SouthWest Structural Biology Consortium Meeting
8-9 April 2003
University of Reading
Report to CCP4 Executive
1
SouthWest Structural Biology Consortium Meeting - University of Reading 2003
Summary Report
TheSouthWest Structural Biology Consortium Meeting was held on 8-9 April 2003 in the School of Chemistry, The University of Reading. Groups from the universities of Bath, Bristol, Exeter and Southampton took part, with typically three speakers from each university representing their groups. Speakers were exclusively postgraduate students or young postdoctoral workers, with the only research leaders making presentations being those who did not speak last year in Exeter. There was a very good turnout and enthusiastic participation from students, showing that cost is the main factor deterring students from attending and contribution to scientific meetings. There was an audience of 50-60 for the talks, giving the speakers an important opportunity to present their work and that of their groups to a knowledgeable yet friendly audience. This meeting therefore fills an important gap between the departmental seminar and the national level. This was the second meeting of this group and also notable for the sense that a genuine ‘group’ was emerging, important for the young people to get to know each other. There were also about 30 poster presentations, and a lively poster session with a book token prize for the best poster kindly donated by Syngenta Ltd. The prize was won by Jodie Guy (Exeter) for her poster entitled The Structure of a hyperthermophilic Alcohol Dehydrogenase from Aeropyrum pernix.
Attached to this report are the attendance list, the summary timetable and abstracts for the oral and poster presentations. Professor Gerd Materlik, CEO Diamond , kindly accepted an invitation to present the Diamond project to this largely student audience, most of whom had not had the opportunity to hear him speak before. He talked to the students in the poster session and stayed for the dinner, and seemed genuinely pleased to be involved. The photo shows him talking to Dr Andrew Pannifer of Syngenta.
Timetable Summary
Tuesday 8 (LT1) / Wednesday 9 (LTG)09.00-09.30 / Arrive / Susan Crennell (Bath)
09.30-10.00 / Misha Isupov (Exeter)
10.00-10.30 / Andrew Dalby (Exeter)
10.30-11.00 / Coffee / Coffee
11.00-11.30 / Jennie Littlechild (Exeter) / Steve Wood (Southampton)
Stefan Bagby (Bath) / Andrew Martin (Reading)
11.30-12.00 / Gus Cameron (Bristol) / Darren Thompson (Southampton)
12.00-12.30 / Jim Thorpe (Reading) / Ramanathan Natesh (Bath)
12.30-13.00 / Daniel Holloway (Bath) / Kay Wilkinson (Bristol)
13.00-13.30 / Lunch Lunch
13.30-14.00
14.00-14.30 / Jodie Guy (Exeter) / Alison Cuff (Reading)
14.30-15.00 / Andrea Hadfield (Bristol) / Liam Smith (Bristol)
15.00-15.30 / Cerys Huggins (Reading) / Mark Montgomery (Southampton)
15.30-16.00 / Poster Session & Coffee / Depart
16.00-16.30
16.30-17.00
17.00-17.30 / Gerhard Materlik (Diamond)
17.30-18.00 / Drinks, Poster Session & Prize
18.00-18.30
18.30-19.00 / Free Time
19.00-19.30
19.30-00.00 / Conference Dinner & Bar
Meeting Organisation
Local Organising Committee
Dr Christine Cardin
Mr Ben Gale
Ms Susana Teixeira
Dr James Thorpe
Mrs Nicola Young
Local Helpers
Ms Yu Gan
Ms Isabel Moraes
Delegate List
Ms Karen
Dr Stefan
Dr Matthew D
Dr Mark
Professor Leo
Dr Gus
Dr Christine
Ms Marta CarvalhSouthampton
Dr Antonio Cavallo
Mr Stephen
Dr Jon
Mr Steve
Ms Elizabeth E
Dr Susan J
Ms Alison Cuff
Dr Andrew
Dr Ashlesha
Ms Hazel
Ms Helen Fooks
Mrs Yu
Ms Esther
Ms Jodie E
Dr Andrea
Mrs Michelle C
Dr Daniel E
Dr Cerys
Dr Misha
Dr Shalini
Michelle JenveySouthampton
Professor Ian
Ms Liz
Mr Arun
Dr Tim
Dr Gail Hutchinson
Dr Claudia
Mr Samuel
Dr Claire
Ms Kirsty
Professor Jennifer A
Ms Lucy E
Dr Andrew Martin
Mr David
Mr Mark
Mrs Isabel
Dr Ramanathan
Mr Stuart
Mr Didier
Dr Alan
Mr Phil
Dr Lowrie
Mr Liam
Ms Danielle Talbot
Ms Susan
Dr Darren
Dr Jim
Dr Becky
Mrs Kathryn
Dr Abhishek
Ms Samantha J
Dr Kimberly
Mr Clive
Dr Kay
Mr Chris
Mr Steve J. Wills
Professor Steve
Mr Huan-Lin
Detailed Timetable & Talk Abstracts
Tuesday 8 April
All lectures will be held in LT1 on the first floor
11.00 - 13.00 Scientific Session 1
11.00 - 11.15Jenny Littlechild - University of Exeter
The Exeter Biocatalysis Centre:-Project Overview and Future Directions
J.Littlechild, University of Exeter, School of Biological and Chemical Sciences, Exeter, UK
The Centre building will be completed in July this year and the research to be carried out there will be built around the existing and new collaborations between the School of Biological and Chemical Sciences, the School of Physics, the School of Computing and Engineering and the Peninsula Medical School.
