The Department of Biology and Chemistry at Birkbeck recently hosted as a seminar speaker Dr Geoff Coxon, from the University of Strathclyde, Glasgow. Dr Coxon described a series of compounds that his group had synthesised, some of which are promising candidate drugs against tuberculosis. These are inhibitors of the enzyme beta-ketoacyl-ACP synthase (otherwise known as FabH) with a novel chemical scaffold.
Mycobacterium tuberculosis now infects over a third of the world's population, and tuberculosis kills two million people a year. The threat of tuberculosis is growing largely because of two reasons: a synergy between HIV and TB infections, and the growth of drug resistant strains of the bacterium. Multi-drug resistant (MDR) TB is defined as TB that is resistant to the two most commonly used anti-TB drugs, and extensively drug resistant (XDR) TB as TB that is resistant to four drugs including a fluoroquinolone. The latter is particularly hard to treat. More information on this is available from the TB Alliance.
In order to combat drug resistance it is essential to keep introducing new drugs, and preferably drugs with novel targets and mechanisms. Ideally, a drug should be active against active, replicating TB and persistent TB, which can resist treatment by remaining dormant in macrophages. Much work in anti-TB drug development has focused on enzymes involved in synthesising the very complex cell wall of the bacteria.
The genome sequence of M. tuberculosis (Camus et al., 2002, and material in PPS section 6) revealed a number of enzymes involved in cell wall synthesis. Coxon's group is focusing on the synthesis of one cell wall component, mycolic acids. These are long chain, 2-alkyl 3-hydroxyl fatty acids with between 60 and 80 atoms in their hydrocarbon chains. Two enzyme systems are involved in their synthesis: FAS-1 synthesises the main chain and FAS-2 adds the alpha branch. FAS-2 is found only in plants and mycobacteria.
The FAS-2 system includes a large number of enzymes, all involved in the complex, cyclic process of synthesising a long lipid chain. Inhibiting any of these enzymes will prevent the synthesis of the mature long chain mycolic acids. Coxon's group has been targeting one key enzyme in this process, known as FabH, which connects the FAS-1 and FAS-2 systems. The reaction it catalyses is an extension of the lipid chain by two carbon units.
This enzyme is a homodimer with a tunnel-shaped active site; the chain extension takes place after the substrate has moved into the tunnel. Its structure was first solved in 2001 by Scarsdale and co-workers (PDB code 1HZP). Coxon and his co-workers started their search for a specific inhibitor of this enzyme from the natural antibiotic, thiolactomycin (TLM). This, however, is a relatively weak inhibitor and a chiral compound that is extremely challenging to synthesise. He used fragment-based chemical libraries to develop a range of likely inhibitors with similar skeletons but that would be simpler to synthesise.
Some of the first compounds investigated were better inhibitors of the enzyme but not active against the whole FAS-2 system or M. tuberculosis itself. It appeared that another enzyme, known as KasA, may sometimes take the place of FabH if that is inhibited. They have now produced a series of compounds based on a 2-aminothiazole-4-carboxylate scaffold that includes some very active ones. However, the compounds in this series that are the most potent inhibitors of the enzyme are not the most active against the intact bacterium, and vice versa - there are complex interactions going on that are not yet entirely understood. It is likely, even, that FabH is not their most important protein target.
A good medicine against tuberculosis must be active against both resistant and dormant forms of the bacterium, with few side effects and few interactions with other drugs, and preferably orally available, and it must be cheap, and therefore easy, to synthesise. These 2-aminothiazole-4-carboxylates are easily synthesised and some are effective against M. tuberculosis in vivo, but much more work is needed to determine whether they will fulfil the other criteria.
See Al-Balas, Q. et al. (2009) PLoS ONE 4(5) (open access) for more information about this work.
Dr Coxon also works in TB Drug Discovery UK, an alliance of scientists involved in developing new treatments for tuberculosis.
Thursday, 18 June 2009
Monday, 9 March 2009
Structure of the trypanosome microtubule cytoskeleton
Twice a term, the School of Crystallography hosts a seminar for the whole of the Institute of Structural and Molecular Biology, which consists of research departments in related disciplines from both Birkbeck and neighbouring University College London. Tom Blundell's (see previous post) was an ISMB seminar; today, we heard one from Professor Keith Gull of the Sir William Gunn School of Pathology, University of Oxford.
