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

New protein structures presented at the 2013 BCA Winter Meeting

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

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

Maurice Wilkins' original DNA model

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

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

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

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

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

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

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

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

From Genome to Proteome: BCA Winter Meeting 2012

The British Crystallographic Association is the main UK organisation supporting the science of crystallography in all its forms. Every year, its Biological Structures Group holds a meeting in the run-up to Christmas to discuss and celebrate recent developments in structural biology research. In 2012, this Winter Meeting was held at the MRC Laboratory of Molecular Biology at the University of Cambridge.

The LMB, as it is usually known, is one of the birthplaces of modern structural and molecular biology. It moved into its current building in 1962, the year when four of its most famous scientists were awarded two Nobel Prizes for some of the most important discoveries in twentieth-century biology: James Watson and Francis Crick or the structure of DNA, and Max Perutz and John Kendrew for the very first three-dimensional structures of proteins (myoglobin and haemoglobin, respectively).

It was appropriate, therefore, that the theme of this year's Winter Meeting was "From Genome to Proteome". The basic molecular processes that underlie all of life - DNA replication, transcription of DNA into RNA and translation of RNA into protein - are all, now, quite well understood. These processes are all very complicated and require numerous proteins, many of which interact together to form complexes and "molecular machines" that are quite large, at least in molecular terms. Scientists presenting at the meeting discussed recent, innovative studies of the structures of many of these proteins and the nucleic acids that they interact with. Many of these processes will be discussed in some detail in section 8 of the PPS course, "The Protein Lifecycle".

The meeting programme was divided into three sections, corresponding respectively to DNA synthesis and repair, RNA transcription and protein translation.

DNA Replication and Repair

DNA synthesis and repair are not even mentioned in the famous Central Dogma of Molecular Biology (put very simplistically, DNA makes RNA makes protein) but they are, of course, essential for it. The first speaker in this session, and therefore in the meeting as a whole, was Luca Pellegrini from the University of Cambridge. He described structural studies of the first part of this process: the initiation of DNA synthesis. In all organisms, this process involves an enzyme called primase, which is found at the DNA replication fork - the point at which the strands of the original DNA helix divide so that a new strand can be synthesised on each of the template strands. Pellegrini and his group have solved the structure of several of the subunits of yeast primase, alone and bound to part of the DNA polymerase Pol alpha, and are using these structures to deduce the precise mechanism of this vitally important process.

Then Neil Kad of the University of Essex described the techniques he has developed for visualising individual molecules, and how he is applying them to the study of DNA repair by nucleotide excision. Briefly, this technique involves stretching a single molecule of DNA between two positively charged silica beads, and tagging individual molecules of DNA-binding proteins using fluorescent quantum dots so that their binding to and progress along this DNA "tightrope" can be monitored. He has discovered that although single subunits of the Uvr DNA repair protein complex may bind DNA and search it for errors, a complex between the subunits UvrA and UvrB is required for quick and efficient searching.

Schematic diagram of a "DNA tightrope" with labelled proteins bound. (c) Neil Kad, from the Kad Lab homepage

Transcription

The spliceosome is a "molecular machine" comprised of protein and small nuclear RNA (snRNA) subunits that found only in eukaryotes and that catalyses the removal of introns from the messenger RNA precursor molecules that are initially transcribed from DNA. Chris Oubridge, a member of Kiyoshi Nagai's group at the MRC Laboratory of Molecular Biology in Cambridge (and therefore one of the "home team") described an atomic resolution structure of a complex known as U1 that forms a major part of the soliceosome. This "small nuclear ribonucleoprotein" (snRNP) comprises the snRNA molecule U1 bound to ten proteins. This technically challenging exercise in X-ray crystallography is yielding important insights into the function and mechanism of this important part of the spliceosome.

Structure of the U1 ribonucleoprotein, from Kiyoshi Nagai's web pages at the MRC-LMB.

Another interesting presentation in the Translation section was given by David Lilley from the University of Dundee, who described the structures of kink turns in RNA molecules, and how these structural motifs interact with proteins.

