Atoms and Empty Space: the Structural Biology of Specificity

The eminent structural biologist Laurence Pearl, Professor of Structural Biology in the Genome Damage and Stability Centre at the University of Sussex, has strong links with Birkbeck. He studied for his PhD under Professor (now Sir) Tom Blundell at Birkbeck in what was then the Department of Crystallography (and the only full university department so named in the UK). The latest of the many honours and awards that he has received is the Biochemical Society's prestigious Novartis Medal for 2018. This is an annual award, made "in recognition of contributions to the development of any branch of biochemistry" and the winner is invited to present a lecture either at the Society's London headquarters or at one of its many conferences. Pearl gave his Novartis Medal Lecture, entitled Atoms and Empty Space: the Structural Biology of Specificity at the London office of the Biochemical Society on 13 November 2018.

This lecture was live-streamed on Facebook. It is linked from the Biochemical Society Facebook page, but you don't need to have a Facebook account to view it; just click on the video link below. (It is about an hour long.)

Video © Biochemical Society 2018

To summarise the lecture briefly, Pearl began with a well-known but still controversial quote from the Greek philosopher Democritus: "Nothing exists except atoms and empty space. All else is opinion." He then summarised the beginning of his scientific career as a postgraduate student at Birkbeck. During his PhD he learned computer programming and wrote - or contributed to - some of the first structural bioinformatics programs, as well as studying the structure of the aspartic protease, endothiapepsin (PDB 4APE). This was in the early 1980s, when the world was becoming aware of the scourge of AIDS and the necessity of targeting its viral cause, HIV. Sequencing the tiny HIV genome revealed a protease with similar sequence motifs to aspartic proteases, but less than half as long. It was Pearl, by then a postdoc at the Institute of Cancer Research in London, and Willie Taylor from Birkbeck who predicted, years before the structure was solved, that HIV's protease would be active as a dimer. (There is more about this in section 7 of PPS, on Quaternary Structure.) The HIV protease structure - exactly as Pearl and Taylor had predicted it - became an important tool for the discovery of protease inhibitors as drugs against AIDS. The first protease inhibitor to enter the clinic was named saquinavir after the amino acid motif SQNI that led them to predict the enzyme's dimeric structure.

Not surprisingly, this discovery also proved to be a gateway to Pearl's career as an independent researcher. He set up his lab in 1989 in the biochemistry department at UCL, with research projects in three areas: DNA repair, signal transduction, and a group of unrelated proteins that help other proteins to fold and that have collectively been named chaperones. He spent the rest of his lecture describing just a few of the many proteins that his group has studied during the last 30 years: first at UCL, then back at the Institute of Cancer Research and since 2009 at Sussex. These included the enzyme uracil DNA glycosylase, which is involved in the specific repair of cytosine residues in DNA that have been deaminated to form uracil. His co-worker on this project was Renos Savva, who is now a senior lecturer at Birkbeck and director of our MSc course in Biobusiness. The structure of this enzyme (PDB 1UDI) explains the exquisite specificity of this enzyme. Another, much smaller DNA repair enzyme, mismatch uracil glycosyase, was found to have the same fold. His more recent work, which uses electron microscopy as well as X-ray crystallography, includes the structure and mechanism of glycogen synthase kinase, and the structure and dynamics of further DNA damage and repair systems. Listen to the lecture if you want to find out more!

Welcome to new PPS students - and a few more links...

This post is very like those I have written at the beginning of the academic year for the past few years; 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. Welcome too if you are thinking that you might want to study with us in the future, or if you are just interested in learning more about a fascinating and fast-moving area of research in molecular biology.

I run this occasional 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 2018 has the intriguing title of 'Mischievous Microbes'; its themes of microbiology and infectious disease biology have links to some of the later sections of the course. Other posts may be reports from conferences (such as this one at Imperial College, London in December) or summaries of recently published papers in protein structure and allied areas/

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!

I also work part-time as a freelance science writer, and sometimes I even have a chance to write about structural biology. You might like to follow a set of blog posts I wrote from the International Union of Crystallography's triennial meeting in Hyderabad, India last summer (posts from 22 August - 6 September). The first entry, featuring a talk by Sir Tom Blundell, a former head of the Department of Crystallography at Birkbeck (now part of the Department of Biological Sciences) is perhaps most relevant to PPS. Sir Tom was involved 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. This year I reported on a meeting much nearer at hand (in Liverpool) and, specifically, on one of the most exciting advances in structural biology by X-ray crystallography for some years: X-ray free electron lasers.

Finally, the best of luck to new students 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
Tutor, Principles of Protein Structure

Rosalind Franklin Lecture 2018: Eva Nogales, electron microscopist

Since 2016, Birkbeck has held an annual lecture named in honour of perhaps the most famous woman scientist ever to work there: Rosalind Franklin, whose extraordinary, meticulous experimental work was a necessary part of solving the structure of DNA. This lecture is part of Birkbeck’s commitment to the Athena SWAN equality initiative, and is it given by a woman scientist distinguished in one of the disciplines represented there.