Much of Keith Gull's work concerns the single-celled parasite Trypanosoma brucei, which is endemic in large parts of Africa and causes African trypanosomiasis, otherwise known as sleeping sickness. Diseases like this one attract relatively little research funding even though they are important causes of morbidity and mortality in many poor countries; they are classed as neglected tropical diseases. Keith and his colleagues in Oxford are studying many aspects of the molecular biology and genetics of this parasite.
The trypanosome cell surface is covered by a dense coat made up of very many copies of a single protein, called variable surface glycoprotein (VSG). A glycoprotein is a protein with carbohydrate (sugar) groups attached to one or more amino acid sidechains; the word "variable" is used because there are about 1000 variants of this protein. Each of these variants is encoded by a different gene, so the VSG genes account for about 10% of the trypanosome's genome. However, only one of these is expressed at any time, so the trypanosome coat (almost) always consists of multiple copies of a single protein. (There will be times when two proteins are present because the expressed variant is in the process of changing.) Although the sequences of VSG proteins differ considerably, their structures are very similar: they are anchored to the cell membrane and the large extracellular domain consists of an antiparallel coiled coil of alpha helices (see PDB file 2vsg).
Most of Keith's talk, however, concerned the structure of the trypanosome cytoskeleton, which consists of microtubules. Very basically, these are composed of polymers of a protein called tubulin and they are involved in maintaining the structure of components of many types of cells. Keith and his group have used the technique of electron tomography - a type of transmission electron microscopy - to obtain exquisite pictures - if at much lower than atomic resolution - of the structure of this cytoskeleton and begin to understand its function and role in trypanosomal cell division. Structures of these multi-protein complexes are not - yet - available in the PDB.
Trypanosomes have flagella - whip-like structures attached to the cells that can propel them through the host bloodstream. Flagella are found in many cell types, and if they have receptors bound they can also be used to sense cells' surroundings. The trypanosome flagellum is attached to the cell body via a filament and this point of attachment is within a pocket that, interestingly, is the end point for all vesicular traffic within the cell. This means that this pocket needs to contain a large number of proteins vital to the parasite's survival, including its transferrin receptor and haemoglobin receptor. Imaging the structure of the trypanosome cytoskeleton, particularly in the pocket where the flagellum attaches, has also given insight into the rather unusual process through which this cell conducts mitosis (cell division).
Much of Keith Gull's work concerns the single-celled parasite Trypanosoma brucei, which is endemic in large parts of Africa and causes African trypanosomiasis, otherwise known as sleeping sickness. Diseases like this one attract relatively little research funding even though they are important causes of morbidity and mortality in many poor countries; they are classed as neglected tropical diseases. Keith and his colleagues in Oxford are studying many aspects of the molecular biology and genetics of this parasite.
The trypanosome cell surface is covered by a dense coat made up of very many copies of a single protein, called variable surface glycoprotein (VSG). A glycoprotein is a protein with carbohydrate (sugar) groups attached to one or more amino acid sidechains; the word "variable" is used because there are about 1000 variants of this protein. Each of these variants is encoded by a different gene, so the VSG genes account for about 10% of the trypanosome's genome. However, only one of these is expressed at any time, so the trypanosome coat (almost) always consists of multiple copies of a single protein. (There will be times when two proteins are present because the expressed variant is in the process of changing.) Although the sequences of VSG proteins differ considerably, their structures are very similar: they are anchored to the cell membrane and the large extracellular domain consists of an antiparallel coiled coil of alpha helices (see PDB file 2vsg).
Most of Keith's talk, however, concerned the structure of the trypanosome cytoskeleton, which consists of microtubules. Very basically, these are composed of polymers of a protein called tubulin and they are involved in maintaining the structure of components of many types of cells. Keith and his group have used the technique of electron tomography - a type of transmission electron microscopy - to obtain exquisite pictures - if at much lower than atomic resolution - of the structure of this cytoskeleton and begin to understand its function and role in trypanosomal cell division. Structures of these multi-protein complexes are not - yet - available in the PDB.
