4th ISMB Symposium 17-18 June 2010

Write-up of the event

This article was written by Dr. Clare Sansom (Department of Biological Sciences, Birkbeck, University of London).

The Institute of Structural and Molecular In introducing the symposium, Mary Collins, Biology holds a research symposium every two the Executive Dean of the Faculty of Life years. Its fourth such symposium was held at Sciences at University College London, one of its constituent institutions, University congratulated ISMB’s director, Gabriel College London, on June 17 and 18, 2010. As Waksman, on putting together “one of the before, the symposium brought together all highest quality meetings” researchers and postgraduate students of the that she had been to in a long time. five departments from UCL and Birkbeck Waksman, whose many honours include College making up the ISMB together for a Fellowship of the Academy of Medical two-day lecture programme. The 2010 Sciences and membership of the European symposium, attended by all researchers and Molecular Biology Organisation, is the head of postgraduate students of the five UCL and both the Department of Biological Sciences at Birkbeck departments making up the Birkbeck and the Research Department of Institute, as well as many external scientists, Structural and Molecular Biology at UCL. featured twelve excellent talks. Three each were taken from four of the Institute’s six The first programme featured structural disciplinary programmes: structural biology, biology. Its speakers may have been chosen to biophysics, chemical biology and complement each other, with one . Each three-lecture slot representing each of the three main structural featured one speaker from the ISMB, one from biology techniques: X-ray , the UK and another from overseas: the nuclear magnetic resonance, and electron external speakers were invariably established microscopy. The first to speak was X-ray research leaders, whereas most ISMB speakers crystallographer Laurence were promising principal investigators nearer Pearl, who moved very the beginning of their research careers. recently from London’s Institute of Research to head the School of Life Sciences at the , UK. Pearl has a long association with both Birkbeck and UCL as student, lecturer and professor. He described structural studies of a known as heat shock protein 90 (Hsp90), which is a molecular chaperone. The concept of chaperones is a very familiar one at Birkbeck, thanks to Helen Saibil’s ground-breaking work on the bacterial chaperone GroEL; they are that help

