Thursday, 12 April 2018

A Visit to Diamond Light Source

Last February, a group of Birkbeck students were given the opportunity to visit the UK's synchrotron light source, Diamond, located near Didcot in Oxfordshire. Most of the students on the trip were taking the Techniques in Structural Biology (TSMB) course as part of a Master's degree. The majority were studying TSMB as a module of the face-to-face M.Sc. in Analytical Bioscience; one was on the distance-learning Structural Molecular Biology M.Sc and a few were studying Bio-Business (which does not include TSMB). This photo shows most of the group with Professor Nick Keep, director of the Structural Molecular Biology course and Dr Katherine Thompson, director of Analytical Bioscience, with part of Diamond's main building in the background.

Birkbeck staff and students in front of the 'doughnut' that houses the Diamond ring system

Dr David Price from Diamond gave the students a short lecture to introduce Diamond and the types of experiment that can be carried out there.

He explained that Diamond is a synchrotron radiation source, which means that it accelerates beams of electrons around the rim of a large doughnut-like structure so they reach speeds approaching that of light and give out extremely intense electromagnetic radiation in the form of X-rays 100 billion times more intense than the sun's rays. X-rays have wavelengths that range from 10-8 to 10-11 m, and those in the middle of this range, with wavelengths close to the length of inter-atomic bonds. These are ideally suited to probe the structure of matter on the molecular level, and they can do so in a lot of different ways.

Price explained briefly how a synchrotron radiation source works. Electrons are fired from an 'electron gun' into a linear accelerator and then into a small ring known as the booster synchrotron for further acceleration. They then enter the large storage ring, which has a circumference of about half a kilometre; it is this that gives the overall structure its doughnut-like shape. Strictly speaking, however, it is not a smooth ring but a polygon with 48 straight sections. The electron beam is bent by magnetism at each of its vertices, and it is this process that emits the intense X-rays. The X-rays produced at each vertex are emitted through a 'hole in the wall' at that vertex. They are then filtered and focused using complex optical equipment to give them the exact properties that are needed for a particular experiment. Each of these 'beamlines' is essentially a small laboratory with its own optics and analysis equipment and its own staff team. As the synchrotron structure has 48 vertices, there are 48 possible beamline stations, although they are not all operational at the same time. The Diamond website currently lists 31 operational beamlines, of which seven have been optimised for macromolecular X-ray crystallography. Some of the other techniques available are absorption and fluorescence spectroscopy and small-angle X-ray scattering.

There are more than 50 synchrotron radiation sources ('lightsources') in the world; Diamond is one of the more powerful and versatile of these, but it is the only one in the UK. Most - about 90% - of its users are academics who are required to publish their work in open access journals; over 1000 publications each year cite Diamond Academics apply for time on a specific beamline, and these applications are peer reviewed. About 10% of users come from industry and pay for access.

Price also highlighted briefly a few recent examples of research carried out at Diamond, focusing mainly but not exclusively on structural biology. These included the structure of a peptide hormone, GLP-1 (PDB 5NX2), that stimulates the secretion of insulin by beta cells in the pancreas and that could is an important target for drugs to treat type 2 diabetes. The structure of the EV71 virus (PDB 4CDQ), which causes the potentially fatal hand, foot and mouth disease, was solved at Diamond in 2012 and this is now being used to design potential drugs for this occasionally fatal disease. Away from structural biology - but not away from medicine - he described structural studies of metal alloys that can help understand why hip replacements can be rejected and inform the design of improved materials.

After the talk, the Birkbeck party was divided into two smaller groups to tour of the main site. Both groups visited one of the beamlines devoted to macromolecular X-ray crystallography, I24, where the structure of GLP-1 was solved. This beamline includes facilities for determining structures of viruses and membrane proteins, and for working with very small crystals (down to about 1.5 microns in diameter).

Looking down on a small part of the storage ring

Birkbeck staff (Prof. Nick Keep, far left and Dr. Clare Sansom, next left) and students in the control cabin of beamline I24, one of those used for macromolecular X-ray crystallography

The trip ended with a short visit to the UK's high-energy neutron and muon source, located on the same campus as Diamond and named ISIS after Oxford's river. These beams are also used to probe the structure of matter, using techniques such as neutron diffraction. We would like to thank all Diamond and ISIS staff involved for their contributions to a fascinating day.

PPS students will learn more about X-ray diffraction in the second-year module Techniques in Structural Molecular Biology (TSMB), and much more still if they choose to take the specialist Protein Crystallography course. And students on either course may get a chance to visit Diamond fir themselves, as we hope to run the trip again next year.

