the Research Department of Structural and Molecular Biology at UCL. Professor Waksman’s work for many years has focused on the complex structures through which bacteria interact with the outside world. Pathogenic bacteria cause problems for their hosts only when they interact with them, by secreting toxic substances into their environment or attaching to host cells. Now, when bacteria are rapidly developing resistance to many traditional antibiotics and more antibiotics, particularly with novel mechanisms of action, are desperately needed, some of these mechanisms are at last becoming understood.

Gram negative bacteria, which have double cell walls, often carry hair-like fibres or filaments known as pili on their surfaces. Some bacteria use these to bind to receptors on the surfaces of host cells, a process that can trigger the host cell surrounding and engulfing the bacteria in infection. Different forms of bacteria even from the same species carry different pili that bind to different cell receptors: for example, some E. coli bind to and infect bladder cells causing cystitis, while others infect kidney cells causing pyelonephritis. E. coli can also use pili to bind to each other, forming colonies around the bladder that are particularly difficult to treat.

The type of E. coli that infects the kidney carries a large number of so-called P pili on its outer membrane. These consist of a relatively thick rod near the cell wall and a thin filamentous tip. The whole pilus is made up of thousands of similar protein subunits encoded by genes within the Pap gene cluster. Almost all are identical PapA subunits, and these form the rod: the tip consists of just a few homologous PapE subunits, tipped by the sensor, PapG, which recognises and binds to kidney cells.

Pilus subunits, like all proteins, are synthesised in the cytoplasm; they need to be transported to the outer membrane and polymerise to form the pilus structure, and this complex task is achieved by other Pap proteins. When a pilus subunit is synthesised it is first translocated across the inner membrane into the periplasm, where it will be immediately degraded unless it can bind to a PapD protein. This acts as a chaperone, moving the subunit to the outer membrane where it docks with the membrane-bound PapC. This latter, or “usher” protein, is the core of the pilus biosynthesis molecular machine, and PapC and PapD together give the process its name: the chaperone-usher pathway. PapC has a large central pore through which the intact pilus is secreted.

Over many years, Gabriel Waksman and his group have solved the structure of many of these Pap proteins, and they have now built up an accurate picture at atomic resolution of how the chaperone-usher pathway works. The first structure to be solved was that of a binary complex of one pilus subunit, PapK, bound to the chaperone PapD (PDB 1PDK). PapK – and, subsequently, each of the pilus subunits – was found to have an immunoglobulin type fold, but with one beta-strand missing. This structure can only be stabilised when another protein, either the chaperone or a second pilus subunit, completes this fold with a strand of its own. One by one, starting with the tip subunit PapG, chaperone-subunit complexes migrate to the PapC usher, where the chaperone strand is replaced by a strand from another subunit in what has been termed a “donor strand exchange” model of polymerisation. The pilus fibre therefore forms from a series of “typical” immuno-globulin-like subunits in which each subunit is completed with a single strand from the next nearest subunit. The pilus biogenesis process only stops when a PapH, or “terminator” subunit is incorporated.

The usher forms a wide pore in the outer membrane (PDB 2VQI) and acts both to synthesise and to secrete the pilus polymer. It is a long, multi-domain protein. Using X-ray crystallography, Waksman’s group first determined that the pore comprises a very large beta barrel derived from the central domain of the usher, with a small sub-domain embedded within this domain forming a “plug” that blocks the pore when it is not being used. The usher also has a short N-terminal domain that dangles down into the periplasm and grabs on to chaperone-subunit complexes. The function of the C-terminal domain, however, remained unknown until the group solved the structure of an intact usher-chaperone-subunit complex.