ISMB Symposium 2010 www.ismb.lon.ac.uk/symposium2010.html other proteins to attain and maintain their leaves the tunnel. NMR can give useful folded conformation. Hsp90, which enables information about the dynamics of these protein stability at a late stage of folding, is structures, and hence about the process of essential in eukaryotes. in vivo. Christodoulu described how “fingerprint” NMR spectra obtained at Pearl has studied the structure of this protein different points during the protein synthesis and its constituent domains since his time at process showed significant differences UCL in the 90s. The first structure of its N- between the folded and unfolded states of the terminal domain, which binds ATP, was emerging protein chain. He has used this published in 1998. Since then, he has been technique to investigate the folding of establishing the mechanism by which ATP proteins involved in diseases of protein binding drives a conformational change that aggregation, such as alpha-synuclein, which is leads to Hsp90 forming a dimer. When ATP implicated in Parkinson’s disease, and alpha-1 binds, a “lid” of protein structure folds down antitrypsin. He has obtained a high resolution over its phosphate groups, opening up a structure for the stalk protein on the intact hydrophobic surface that can be stabilised by ribosome and compared this to X-ray dimer formation. The chaperone’s substrate structures of this protein in isolation. He is proteins bind to the dimer, and ATP can be now developing cell-free methods for hydrolysed into ADP, after which the protein obtaining NMR samples of ribosomes that can relaxes back into its monomeric state. generate high-quality data faster and more Substrates that bind to Hsp90 include many cheaply than conventional methods, and is protein kinases, which are key targets for aiming to use these to probe the mechanism drugs against cancer; Pearl has published of protein folding – one of the “holy grails” of structural studies of Hsp90-kinase complexes structural biology – even more closely. in collaboration with Birkbeck electron microscopist Cara Vaughan. The last part of The name of Wolfgang Baumeister of the his talk was devoted to unpublished studies of Max-Planck Institute of , Hsp90 from plants in complex with co- Martinsried, Germany, is synonymous with chaperones that are necessary for its activity, technical developments in electron and he explained how these structures are microscopy and, particularly, the leading, in the light of the ATP “switch” development of electron tomography – described earlier, to new insights into the imaging in sections - as a tool for chaperone’s mechanism of action. understanding cell biology. This can The ISMB contribution to the structural produce relatively high biology programme was given by John resolution images of Christodoulou, who took up his position as a non-repetitive lecturer in the Research Department of structures the size of Structural and Molecular Biology at UCL in cells in their natural 2007. He uses NMR spectroscopy to study the environment. mechanism of protein folding on the Baumeister started his ribosome. These NMR studies of a large, talk, however, with an complex molecular machine in motion are all overview of more the more remarkable as, traditionally, NMR is recent work in his laboratory using single seen as a technique most suitable for studying particle analysis. He used as examples two relatively small molecules, and as the involved in the protein degradation emerging protein chain is still rarely visible pathway, the proteasome, which chops even in X-ray structures of ribosomes. unwanted proteins into about 8-12 amino acids long, and the TPII , Each ribosome is formed from one large and which breaks down those short peptides one small subunit, which only come together further. He can now obtain high quality to form the intact structure during protein structures of the proteasome, a cylindrical synthesis. The newly synthesised protein chain structure with a “cap” at each end, quickly emerges through a tunnel in the large subunit, and almost routinely, and is exploring its beginning to fold into its stable structure as it mechanism further with structural studies of ISMB Symposium 2010 www.ismb.lon.ac.uk/symposium2010.html the complex bound to proteins that intact, fully assembled fibrils using proteomics experiments have suggested as infra-red spectroscopy and electron interaction partners. microscopy. She discovered that structurally identical fibrils of different lengths form from Electron tomography explores the structures the assembly of globular subunits. The lengths of proteins in the context of intact cells or and physical properties of the fibrils depend organelles. Baumeister presented his studies on the environment in which they form, with of the nuclear pore complex through which slower growth producing longer fibrils. proteins are transported into and out of the Interestingly, she suggested that cell nucleus. Higher resolution structures of intermediates and short fibrils are more toxic protein components of the pore complex have to cells than the inert long fibrils; this insight been docked into a lower resolution structure may suggest new targets for intervention in of a section across the intact complex to amlyoid disease. produce a full model. He is now turning his attention to the human ribosome, which is Mark Williams, a lecturer in the Department larger than those of lower organisms and less of Biological Sciences at Birkbeck and tractable to other structural techniques. director of the ISMB Biophysics Centre, represented the “home team” in the The second programme, on biophysics, was biophysics section. His talk, intriguingly opened by Sheena Radford of the Astbury entitled “A Wet Walk in the Thicket of Centre for Structural Molecular Biology and Thermodynamics”, described some uses of Institute of Molecular and Cellular Biology, thermodynamics - the study of energy , UK. Radford’s research, conversion and heat changes - to aid the like Christodoulou’s, focuses understanding of protein complex formation. on protein folding, Whenever molecules interact, by either misfolding and disease, and covalent or non-covalent binding, heat is she is no stranger to the gained or lost. These gains and losses can be ISMB, having established measured and monitored over time using a productive collaborations technique known as isothermal titration with Gabriel Waksman and calorimetry (ITC). Helen Saibil. Early protein folding theories proposed a Williams illustrated the use of ITC using single mechanism for folding each sequence examples from the Hsp90 chaperone system into a single low-energy structure with a described by Pearl. The switch mechanism of single function. Folding is now known to be Hsp90 is driven by a conformational change much more complex, with folded and that closes a lid when ATP binds. Williams unfolded structures in some kind of used the measurement of heat capacity when equilibrium. Many if not most proteins, can ATP and its analogues bind to answer the form an amyloid-type structure consisting of question of whether this alone is sufficient to ladders of beta sheets as well as their close the lid, concluding that lid closure is at functional folds, and amyloid aggregation is a least partially driven by ATP. In collaboration cause of many diseases. Understanding the with other ISMB researchers, he has set up process that drives proteins into either the SCORPIO, a database of the thermodynamics folding or the aggregation pathway is crucial of about 250 different protein-ligand to understanding these diseases. Radford complexes. An analysis of these data shows presented two pieces of work focusing on that that the balance between enthalpic and different parts of the aggregation pathway. entropic contributions to energy change is One, which is not yet published, uses trapped often very close, and that whereas the free intermediate structures of mutant proteins to energy of binding is strongly correlated with identify and characterise the folding hydrophobic surface burial, this free energy is precursors of amyloid aggregates in beta-2 not clearly derived from the change in microglobulin. entropy to which it is traditionally attributed.