Monday, 29 January 2018

The Joy and Pain of Structural Biology

The British Crystallographic Association is, as its name implies, the main organisation supporting crystallograpy and crystallographers in the UK. Theirs is a multi-disciplinary science, and the different needs of the eclectic group of people who call themselves crystallographers are met by the Association's special interest groups: among them, the Biological Structures Group for structural biology. The annual meeting of this group, known as the Winter Meeting, takes place in December, often just a few working days before Christmas. The BSG Winter Meeting has featured on this blog on several occasions: in 2016, it was held at Birkbeck and celebrated the work of one of our distinguished emeritus professors, Steve Wood.

The 2017 meeting, held in the University of Cambridge's famous Cavendish Laboratory, had a rather unusual theme. The organisers asked each of the invited speakers to talk about the ups and downs of their scientific career - the 'joy and pain' of the meeting title - by focusing on one challenging or important piece of work, perhaps described in a single published paper. Not every speaker managed to keep to just one paper, but all the talks gave useful and at times inspiring insights into how structural biology is done.

First of all, however, Malcolm Longair, head of the Cavendish Laboratory from 1997 to 2005 and perhaps the only astrophysicist to address the Biological Structures Group, gave a short history of the university's Physics department that was based there and its links to structural and molecular biology. That early history was quite extraordinary; many of the most important advances in atomic and nuclear physics, including the discoveries of the electron and the neutron and the first controlled nuclear disintegrations, were made there. A lab photo taken in 1932 includes no fewer than nine Nobel laureates.

Crystallography, in those early years, was thought of as part of physics; J.D. Bernal and his group joined the lab in 1931, and the younger Bragg became head of the department in 1938. The rest, as Longair said, was history: seeds of the discipline we now know as structural molecular biology were sown in Bragg's time with Perutz and Kendrew's work on globin structure as well as Watson and Crick's on that of DNA. By the time those studies reached their triumphant conclusion, however, the crystallographers were no longer strictly part of the Cavendish. The 'Unit for Research on the Molecular Structure of Biological Systems’, set up by the Medical Research Council, moved out of the main lab in 1957 into a building known as the 'MRC Hut'. This was the first home of the MRC Laboratory for Molecular Biology (MRC-LMB) at Cambridge with its enduring reputation for excellent structural biology research.

The next speaker, Cambridge University's Tom Blundell, began by describing his early career in 'the Other Place': Dorothy Hodgkin's lab at Oxford, where he had shared some of the glory of the insulin structure. He had considered talking about insulin at this meeting, but, he explained, "Dorothy had had the pain of trying to solve the structure for 30+ years... I had the joy of a paper in Nature!" The story he told instead was his group's own: solving the structures of proteins involved in DNA repair. This was a long story, taking in 15 years' worth of papers in Nature (2002, 2010) and Science (2017) and culminating in the 'great joy' of discovering inhibitors validated against an important protein target for oncology.

DNA damage taking the form of simultaneous breaks in both DNA strands (double-strand breaks) are common but can lead to cell death or cancer. Fortunately, they are easily repaired in healthy cells, mainly through the mechanism of non-homologous end joining (NHEJ). Blundell's group have studied the proteins involved in this complex mechanism for many years. It is a three-stage process, in which the component proteins assemble on the DNA molecule either side of the break; the ends are 'pruned' by adding or removing nucleotides to restore the original sequence and finally, the ends are joined through DNA ligation. One of the proteins involved is a kinase, DNA-PKcs, that exists as a single polypeptide chain of 4128 amino acids. Blundell's group published the structure of this huge molecule in 2010 (PDB 3KGV) and it is still the longest single-chain protein to have been solved by X-ray crystallography. Blundell explained that the chain folds into a flexible, circular 'cradle' like structure that can support the DNA double helix, with the ligation taking place inside. The mechanism requires proteins to work as 'stages, scaffolds and steps' to hold the complex together for repair, and his group has solved structures of many other components including the Ku70-Ku80 heterodimer that recognises and binds to the break, initiating the repair, and a nuclease named Artemis with 'a nice pocket for drug discovery'.

Two talks on structural biology as applied to drug discovery followed. The first was by Pamela Williams from Astex Pharmaceuticals, a company founded by Blundell with Harren Jhoti in 1999 that has just registered its first drug - a kinase inhibitor, Kisqali® (ribociclib) - for clinical use in breast cancer. Williams' talk highlighted another protein family that is just as important in pharmacology as the kinases: cytochromes P450. We have about 50 different P450 subtypes in our livers, and they catalyse reactions that modify drug molecules so they can be more easily removed from our bodies. A handful of these - the subtypes known as 1A9, 2C9, 2C19 and 3D6 - metabolise most prescription drugs. Human (and all eukaryotic) P450s are monotopic membrane proteins with flexible active sites, which allow them to bind a wide variety of substrates but which make the structures hard to solve. Williams' involvement with P450 structural biology began with the first mammalian structure, rabbit cytochrome 2C5, and she joined Astex from California to work on the first human structure, the subtype 2C9. This was published in 2003 (PDB 1OG2); a large number of other human structures have followed, yielding useful insights into drug metabolism.