It was only when this intact structure was solved that Waksman’s group really began to understand the mechanism of this complex “molecular machine”. For this, the group used a homologous system in which the usher is a protein known as FimD, the chaperone is FimC and the bound subunit FimH. This structure was a “first” in several ways, not least because it was the first time that an intact, folded protein was observed inside the pore of another protein structure. In this structure, the C-terminal domain of the FimD usher was seen to bind to the chaperone-subunit complex. It appears that, once the N-terminal usher domain has grabbed on to a chaperone-subunit complex and moved it into the usher, that complex will move up the usher structure to the binding site on the C-terminal domain, freeing the N-terminal domain to capture the next subunit.

This work, the culmination of fifteen years’ study of this secretion system, has just appeared in Nature (published online ahead of print 1 June 2011). More importantly, however, this elegant piece of structural biology may be exploited in the war against bacterial infection. A drug that bound to the usher and prevented pilus biosynthesis – a “pilicide” – would not kill the bacteria, but it would prevent them from binding to their target cells and also from forming the antibiotic-resistant colonies that can remain in the urinary tract for years and that lead to persistent infection.

Structural Biology in the Fight against TB

About a third of the world's population - more than two billion people - are believed to be infected with Mycobacterium tuberculosis, the bacterium that, as its name implies, causes tuberculosis (TB). In most people the infection remains latent, but about 10% of cases develop into causes almost two million deaths a year. Strains of extensively drug-resistant TB (XDR-TB), which are resistant to two of the most effective first-line drugs and to at last three 0f the second-line drugs used against TB, have been found in many countries.

The Stop TB Partnership marks March 24 each year as World TB Day. 2011 is the second year of a two-year campaign to inspire innovation into TB research and care, On the move against tuberculosis. On March 24 2011 the Department of Biological Science at Birkbeck held an afternoon symposium featuring some of the department's tuberculosis research. This was organised by Dr Sanjib Bhakta, head of the ISMB Mycobacteria Research Laboratory and a senior lecturer in the department. Dr. Bhakta's research focuses on the discovery and validation of novel drug targets within the Mycobacterium tuberculosis proteome (link is to the TB proteome page in section 5 of PPS). Structural biology forms a crucial part of this work.

Birkbeck's Stop TB Day research symposium started with a keynote lecture given jointly by Dr Bhakta and Professor Edith Sim of Kingston University and the University of Oxford. Professor Sim is a member of the core group of TBD-UK, an organisation of UK researchers involved in the discovery and development of novel drugs for tuberculosis. After an introduction by Dr Bhakta, she described research in her group into the characterisation of a group of proteins that are necessary for the survival of the M. tuberculosis bacterium within cells. The enzyme NAT metabolizes and inactivates isoniazid, which is one of the first-line drugs used against TB. Researchers in Sim's group have developed inhibitors of this enzyme, some of which have been licensed to pharma company Eli Lilly for further development.

Sim's group is now focusing on a related family of proteins encoded by the Hsa genes which are involved in the metabolism of cholesterol and are also necessary for the bacterium to survive in macrophages. They have recently solved the structure of one of these enzymes, HsaD, which catalyses the cleavage of a carbon-carbon bond in one of the breakdown products of cholesterol. Structures of a mutant form of this enzyme alone (PDB code 2WUD) and with inhibitors (e.g. PDB code 2WUE) are yielding important insights into the mechanism of action of this enzyme. Both NAT and HsaD may prove useful targets for the design of anti-TB drugs that are likely to have novel mechanisms of action and that may therefore be active against resistant strains of the bacterium.