Translation

Since the modern Laboratory o Molecular Biology was constituted as the "Unit for Research on the Molecular Structure of Biological Systems'" in 1947, nine Nobel prizes have been awarded to scientists working there. Its most recent laureate, Venki Ramakrishnan, shared the 2009 chemistry prize with Tom Steitz from the US and Ada Yonath from Israel for determining the first atomic resolution structure of the ribosome. Israel Sanchez from Ramakrishnan's lab at the LMB gave a presentation on the mechanism by which stop codons, which give the signal to terminate protein synthesis, are decoded on the ribosome. This process, which occurs when one of the stop codons (UAA, UAG and UGA in the standard genetic code) binds to the ribosomal A site, is still less well understood than the process through which "sense" codons are decoded into amino acids. Sanchez and his colleagues are studying the structure and function of ribosomes bound to modified RNA in which the uridine in the first position of a stop codon has been substituted by pseudo-uridine. They have discovered that the decoding centre of the ribosome is more flexible than they had originally thought, an insight that may help the understanding of the termination of protein synthesis further.

The final speaker was Birkbeck's own Cara Vaughan. She discussed some of her recent research using a combination of X-ray crystallography and electron microscopy to decipher the assembly of the kinetochore. This is a structure that forms in eukaryotic cells during cell division and that links the dividing chromosome to the mitotic spindle. Vaughan's research concerns a protein called Hsp90 that activates many signalling proteins. This protein is a member of a class of proteins termed the chaperones, which are generically involved in the folding, unfolding and activation of other proteins. Vaughan and her co-workers have solved the structure of two interacting proteins found in yeast, Sgt1 and Skp1, which togethe3r seem to hold Hsp90 in an open conformation that enables other kinetochore proteins to bind.

Image of a dividing eukaryotic cell. The chromosomes are shown in blue, the microtubules of the mitotic spindle in green, and the kinetochores in pink. Image from Wikimedia Commons.

The annual Winter Meeting is the most high profile event organised by the Biological Structures Group of BCA. The association as a whole organises many other events, including, this year, the annual European Crystallographic Meeting. ECM 28 will be held at the University of Warwick from 25-29 August 2013; it will provide an opportunity for British and European crystallographers to celebrate the origin of their science with the discovery of X-ray diffraction by father and son William Henry and William Lawrence Bragg, almost exactly a hundred years ago.

BCA Winter Meeting: Structures of Supramolecular Assemblies

The British Crystallographic Association (BCA) is a national organisation set up to support all types of crystallography in the UK. It is affiliated to the International Union of Crystallography and has five special interest groups, one of which, the Biological Structures Group, is devoted to the study, by crystallography, of proteins and other biological macromolecules. One of the Biological Structure Group's main activities is its annual Winter Meeting, which conventionally takes place just before the Christmas break. This is always well attended by students and post-docs; the speakers, however, are generally senior scientists presenting some of their most exciting recent research in protein structure.

The 2011 BCA Winter Meeting was held at the Diamond Light Source, the country's national synchroton facility (which is located at Harwell, near Didcot in Oxfordshire. When it opened in 1997 it was the largest scientific facility to be built in the UK for over thirty years. Synchrotons like Diamond generate highly intense, monochromatic (single-wavelength) beams of electromagnetic radiation that are used for many types of scientific experiment, including X-rays for crystallography. The theory behind how X-ray diffraction is used to solve the structuresof proteins is not covered in PPS, but it is in both the courses that can be taken for the second year of the MSc: fairly briefly in the general Techniques for Structural Molecular Biology course and very extensively in a specialist course.

This meeting took "Structures of Supramolecular Assemblies" as its theme and featured nine distinguished speakers, discussing complex structures, biological mechanisms and protein-protein interactions. And the first speaker was none other than the head of the Department of Biological Sciences at Birkbeck, Professor Gabriel Waksman. His ground-breaking work on the structure and mechanism of fibres that form at the outer membrane of bacteria such as E. coli to attach to the host cells has been discussed previously on this blog (e.g. in June 2011) and so will not be described in more detail here.