The main areas of structural biology research currently carried out at Exeter fall into two main groups. The first is the study of structure and mechanism of enzymes that have applications for industrial biocatalysis. We are particularly interested in thermostable archaeal enzymes due to their robust properties and evolutionary implications. The second area is concerned with human enzymes, human disease and drug development.
Four projects that fall into the first category are the gamma lactamase enzymes that have been used to produce optically pure gamma lactams that are used in new anti-HIV drugs, thermophilic alcohol dehydrogenase enzymes for the production of chiral alcohols, Baeyer-Villiger monooxygenases and vanadium haloperoxidases. Many of enzymes are from protein classes that have no known structural information available. We will be talking about all of these projects over the next two days.
In the second area we are interested in type 2 diabetes and human glucokinase and the design of inhibitors for human pyroglutamyl carboxypeptidase and its potential role in Alhzeimers disease.
Exeter has a strong presence in the area of bioinformatics with a masters course and several research projects which will be summarised.
We are keen to develop links with other members of the SWSBC to address major structural biology grant initiatives.
11.15 - 11.30Stefan Bagby - Bath
Overview of NMR and X-ray crystallography studies of proteins from bacterial pathogens
Stefan Bagby and Jean van den Elsen, Dept of Biology & Biochemistry, University of Bath
We are studying virulence proteins from Salmonella enterica, Burkholderia pseudomallei and Staphylococcus aureus. I will consider some general features of pathogenesis mechanisms in these bacteria and describe the current status of some of our projects.
11.30 - 12.00Gus Cameron - Brisol
Lactate dehydrogenase from Plasmodium falciparum, the causative agent of the vast majority of fatal malaria infections, is the target enzyme of a large drug development programme. Crystal structures and kinetic data show the enzyme undergoes major loop shifts on substrate binding to create a very restricted active site. Inhibitors with good drug-like properties have been developed that fully occupy the cognate active site. In order to extend this family of inhibitors, a structure-driven approach has been used to design new compounds that utilise the inherent flexibility of the loops. While structure based drug design typically considers the target to be spatially constrained we show that it may be productive to identify regions of pliable secondary structure that allow molecules with little similarity to the native substrate to bind.
12.00 - 12.30Jim Thorpe - Reading
DNA studies within the School of Chemistry
Within the School of Chemistry at the University of Reading we have been focussing our work on DNA structure and function into a number of main areas. These include the structure and cation stabilisation of the DNA Holliday junction, looking closely at how the crystallographic four-way junction is stabilised through its interactions with metal cations, vital to the junction inter-conversion cycle during the recombination process. As a result of this project we have been able to propose the use of the group II metal strontium as an excellent means of derivatising calcium binding macromolecules for use in anomalous measurements. Following on from these studies is the metal stabilisation and distortion of DNA quadruplex structure where we are preparing a ranging of metal - quadruplex complexes as a means of investigating the differing modes of metal binding and structural perturbation of quadruplex structure. Further to these studies our work continues looking into the complexes formed between DNA and experimental anti-tumour agents where we have successfully shown the model of intercalation observed in the majority of texts to be far more complex, with atomic and high resolution studies revealing the messy multiplicity of both the chromophore and side chain modes of binding. These studies have led us to look at the stabilisation of higher order DNA structure through the binding of these agents, where we have successful shown a range of intercalating drugs to bind within an expanded intercalation site, and of particular interest is the observation of a bis-intercalator shown to bind in a threading mode rather than the more regularly observed sandwich motif. These studies are aiding in our understanding of higher order DNA structure and the modes of binding of experimental anti-tumour agents to both B-DNA and higher order DNA structure, with the potential of aiding in the subsequent design of the next generation of anti-cancer drugs.
1). J Mol. Biol., (2003), 327, p97-109, "Conformational and hydration effects of site-selective sodium, calcium and strontium ion binding to the DNA Holliday junction d(TCGGTACCGA)4".