The 2018 lecture differed from its predecessors in forming part of both Birkbeck’s annual Science Week and the eighth ISMB Symposium. The Institute of Structural Molecular Biology (ISMB) is a centre of excellence, founded in 2003 to promote and integrate multi-disciplinary research in molecular, cell and structural biology in Birkbeck and its much larger neighbour, UCL. It holds a varied programme of events for faculty members, research staff and students; symposia, held in ‘even years’, are intensive conferences, generally held over two days and featuring talks from international research leaders.

This symposium was held over two afternoons on Monday 18 and Tuesday 19 June, with the Rosalind Franklin lecture as the last one on the first day. In planning the symposium, its organisers chose to highlight one technique among all those available for researchers at the ISMB: electron microscopy, as used to study the atomic structures of large protein complexes and ‘molecular machines’. The Institute’s director, Gabriel Waksman, highlighted Birkbeck’s acquisition of a new and very powerful electron microscope – a Titan Krios – in his introduction as ‘something to celebrate’. According to the School of Science Facebook page, Birkbeck’s Department of Biological Sciences is the smallest UK university department to house such a powerful microscope, and it is only through the ISMB that it is able to punch so far above its weight. And the Rosalind Franklin lecturer, Eva Nogales from the University of California in Berkeley, was only one of several distinguished proponents of this technique to present their research during the symposium.

Eva Nogales with Prof. Nicholas Keep, Dean of the Faculty of Science at Birkbeck. Photo © Harish Patel, Birkbeck

Few women have achieved as much in electron microscopy research as Nogales. Following a short introduction by Professor Nicholas Keep, Dean of the Faculty of Science at Birkbeck, she began her Rosalind Franklin lecture with thanks. She paid tribute to two of the distinguished researchers present, Helen Saibil and Ken Holmes, describing Saibil, the Bernal Professor of Structural Biology at Birkbeck, as an ‘inspirational’ pioneering woman in electron microscopy. Holmes, who had given one of the previous talks at the symposium, worked with Rosalind Franklin as a PhD student at Birkbeck in the 1950s and went on to make ground-breaking discoveries about the structure of the muscle protein, actin.

Nogales’ main theme during her lecture was her lab’s efforts to decipher the structures of several large, multi-protein complexes that are involved in the process of gene expression. The different types of cells in our bodies – with a few odd exceptions, such as cancer cells – all contain exactly the same DNA in the chromosomes in the cell nucleus. What makes a brain cell differ from a bone cell or a heart call is how the information carried by the genes on those chromosomes is expressed in the functional molecules, mainly proteins. Only a fraction of the genes in a genome are expressed in a given cell at any particular time. Gene expression is the term given to this incredibly complex and exquisitely sensitive process, which can be divided into two stages expressed simplistically as ‘DNA to RNA’ and ‘RNA to protein’. Work in the Nogales lab focuses on two protein complexes that are involved in the first sub-process, the transcription of the DNA sequences of genes into RNA. These bear the rather cumbersome names of polycomb repressive complex 2 (PRC2) and transcription factor II D (TFIID).

If the DNA in each human chromosome could be stretched out it would measure tens of centimetres in length. It is packed and compressed to fit into the microscopic cell nucleus by winding around histone proteins to form circular units of structure called nucleosomes. Proteins in the ‘transcription machinery’ can only access the DNA to start gene expression if these are loosely packed. PRC2, as its full name implies, represses this process: it does so by adding methyl groups to the alkaline lysine residues of the histones, making the nucleosomes pack more tightly together. The protein complex therefore forms an ‘on-off switch’ for gene expression. Disrupting its function can lead to the uncontrolled cell growth and multiplication that is characteristic of cancer cells and it is therefore a useful target for the design of anti-cancer drugs.

Nogales explained that PRC2 is a very large protein complex and that determining its structure using electron microscopy presented a considerable challenge. The first structures, obtained before the ‘resolution revolution’ in this technique, could only show separate protein molecules as ‘blobs’: later, better structures that revealed the positions of individual atoms proved that these were ‘accurate but not very precise’. The complex is now known to exist in several distinct structural states and to be able to add methyl groups (‘active’) in two of them. The main difference between these is in the position of one helix, which is bent against the rest of the molecule in the ‘compact active’ conformation but straightens away from it in the ‘extended active’ one. PRC2 binds to two protein co-factors in ways that mimic the binding of the flexible ‘tails’ of the histone proteins in methylated and unmethylated forms respectively.

She then showed some even more impressive structures to explain how the complex interacts with nucleosomes. One complex binds between a pair of nucleosomes, and as long as the DNA that links the two is the right length, binding the first nucleosome positions the second so that the right amino acids are brought into the right position in the PCR2 active site for methylation to occur efficiently.

The second complex discussed, TFIID, is active exactly when PRC2 is not, as its presence is necessary to begin the process through which DNA is transcribed into RNA. This begins with the step-by-step assembly of proteins close to the position on the DNA where transcription is due to start, forming a ‘preinitiation complex’. TFIID is the first component of this complex to assemble, and this ‘nucleates’ the complex by recruiting other transcription factors so RNA synthesis can begin. Nogales described distinctive structures of parts of the preinitiation complex obtained by members of her group, finishing by showing some unpublished work on its structure and dynamics that included this vital component. If this fascinating lecture has inspired the many young electron microscopists in the audience as much as Helen Saibil’s work inspired Nogales, then the future of the discipline will be in good – and often female – hands.

This has been cross-posted with minimal alterations from Birkbeck's Events Blog

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.

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.