Waksman's talk was followed by two more describing proteins and protein complexes that are embedded in the membranes of bacterial cells. The first of these was given by Colin Kleanthous from the University of York, who described proteins involved in signalling through porins, proteins that form pores in the outer membranes of Gram negative bacteria. Porins were the first transmembrane proteins to be discovered where the membrane-spanning region forms a beta barrel rather than a bundle of alpha helices. Very many alpha-helical membrane proteins pass signals into cells from their environment, with the signal arising when ligand binding triggers the receptor to either dimerise or make a subtle change in its structure (conformation). Colicins are protein antibiotics that are synthesised by bacteria, often to kill very similar bacteria that occupy the same ecological niche. They do this by "parasitizing" the porins through which nutrients enter the target bacteria, a particularly difficult task as porins act as filters that generally allow only "nutrient-sized" molecules (less than about 600Da) to enter cells. Kleanthous described the structures and mechanisms of the "outer membrane translocation" domains of several colicins bound to porins; his group is now trying to solve the structures of intact colicin-porin complexes using both crystallography and electron microscopy.

Leo Sazanov from the MRC Laboratory of Molecular Biology in Cambridge then described the structure of respiratory complex I, which is embedded in the bacterial inner membrane.. This is the first enzyme in the respiratory chain, and it is found in mitochondria as well as bacteria: the respiratory complex I in human mitochondria has been implicated in the pathology of Parkinson's disease.  The bacterial enzyme complex is smaller and simpler than the human one and is often used as a model system. Sazanov and his group crystallised the complete complex from Thermus thermophilus, a "thermophilic" bacterium that can live in extremely hot conditions. Proteins from thermophilic bacteria are often more stable and easier to crystallise than their equivalents in other species. The complete structure was found to contain no fewer than 63 transmembrane helices, 14 in each of three similar subunits. Sazanov discovered that this protein's mechanism involves a quite substantial conformational change between its oxidised and reduced form; an analogy with coupling rods has led the protein to be described as the "steam engine of the cell". It featured, in similar terms on the front cover of the issue of Nature in which the structure was described (Efremov et al. (2010), Nature 465, 441-5).

Nature cover illustrating the structure of respiratory complex I

About 5% of all proteins in an "average" bacterium are synthesised in response to heat or other stress signals. Stresssosomes are large multi-protein complexes found in bacteria and that control this stress response through the release of another protein, known as the sigma factor. Rick Lewis from the University of Newcastle described how the overall structure of a stressosome from Bacillus subtilis has been solved by electron microscopy and structures of some of the individual components by crystallography.  He is using these structures to explore the mechanism through which the stressosome senses the presence of stress conditions. Although several pieces of the stress response pathway still remain to be discovered, his group has shown how the system could respond to differences in levels of light and oxygen (the latter through the presence of a globin domain in one of the stressome proteins), how it could regulate the production of diguanylate cyclase, and how the system is re-set through the action of a serine/threonine phosphatase (the structure of which was solved at Diamond).

Helen Walden of Cancer Research UK described how her group's structural studies are shedding light on the mechanism of a DNA repair pathway that is damaged in Fanconi anaemia, a rare genetic disorder that causes, among other things, a greatly increased susceptibility to one form of leukaemia. This repair pathway, which fixes cross-links in DNA, is triggered by the single ubiquitinylation of a DNA repair protein. (This is the fusion of a small protein known as ubiquitin with a target protein.) The process is triggered when cross-links cause DNA to stop replicating; the first step is the assembly, in the nucleus, of eight proteins into a "core complex" in the nucleus. The core complex then activates another protein, known as FANCL, and this catalyses the fusion of ubiquitin with the DNA repair protein, activating it. The structure of FANCL was recently solved in Walden's group by Ambrose Cole, who is now a post-doc at Birkbeck (PDB 3ZQS). Interestingly, the structure of this protein is not the beta-propellor that was predicted by sequence analysis; instead, it contains two domains similar to the ubiquitin conjugating enzyme UBC. Mutations that abolish ubiquitin binding are known to cause disease.

These are only a few highlights of  a fascinating day's science. Other, no less interesting, structures presented there included several viral proteins: the HIV integrase bound to some of its inhibitors, presented by Peter Cherepanov (Imperial College London); the nucleoprotein from the virus that causes Lassa fever, described by Chang-jing Dong (University of St. Andrew's); and the NS1 protein from the influenza virus, described by Phil Kerry, also from St. Andrew's