Radford also presented her group’s investigation of the structure and function of ISMB Symposium 2010 www.ismb.lon.ac.uk/symposium2010.html

The final biophysics talk was given by Steve Xiaolin Wang from John Christodoulou’s lab, Block of Stanford University in California, and showed each of them taking on the USA. Block is a pioneer in the emerging field persona of one component of the ribosome of single-molecule biophysics: studying and interviewing each other about their proteins or nucleic acids, literally, one function. The second-placed video, made by molecule at a time. Block’s group has Lamya Al-Haj and Linda Dekker from Saul developed a novel technique known as the Purton’s group at UCL, explained the use of optical trap or optical cyanobacteria in biofuels using a rap. tweezers, which can exert exceptionally small forces First to speak in the chemical biology section via focused laser light, was Ben Davies from the Department of and has deployed this to Chemistry at Oxford University, UK. Davies measure the tiny force has been interested in carbohydrate chemistry that is caused by the and carbohydrate-protein interactions for actions of a single many years; his work in this field has molecular machine such generated many honours, including the as RNA polymerase. RNA Wolfson award for Research Merit, and he is a transcription is the key founder of the biotech company Glycoform. nexus of gene control; it is a highly regulated He linked his presentation to many earlier and extremely complex process, particularly ones through the idea that chemistry can be in eukaryotes. In the double optical trap, the used as a tool to force arising from the motion of a single RNA understand biology. By polymerase molecule along the DNA strand analogy with synthetic draws two beads of the optical trap together, chemistry, the and the passage of the polymerase can be characterization, tracked by measuring this change. Reaching synthesis and artificial the precision necessary to measure the almost variation of biological infinitesimal forces represented by the molecules in “synthetic passage of a single DNA base has been an biology” can be used enormous technical challenge. The latest effectively in drug versions of the assay, however, can, indeed, discovery. measure the movement of 3.4 Å that occurs during the synthesis of a single base of RNA. At the beginning of the “genome era”, the Block’s most recent work, however, has fact that the complex biology of higher focused on the motions involved in the folding eukaryotes can be generated from relatively and unfolding of RNA secondary structures few genes was considered a paradox. We now including simple hairpins and the natural RNA know that protein complexity can be aptamers that control gene expression by generated post transcription and post selectively binding regulatory regions of translation. Glycosylation is one very common genes. The double optical track has allowed post-translational modification, and one him to track the folding of an RNA aptamer in protein may exist in many “glycoforms”. real time, showing that folding and binding Davies’ group has developed methods to occur on the same timescale and that one generate synthetic mimics of common RNA loop, the P2 loop, always forms first. glycosylations, for example by replacing an oxygen bridge by a disulphide. This “tap and This section also included the final of a video modify” strategy allows for a wide range of competition for postgraduate students in molecules to be bound to proteins with ISMB. This was funded through the Roberts exquisite specificity. He is exploring synthetic Fund for innovation in training for early-stage models for therapeutic proteins in researchers. The four finalists all produced collaboration with Glycoform, and has entertaining videos explaining their research developed synthetic proteins that mimic to non-scientists. The winning video, chosen specific protein-protein interactions, giving an unanimously by a panel made up from the antibody-like response. non-ISMB speakers, was made by Luke Goodsell, Maria Karyadi, Hélène Launay and ISMB Symposium 2010 www.ismb.lon.ac.uk/symposium2010.html

Derek MacMillan from the Department of development using, as an example, the Chemistry at UCL continued the themes of transcription factor NOTCH, which is mutated glycosylation and complex biomolecule in a form of acute leukaemia known as T-ALL. synthesis with a presentation describing a The crystal structure of NOTCH with its novel method for synthesizing and interaction partners shows its binding site to protein thioesters. This reaction will cleave be occupied by a long alpha helix. Taking this proteins at sites adjacent to cysteine residues helical sequence, and mutating two residues within peptides and proteins, with the amount not involved in interactions with NOTCH to of thioester produced dependent on the form a staple, Verdine has been able to amino acid preceding the cysteine in the generate a stable, functional peptide that can sequence: cysteines immediately following a repress NOTCH-driven DNA transcription in histidine residue are the most readily vivo and that may well have value as an anti- targeted; those preceded by another Cys or by leukaemia agent. Gly also give fairly good yields. MacMillan ended his talk by describing some unpublished The final symposium programme covered work, in which he demonstrated that it is bioinformatics, and its first speaker was Mark possible to synthesise cyclic analogues of anti- Sansom of the Department of Biochemistry, microbial peptides using this new chemical . Sansom’s long and reaction. distinguished research career has focused on modeling the structure and function of Gregory Verdine, Irving Professor of membrane-bound proteins, which are far less Chemistry at Harvard University and the easily studied experimentally Dana-Farber Cancer Institute, Cambridge, than soluble ones, using Massachusetts, shares with MacMillan an computational techniques interest in designing molecules to interact including molecular dynamics with intractable protein targets; he is the simulation. This is an founder and chair of several important topic in drug biotech companies. In an discovery as the 20% of entertaining talk concluding the protein products of the chemical biology section, he human genome that are outlined the theory that a new membrane-bound make up 40% of all known approach to is drug targets. needed to access the full range of potential targets. Currently, for a protein to be Long molecular dynamics simulations are the “druggable” it needs either to be most instructive, but these are very expensive extracellular, and so accessible to biological in memory and CPU time. Sansom’s group has drugs, or to contain a binding site suitable for developed a form of “coarse grain” modeling small molecules; most proteins fit neither that speeds up calculations by grouping atoms category. Verdine’s work involves creating together, switching back to fully atomic constrained and stabilized peptides that can simulations when the system is stable. He has mimic a partner protein’s binding to a used this to model the self-assembly of protein-protein interaction site. Many of these proteins in a membrane, starting with a protein recognition elements are alpha simple model system: the helix dimer of helices, which can be constrained using glycophorin. This was shown to assemble into covalent bonds or “staples” between adjacent its experimentally determined structure turns of the helix; molecular dynamics during the simulation, and the technique then simulations have shown them to be applied to more complex systems, including conformationally stable. the dimer assembly of epidermal receptor (EGFR) which has extracellular Verdine illustrated his theory by showing, and intracellular domains joined by a single firstly, that stapled peptides do, unlike the helix. The simulated structures of the macromolecules from which they are derived, glycophorin dimer and other systems including enter cells, and that they can be used to the M2 proton channel from the influenza target particular organelles. He also virus have been shown to correlate well with demonstrated their potential in drug experimental evidence. ISMB Symposium 2010 www.ismb.lon.ac.uk/symposium2010.html