Trypanosomes have flagella - whip-like structures attached to the cells that can propel them through the host bloodstream. Flagella are found in many cell types, and if they have receptors bound they can also be used to sense cells' surroundings. The trypanosome flagellum is attached to the cell body via a filament and this point of attachment is within a pocket that, interestingly, is the end point for all vesicular traffic within the cell. This means that this pocket needs to contain a large number of proteins vital to the parasite's survival, including its transferrin receptor and haemoglobin receptor. Imaging the structure of the trypanosome cytoskeleton, particularly in the pocket where the flagellum attaches, has also given insight into the rather unusual process through which this cell conducts mitosis (cell division).
Friday, 13 February 2009
Genomes, Structural Biology and Drug Design
Professor Sir Tom Blundell, head of the School of Crystallography at Birkbeck from 1977 to 1995, returned last Monday to give an extremely well attended seminar - a tour de force of the relationship between structural biology and drug design Tom's distinguished career has also included a time as the head of the research council that funds non-medical biological research in the UK, BBSRC; he moved from Birkbeck to become head of Biological Sciences at Cambridge; and he is a director of a biotech company, Astex Therapeutics, which he founded in 1999.
Tom started his talk with a brief history of structural biology and its role in drug discovery. His personal involvement in the discipline goes back to the 1960s, when he, as a Ph.D. student, visited companies such as Eli Lilly, which manufactured insulin, with his supervisor, Dorothy Hodgkin - who solved the insulin structure, but won her Nobel prize (Chemistry, 1964) for structures of penicillin and vitamin B12.
The intervening decades have seen trends in drug discovery come and go. In the 1990s, it seemed that increases in the speed of synthesising and screening large numbers of small molecules against drug targets had made the more targeted approach of the academic structural biologists redundant. However, even the millions of compounds that can now be screened represent the tiniest fraction of "chemical space": the number of potential molecules of a size to bind to a drug target is larger than the estimated number of atoms in the universe.
And now, in the so-called age of the genome, structural biology has become an integral part of drug discovery, involved in all steps: target identification and validation, screening, and lead compound identification and optimisation. The organisation of information about sequences and structures in databases - some of which were mentioned in the PPS Bioinformatics section - began when Tom was at Birkbeck, and was spearheaded by his co-workers and collaborators, particularly Janet Thornton (now head of the EBI). The databases set up and curated by members of his Cambridge group - too many to describe properly here - are available from this page.
Some particularly useful insights arise from the relationship between the single changes in nucleic acid sequence (known as Single Nucleotide Polymorphisms, or SNPs) that are collected into databases and the structural biology of drug targets. Sometimes, with simple Mendelian diseases, one such change is sufficient to cause disease; more complex diseases arise (anything from hypertension to breast cancer to bipolar disorder) arise from interactions between many such changes that increase the chance of disease. Mapping changes in protein coding brought about by an SNP to a protein's structure can give insight into disease-causing changes in protein mechanism and lead to the identification of novel drug targets. See Burke et al. (2007), BMC Bioinformatics 8, 301 (this is an open access journal with full text available free of charge).
The drug discovery programme at Astex Therapeutics is based on experimental structural biology, on a technique known as fragment screening. In this, small chemical fragments that bind to drug targets are identified by fast X-ray crystallography. Knowing both the structures of these small compounds - which are too weak as binders to be identified by chemical means - and where they sit in a drug target's binding site enables chemists to build them out to form larger tight-binding molecules that fit into the whole site. The company's pipeline focuses on kinase inhibitors as anti-cancer drugs, and some of its lead compounds have reached early clinical trials.
Tom started his talk with a brief history of structural biology and its role in drug discovery. His personal involvement in the discipline goes back to the 1960s, when he, as a Ph.D. student, visited companies such as Eli Lilly, which manufactured insulin, with his supervisor, Dorothy Hodgkin - who solved the insulin structure, but won her Nobel prize (Chemistry, 1964) for structures of penicillin and vitamin B12.
The intervening decades have seen trends in drug discovery come and go. In the 1990s, it seemed that increases in the speed of synthesising and screening large numbers of small molecules against drug targets had made the more targeted approach of the academic structural biologists redundant. However, even the millions of compounds that can now be screened represent the tiniest fraction of "chemical space": the number of potential molecules of a size to bind to a drug target is larger than the estimated number of atoms in the universe.