The keynote address was followed by some short talks by members of Dr Bhakta's research group at Birkbeck. Two of these, by Dimitrios Evangelopoulos and Dr Antima Gupta, described novel methods for testing drug susceptibility and for screening potential inhibitors respectively. Two others, however, focused again on the structural biology of potential drug targets. Dr Tulika Munshi described the Mur ligases, a family of proteins that are involved in synthesising the bacterium's complex cell wall. This cell wall is extremely rich in peptidoglycan; it is essential for the growth of Mycobacterium tuberculosis and has no homolog in the human proteome, both features that are important in a good drug target. Munshi and her colleagues have solved the structure of a member of this family, the ATP-dependent ligase MurE (PDB code 2XJA), in collaboration with Birkbeck structural biologist Professor Nicholas Keep (who is also the director of the MSc in Structural Molecular Biology) and identified amino acids that are essential for its activity. Another speaker, PhD student Juan David Gusman, described screening compounds recently isolated from Columbian plants as potential inhibitors of this enzyme. This work, published last year in the Journal of Antimicrobial Chemotherapy (link to PubMed) identified 3-methoxynordomesticine hydrochloride as a potential lead compound.

The scientific presentations were followed by a poster session and by an interesting panel discussion on some of the political issues involved in tackling this important public health issue. The take-home message from the day was that important steps are being taken - particularly in the academic and not-for-profit sectors - in elucidating the metabolism of this bacterium and developing badly needed treatments for the disease it causes and that Birkbeck researchers are playing an important part. If these treatments are to make it into clinical use, particularly in the developing world, however, political will as well as research insights will be needed.

Computational Biology - and Bioinformatics - at UCL

People not uncommonly mix up the term "bioinformatics" with "computational biology", or use the two terms interchangeably. This is an easy mistake to make as both disciplines involve a computational approach to life sciences, and both terms have evolved over time. The consensus, however, is that there are significant differences: bioinformatics refers to the analysis of large quantities of (generally molecular) biological data, whereas all research fields that involve the development and use of computational approaches to them study of biological problems can be grouped into coomputational biology. Thus, although this is not precisely correct, it is more accurate to think of bioinformatics as a sub-division of computational biology than the other way round.

Birkbeck's much larger neighbour, University College London (UCL) has over twenty research groups working in areas that fall within the remit of computational biology, scattered across a number of departments and locations. Last week the college's whole computational biology community came together in a one-day symposium to make connections between these diverse research groups and to promote and celebrate the wide range of computational biology research that is carried out at UCL. This post gives a very brief overview of some of the work presented there, with links to the research groups involved.

The symposium was chaired by David Jones, director of the Bloomsbury Centre for Bioinformatics. which includes researchers from both UCL and Birkbeck. It started with a keynote lecture from Steve Oliver of the University of Cambridge, describing his group's ambitious project to understand and model completely the metabolism and behaviour of a simple, single celled organism, the yeast Saccharomyces cerevisiae.

The first UCL speaker was Christine Orengo of the Research Department of Structural and Molecular Biology. In the 1990s, she, with Janet Thornton (now director of the European Bioinformatics Institute) developed the CATH protein structure classification database which is very widely used in PPS. Since then, researchers in her group have built more databases of protein structure and function and prediction tools, and have moved on to the analysis of complete genomes and (as she presented here) functional protein networks. Domenico Cozzetto of the Department of Computer Science then described novel methods of predicting protein function from multiple data sources.

The research presented in both these talks clearly falls within the remit of "bioinformatics", in that it is concerned with the analysis of large quantities of molecular data. The next speakers, however, illustrated just how widely the term "computational biology" is being applied. Peter Hammond trained as a mathematician but is now working at UCL's Institute for Child Health, using imaging techniques and mathematical models to determine the subtle effects of genetic differences on human face shape. These models are already being used to aid early diagnosis of developmental disorders, facilitating both early intervention and genetic counselling. His presentation was followed by two more with a medical focus, by Angus Silver, a neuroscientist who develops mathematical models of neuronal signalling in the cortex, and a physician, Malcolm Finlay from the Heart Hospital, who described the computer simulations that his group has developed to predict the electrochemical responses of individual patients' heart muscles during periods of abnormal heart rhythm (arrhythmia).