2). J. Mol. Biol., (2002), 323, p167-171, "Structural characterisation of bis-intercalation in higher-order DNA at a junction like quadruplex".
3). Nucleic Acids Res., (2003), 31, p844-849, "Crystal structure of the complementary quadruplex formed by d(GCATGCT) at atomic resolution".
12.30 - 13.00Daniel Holloway - Bath
Structure of the Chemokine, Interferon--inducible Protein 10 (IP-10/CXCL10)
Daniel E. Holloway1, G. Jawahar Swaminathan1, Richard A. Colvin2, Gabriele K. Campanella2, Anasstassios C. Papageorgiou1, Andrew D. Luster2, and K. Ravi Acharya1
1Department of Biology and Biochemistry, University of Bath, Claverton Down,
Bath BA2 7AY, U.K.
2Center for Immunology and Inflammatory Diseases, Division of Rheumatology,
Allergy and Immunology, Massachusetts General Hospital, Charlestown MA 02129, U.S.A.
Chemokines are small, structurally-related proteins that play important roles in leukocyte trafficking. Interferon--inducible protein 10 (IP-10; CXCL10) is one such chemokine, secreted by a diverse range of tissues under proinflammatory conditions. It is a key mediator of the interferon response, preferentially attracts activated Th1 lymphocytes to sites of inflammation, and is an inhibitor of angiogenesis. In addition, IP-10 is highly expressed in a wide variety of diseases, including multiple sclerosis, rheumatoid arthritis, cardiac allograft rejection, atherosclerosis, and sarcoidosis, making it a promising therapeutic target.
Many of the physiological effects of chemokines are exerted via interaction with specific G-protein-coupled seven-transmembrane-domain receptors on the surface of target cells. In the case of IP-10, the receptor is CXCR3. Interactions with glycosaminoglycans such as heparin and heparan sulphate are also likely to be important for activity in vivo.
The structural details of IP-10’s mechanism of action are crucial to our understanding of IP-10-mediated processes and to the development of IP-10/CXCR3-targeted therapeutics. To this end, we have determined the structure of IP-10 from three crystal forms: monoclinic, tetragonal and hexagonal (M-, T- and H-forms, at 3.0, 1.92 and 2.0 Å resolutions, respectively) [1]. The crystals provide eight separate models of the IP-10 polypeptide chain. In each case, its topology is that of a typical CXC chemokine, although surprisingly, the main chain trace deviates substantially from that of a monomeric IP-10 mutant examined previously by NMR spectroscopy.
In each crystal form, IP-10 chains form conventional -sheet dimers that in turn form a distinct tetrameric assembly. The M-form tetramer is reminiscent of platelet factor 4, whereas the T- and H-forms feature a novel twelve-stranded -sheet that is hitherto unprecedented among chemokines. The physiological role of these IP-10 oligomers is unclear at present but it is possible that they represent species promoted by the binding of glycosaminoglycans at various sites of action. Furthermore, characterisation of chemokine oligomerisation is of great value to future clinical applications where the local concentration may be high.
The structures have allowed us to map the binding sites for several monoclonal antibodies that abolish the CXCR3-binding activity of IP-10, as well as residues that are important for binding to glycosaminoglycans.
[1] Swaminathan, G.J. et al. (2003). Structure11, in press.
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13.00 - 14.00 Lunch (Chemistry Foyer)
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14.00 - 15.30 Scientific Session 2
14.00 - 14.30Jodie Guy - Exeter
The Structure of a hyperthermophilic Alcohol Dehydrogenase from Aeropyrum pernix
Guy, J.E., Isupov, M. and Littlechild, J.A.
Schools of Chemistry and Biological Sciences, University of Exeter, UK
Alcohol dehydrogenases (ADHs, EC 1.1.1.1) catalyse the cofactor dependent interconversion of alcohols with the corresponding aldehyde or ketone. Reduction of ketones by alcohol dehydrogenases is a potential method for the industrial synthesis of chiral secondary alcohols. Enzymes from the thermophilic archaea are particularly suitable for industrial applications due to their high stability to temperature and organic solvents.
Aeropyrum pernix is an aerobic archaeon with an optimum growth temperature of 90 – 95 °C. A medium-chain ADH has been cloned from A. pernix and overexpressed in Escherichia coli. The protein has been crystallised in its holo form by the method of vapour-phase diffusion.1 Orthorhombic crystals grow in the space group P21212 with cell parameters a = 100.7Å, b = 103.2Å, c = 67.5Å, = = = 90.0.