And now, in the so-called age of the genome, structural biology has become an integral part of drug discovery, involved in all steps: target identification and validation, screening, and lead compound identification and optimisation. The organisation of information about sequences and structures in databases - some of which were mentioned in the PPS Bioinformatics section - began when Tom was at Birkbeck, and was spearheaded by his co-workers and collaborators, particularly Janet Thornton (now head of the EBI). The databases set up and curated by members of his Cambridge group - too many to describe properly here - are available from this page.
Some particularly useful insights arise from the relationship between the single changes in nucleic acid sequence (known as Single Nucleotide Polymorphisms, or SNPs) that are collected into databases and the structural biology of drug targets. Sometimes, with simple Mendelian diseases, one such change is sufficient to cause disease; more complex diseases arise (anything from hypertension to breast cancer to bipolar disorder) arise from interactions between many such changes that increase the chance of disease. Mapping changes in protein coding brought about by an SNP to a protein's structure can give insight into disease-causing changes in protein mechanism and lead to the identification of novel drug targets. See Burke et al. (2007), BMC Bioinformatics 8, 301 (this is an open access journal with full text available free of charge).
The drug discovery programme at Astex Therapeutics is based on experimental structural biology, on a technique known as fragment screening. In this, small chemical fragments that bind to drug targets are identified by fast X-ray crystallography. Knowing both the structures of these small compounds - which are too weak as binders to be identified by chemical means - and where they sit in a drug target's binding site enables chemists to build them out to form larger tight-binding molecules that fit into the whole site. The company's pipeline focuses on kinase inhibitors as anti-cancer drugs, and some of its lead compounds have reached early clinical trials.
Tuesday, 20 January 2009
Designing Magic Bullets
This week's Monday seminar at Birkbeck was given by Jose Saldanha, who works in Willie Taylor's group at the National Institute of Medical Research at Mill Hill, London. Both Jose and Willie are Birkbeck Ph.D. graduates. Jose described his work in using molecular modelling and bioinformatics to design specific antibodies for therapeutic applications.
These antibodies are the "magic bullets" of the title of this blog post. Interestingly, the term was invented by Paul Ehrlich, who won of one of the first Nobel prizes for Medicine - in 1908 - for his work on immunity. The magic bullet, he suggested, would be a molecule that could be targeted directly to a part of the body needing drug treatment, and which a drug molecule could be attached to.
Antibodies (or immunoglobulins) are secreted into the bloodstream in response to the presence of "foreign" molecules (e.g. from bacteria or viruses) and target those molecules in order to destroy the "invader". They do this by binding very specifically to the targeted molecule. Therefore, molecules with essentially the same structure must be able to bind an almost infinite variety of targets.
The mechanism that has evolved for this is a very elegant one. The commonest type of immunoglobulin, Immunoglobulin G (IgG) is a Y shaped molecule made up of four chains, two heavy and two light. Heavy chains contain four copies of the same domain fold (a type of beta barrel) and light chains contain two. The binding site is at the "top" of the "arms" of the Y; the domain of each chain that is closest to the binding site is much more variable in sequence than the others, and is termed the variable domain (the others are constant domains). Each variable domain has three regions that are particularly variable in sequence, termed complementarity determining regions or CDRs, and it is these that bind the target molecule (the antigen). There is a more about immunoglobulin structure on this page from the Arizona Biology Project, and it will be covered in depth in section 11 of PPS.
The only antibody that can be successful as a "magic bullet" is a completely homogenous sample, where every molecule has the same sequence and structure and binds the same antigen. Such identical antibodies, or monoclonal antibodies, can be produced by fusing myeloma (cancer) cells with spleen cells produced by an infected mouse, and selecting and cloning those resulting cells that secrete the required antibody. However, these are mouse antibodies that do not work in humans; they can also cause an immune response because they themselves are seen as "foreign".
In the 1980s, Greg Winter at NIMR developed a process called "CDR grafting" to overcome this, in which the CDR regions from the mouse antibodies are grafted on to a human antibody framework. An alternative technique involves the fusion of a few residues from the mouse antibody with a human one to form a "humanised" antibody. Jose's work at NIMR involves sequence and structure analysis to work out exactly which mouse antibody residues to fuse with which human antibodies for different indications. A "successful" antibody must express well, bind specifically to its antigen and cause no immune or other adverse reaction in human patients. Since starting the project, he has designed more than 30 different humanised antibodies, some of which are in clinical trials.