Ben Bax, who studied for his PhD under Tom Blundell at Birkbeck, has just moved to the University of York after eighteen years at the pharma company GlaxoSmithKline (GSK). His talk described work at GSK to determine the structures of bacterial DNA gyrases. These are members of a large class of enzymes called topoisomerases that catalyse topological transitions in DNA; the gyrase, which catalyses DNA supercoiling, is the target of the widely used quinolone family of antibiotics (e.g. ciprofloxacin). However, quinolone resistance is increasing, mainly through mutations at specific amino acid positions of the target gyrase. GlaxoSmithKline is investing heavily in the development of novel gyrase inhibitors based on oligonucleotides, and Bax' structural biology group has contributed a large number of still unpublished structures of the enzyme with and without inhibitors or DNA bound to this work.

Janet Thornton, emeritus director of the European Bioinformatics Institute, is one of the best known figures in British bioinformatics. Her talk, on what she termed an 'accidental' paper, took the audience back to the basic principles of protein structure. In the late 70s, when she started her career, there were only about fifteen protein structures known but scientists were already examining those structures to determine characteristic patterns. Many of these first structures determined had major inaccuracies, and discovering and correcting these was a major task for early structural biologists. The Ramachandran Plot, now half a century old, was one of the first tools to be developed to gauge the quality of a protein structure, and it is still widely used. Thornton's 'accidental' (and very highly cited) paper described the program PROCHECK, which runs this and other checks on a structure to give a comprehensive assessment of its quality. A PROCHECK record for each structure in the PDB is linked from the database PDBsum.

The final talk provided delegates with a rare opportunity to hear a new Nobel Laureate - in this case, the Laboratory of Molecular Biology's own Richard Henderson - tell the story behind some of his ground-breaking research. Henderson shared the 2017 Chemistry Nobel, for "developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution", with Joachim Frank and Jacques Dubochet. He chose to talk about one structure that he had in some senses made his own: that of bacteriorhodopsin, a proton pump found in Archaea that captures light energy as photons and that has many structural and mechanistic similarities with the G-protein coupled receptors, although the exact evolutionary relationship is unclear. Henderson's studies of this important molecule started in the 1970s with structures that were just about detailed enough to show the cylindrical helices. It took him over 15 years'effort with collaborators in Berlin, Berkeley and elsewhere to improve the technology enough to solve the so-called 'phase problem' and obtain an atomic-resolution structure by electron diffraction. The rest, again, is history.

Friday, 27 October 2017

Welcome to new PPS students - and blogging crystallography

This post is very like those I have written at the beginning of the academic year for the past few years, if posted rather later than usual. This is because what I have to say now is also very similar...

I would like to offer a warm welcome to the Principles of Protein Structure blog to all students who have recently 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 2017 focuses on the molecular biology of cancer; there is some material on this topic in section 5 of this course, 'Towards Tertiary Structure', where we look briefly at the structure and function of kinases. Many of the newer anti-cancer drugs, including Glivec, which has transformed the prospects for patients with chronic myeloid leukaemia, target this class of protein. Other posts may be reports from conferences or summaries of recently published papers in protein structure and allied areas; watch out for one at the end of this year featuring a lecture by the UK's newest Nobel laureate, Richard Henderson.

Some earlier posts 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!

And I hope that you will also be interested in some more blogging of mine, in which I explore crystallography - the most popular experimental technique for determining protein structures - more widely. In August I was lucky enough to attend the congress of the International Union of Crystallography in Hyderabad, India, and to write the conference blog. The topics I covered over the 8 days of the meeting ranged from crystallography in history to crystallography in space, but did also include some structural biology. The first post in this series covers the opening ceremony, including the award of one of crystallography's highest honours, the Ewald Prize, to Sir Tom Blundell, a former head of the Department of Crystallography at Birkbeck (now part of the Department of Biological Sciences). Sir Tom is perhaps best known for his part in solving the structure of HIV protease, target of some of the most successful drugs for AIDS, and he went on to found a drug discovery company, Astex. A later post describes a plenary lecture by John Spence of Arizona State University on imaging proteins in motion.

Finally, the best of luck for the 2017-18 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

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