A later talk by Sally Price of UCL's Department of Chemistry illustrated the value of computational biology to the pharmaceutical industry. She described the use of inter- and intra- molecular forces (to be covered in PPS section 9) to determine which crystalline structures of chemicals, including prescription drugs, would be most likely to form. For me, however, one of the highlights of the day was a talk by Mark Girolami of the Department of Statistical Science that linked computational biology, biostatistics and genetics to archaeology and anthropology. Mark described how a genetic mutation that allows some Europeans to digest milk as adults spread through the continent. His models traced the emergence of the mutation (which would not have remained in the population if it had not conferred significant evolutionary advantage) to a time and a place - about 7-8,000 years ago and in what is now central / Eastern Europe - when cattle replaced sheep and goats as the main domesticated animals.

I hope that this partial overview of a fascinating day's science will give you some idea of the breadth of computational biology research, and the depth of its coverage at UCL.

Peter Murray-Rust, PPS and a Semantic Molecular Future

Yesterday (17 January) I attended a symposium at the Department of Chemistry, University of Cambridge, that was held to celebrate the career and ideas of one of the founders of the PPS course: Peter Murray-Rust. Since 2000, Peter has been based in Cambridge, where he is a Reader in Molecular Informatics.

The symposium was opened by Professor Sir Tom Blundell, a former head of the crystallography department at Birkbeck and now emeritus professor of biochemistry at Cambridge. Tom told the audience that when Peter came to Birkbeck in the mid-90s he already had a distinguished career in molecular science behind him. He had been a PhD student at Oxford in the 1960s, working with Keith Prout on the crystal structures of inorganic molecules. Tom, who worked with Dorothy Hodgkin on some of the early crystal structures of insulin, was a fellow student. Peter then worked as a lecturer at the University of Stirling, Scotland, and at pharma giant Glaxo (now part of GSK). He joined Birkbeck at a time when the Web was just beginning to open up the world of the Internet to the wider community. He, with David Moss, then a reader in crystallography at Birkbeck, and research associate Alan Mills, saw the potential for the web to extend the department's specialist teaching beyond the reach of those who could readily commute to central London, and PPS was born. The first, experimental course was delivered, free of charge, in 1995; within six years it would be incorporated into the full online-only MSc course.

Peter has never been one to let the grass grow under his feet. While he was busy creating and launching the PPS course, he was already thinking about how the still infant Web could be harnessed to allow data and information to be manipulated and understood rather than simply displayed. With Henry Rzepa from Imperial College, London, he developed Chemical Markup Language (CML) as a version of XML ("extensible markup language") for chemists. This is now very widely used. The first scientific paper written entirely in XML was published in 2001, although the editors of the journal concerned described it as "an interesting exercise, but [not] easy to deal with by any means". It is now being used for Microsoft's new chemistry add-in for Word: Chem4Word.

CML is described as a semantic language. The term "semantic web" was coined by WWW developer Tim Berners-Lee as "a group of methods and technologies to allow machines to understand the meaning – or 'semantics' – of information". Peter's wide interests include, besides the automatic analysis of data in scientific publications - the development of virtual scientific communities, and he campaigns passionately for all scientific data to be freely available to all. He was one of a small group who drafted the Panton Principles (named after a Cambridge pub) which state that future scientific advances will depend crucially on all science data - not necessarily its interpretation - being made freely available on the Internet. Later presentations, including one by Henry Rzepa, developed these ideas in more detail. The symposium ended with a presentation of software being developed in Peter's group, including an application where a student manipulated an image of a molecule by waving his arms. This may have looked like a fun gimmick, but it must be potentially useful for disabled students who have difficulty with using a mouse.

There was one other delegate at the symposium to whom PPS students owe a debt, although they are probably unaware that they do: Roger Sayle, the developer of Rasmol. Today, we rely on molecular graphics programs that are fast, free, and easy to install and use on any desktop machine. Roger's Rasmol, developed in the mid-90s, was the first of these.

If you would like to know more about Peter and his ideas, and how some of them have been applied in Birkbeck's web courses, you can visit his blog.