The ISMB’s contribution to the bioinformatics for proteins in isolation. Sali described his programme was given by Andrew Martin from method for combining information from many the Research Department of Structural and sources to yield assembly structures that are Molecular Biology at UCL. Martin’s work on as detailed and as functionally correct as the analysis of antibody sequences and possible. This involves a four-stage scheme structures has attracted the attention of not dissimilar to that used for solving NMR pharmaceutical companies and he holds structures. Firstly, data is collected from a several patents. He began his talk, like some variety of sources, and then the structure is others, by illustrating the importance of his modeled using both theoretical and topic with a statistic: the total market in experimentally derived restraints. This therapeutic antibodies will be worth some generates an ensemble of structures that fits $30Bn in 2010. The large number of antibody the restraints as far as possible, and an structures known still represents only a analysis of the ensemble generates more fraction of antibody sequences, so modeling is restraints so the process can be iterated. The extremely important, and standard automatic method is a general one that allows data from alignment and modeling methods may be a wide range of structural studies to be inappropriate for these molecules. Several incorporated if it is available. Sali cited, as an groups have published classifications and example, his partly unpublished work on the residue numbering schemes for human and structure of the nuclear pore complex. This mouse antibodies, and used them to generate analysis combines low resolution data alignments and profiles. With funding from obtained using electron microscopy, protein pharmaceutical giant UCB Celltech, Martin’s localization, affinity purification and other group has assembled all antibody sequence techniques with atomic resolution data from and structural information into a single such X-ray structures of the consistent database, Abysis, which now contains over nucleoporin sub-complexes as are available to 40,000 sequences and over 1,000 non derive models that are “as close as possible” redundant structures. This comprehensive to the complete structure of the whole pore, database is being used in homology modeling or, at somewhat higher resolution, of groups and to test the extent to which any proposed of proteins assembled into sub-complexes. therapeutic antibody resembles a human sequence, and so, whether it can be expected In summing up, Gabriel Waksman thanked all to be free of toxic side effects. speakers and stressed the theme of molecular complexes and machines running through the Andrej Sali of the University of California, four programmes. Almost invariably, San Francisco wound up the bioinformatics biological macromolecules function in session and thus the symposium with a lecture complexes rather than singly; this is on the integration of methods for increasingly becoming the focus of biological characterizing macromolecular assemblies. research and reflects the direction he would He, like Pearl, started his scientific career in like the ISMB to take. Finally, Nicholas Keep, Bloomsbury; during his PhD studies with (now Executive Dean of the Faculty of Science at Sir) Tom Blundell at Birkbeck he developed Birkbeck, thanked Waksman, stressing his the program Modeller, which is still one of the irreplaceable contribution to the leadership of most widely used homology modeling the ISMB and his indefatigable work as head of methods. two departments and as a research leader and mentor. Proteins function in vivo by assembling to form complexes or molecular machines. The proteome of any organism, however complex, will produce only a few thousand such assemblies, involved in a few hundred biological processes. Conversely, much structural information is available only ISMB Symposium 2010 www.ismb.lon.ac.uk/symposium2010.html