The structure of the ADH has been solved to 1.62 Å by the multiple anomalous dispersion technique using the signal from the naturally occurring zinc ions. The enzyme is a homotetramer with 222 point group symmetry. The monomer contains two zinc ions, one catalytic and one with a structural role. The catalytic zinc ion is coordinated to the active site residues Cys, His and Asp. On solution of the structure, additional electron density was found in the active site. This density has been described as the inhibitor octanoic acid, with its carbonyl oxygen forming the fourth ligand of the catalytic zinc ion. The structural zinc ion is coordinated by three Cys residues and one Asp. It is present in the structure at only partial occupancy, and in its absence two of the cysteine residues move closer together to form a disulfide bond. The high thermal stability of the A. pernix ADH is thought to arise primarily from increased ionic and hydrophobic interactions on the subunit interfaces.
1 Guy, J.E., Isupov, M.N. and Littlechild J.A. (2003). Crystallisation and preliminary X-ray diffraction studies of a novel alcohol dehydrogenase from the hyperthermophilic archaeon Aeropyrum pernix. Acta Cryst. D59, 174-176.
14.30 - 15.00 Andrea Hadfield - Bristol
Naphthalene is an important model system for understanding the degradation of polyaromatic hydrocarbons (PAHs) which are priority pollutants. Ralstonia sp. strain U2 was isolated from oil-contaminated soil in Venezuela by selective enrichment on naphthalene as the sole carbon source. In the ‘classical’ route for bacterial naphthalene catabolism, naphthalene is converted to salicylate (2-hydroxybenzoate) which is then routed to central metabolites by the meta-cleavage pathway: the two sequences of reactions (naphthalene to salicylate and salicylate to central metabolites via catechol) are encoded on physically separate operons. In Ralstonia U2, salicylate is also formed from naphthalene but is then metabolised through the gentisate (2,5-dihydroxybenzoate) pathway and the entire set of catabolic genes are in a single operon. The bacterial gentisate pathway is also used for a variety of other pollutant xenobiotics and is chemically analogous to the mammalian homogentisate (2,5-dihydroxyphenylacetate) pathway, through which the amino acids phenylalanine and tyrosine are catabolised to central metabolites. A complete analysis of this particular sequence of enzymes will improve our understanding of bacterial pollution degradation and, since there are a number of genetically inherited mutations associated with the enzymes in the human homogentisate pathway, our study of the related bacterial enzymes could provide insights into the defects in proteins which cause phenylketonuria, alkaptonuria and hereditary tyrosinemia type 1.
We are collaborating with the University of Bangor to study the enzymes of salicylate catabolism from Ralstonia U2 at the level of enzymology and protein structure: the monooxygenase component of S5H (NagGH), its two electron transport proteins
(NagAa and NagAb), the gentisate dioxygenase (GDO, NagI), the maleylpyruvate isomerase (MPI, NagL) and the fumarylpyruvate hydrolase (FPH, NagK). These represent a group of enzymes that are not only metabolically contiguous but, each in its turn, has novel features regarding mechanisms and/or structure which have not previously been described in the literature. All of these enzymes have already been cloned into high expression vectors and simple spectrophotometric assays have been developed for them. Last year we reported the crystallisation of the Maleyl Pyruvate Isomerase. This structure is now refined and gives insights into the glutathione mediated catalysis. We have also crystallised the gentisate dioxygenase and will report progress on the data collection and structure solution.
15.00 - 15.30 Cerys Huggins - Reading
Improving protein expression in baculovirus
Cerys C. Huggins, David A. G. Chapman, Stuart Pengelley, Kevin Dalton and Ian M. Jones.
School of Animal and Microbial Sciences, University of Reading, Whiteknights, Reading, Berks, RG6 6AJ, UK.
The baculovirus expression system, based on the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) has been extensively used for the production of membrane proteins. Yields are often high and the post-translational modifications carried out by insect cells reflect those found in mammalian cells. However, the construction and isolation of recombinants was time consuming and the range of vectors limited. We developed methods that improve both aspects of the system. The first improvement of this system has been the production of a modified baculoviral genome that can be maintained in E. coli and that can only produce viable virus if it undergoes recombination with a baculovirus transfer vector. This modified baculovirus DNA produces no background virus and allows 100% recombinant formation, thereby making the production of viruses faster and more reliable (Zhao et al., 2003).
The second area of our work addresses some of the problems that limit low expression levels. A commercial vector (pTriEX1.1, Novagen) has been modified in a number of ways to attempt to improve solubility and recovery. Folding and purification of complex proteins have been achieved through use of two affinity tags (maltose binding protein [MBP] and polyhistidine) which flank the vector multiple cloning site (MCS). This new vector/virus combination has been used to produce the human cytokine receptor CCR5, a seven trans-membrane domain G protein coupled receptor (GPCR) to a higher level than expression of CCR5 alone. In addition, a variant of the vector (pTriExGFP) incorporates GFP as a marker for the direct measurement of expression level.