There are many structures of immunoglobulins and immunoglobulin fragments in the PDB. The most commonly crystallised fragment is the four domains that make up one of the arms of the Y, which is known as Fab (standing for "Fragment, antibody binding"). Look at this one (1BM3), which binds a peptide antigen. And remember - we will be coming back to this topic later in the course.
These antibodies are the "magic bullets" of the title of this blog post. Interestingly, the term was invented by Paul Ehrlich, who won of one of the first Nobel prizes for Medicine - in 1908 - for his work on immunity. The magic bullet, he suggested, would be a molecule that could be targeted directly to a part of the body needing drug treatment, and which a drug molecule could be attached to.
Antibodies (or immunoglobulins) are secreted into the bloodstream in response to the presence of "foreign" molecules (e.g. from bacteria or viruses) and target those molecules in order to destroy the "invader". They do this by binding very specifically to the targeted molecule. Therefore, molecules with essentially the same structure must be able to bind an almost infinite variety of targets.
The mechanism that has evolved for this is a very elegant one. The commonest type of immunoglobulin, Immunoglobulin G (IgG) is a Y shaped molecule made up of four chains, two heavy and two light. Heavy chains contain four copies of the same domain fold (a type of beta barrel) and light chains contain two. The binding site is at the "top" of the "arms" of the Y; the domain of each chain that is closest to the binding site is much more variable in sequence than the others, and is termed the variable domain (the others are constant domains). Each variable domain has three regions that are particularly variable in sequence, termed complementarity determining regions or CDRs, and it is these that bind the target molecule (the antigen). There is a more about immunoglobulin structure on this page from the Arizona Biology Project, and it will be covered in depth in section 11 of PPS.
The only antibody that can be successful as a "magic bullet" is a completely homogenous sample, where every molecule has the same sequence and structure and binds the same antigen. Such identical antibodies, or monoclonal antibodies, can be produced by fusing myeloma (cancer) cells with spleen cells produced by an infected mouse, and selecting and cloning those resulting cells that secrete the required antibody. However, these are mouse antibodies that do not work in humans; they can also cause an immune response because they themselves are seen as "foreign".
In the 1980s, Greg Winter at NIMR developed a process called "CDR grafting" to overcome this, in which the CDR regions from the mouse antibodies are grafted on to a human antibody framework. An alternative technique involves the fusion of a few residues from the mouse antibody with a human one to form a "humanised" antibody. Jose's work at NIMR involves sequence and structure analysis to work out exactly which mouse antibody residues to fuse with which human antibodies for different indications. A "successful" antibody must express well, bind specifically to its antigen and cause no immune or other adverse reaction in human patients. Since starting the project, he has designed more than 30 different humanised antibodies, some of which are in clinical trials.
There are many structures of immunoglobulins and immunoglobulin fragments in the PDB. The most commonly crystallised fragment is the four domains that make up one of the arms of the Y, which is known as Fab (standing for "Fragment, antibody binding"). Look at this one (1BM3), which binds a peptide antigen. And remember - we will be coming back to this topic later in the course.
Thursday, 11 December 2008
An Enzyme in Histidine Biosynthesis
The School of Crystallography at Birkbeck has a regular programme of research seminars held on Monday lunchtimes during term. Many of these describe recent developments in protein structure, and, from time to time, I will be reporting these here and linking in to course material where relevant. This week's seminar was by Adrian Lapthorn, a protein crystallographer based at Glasgow University, who just happens also to be the external examiner for the MSc in Structural Molecular Biology as well as the second year module TSMB. Adrian's research is concerned with solving the structures of enzymes, including those involved in the biosynthesis of the amino acid histidine.
Histidine is known as an essential amino acid; that is, it cannot be synthesised de novo in humans, but must be supplied from the diet. However, bacteria, fungi and plants all have enzymes that enable them to synthesise histidine from simple chemical precursors. The enzymes in the histidine synthesis pathway are therefore, at least potentially, good targets for novel antibiotics and herbicides as there are no equivalent human enzymes for them to inhibit, so they should be relatively free of side effects.
In bacteria, the histidine synthesis pathway consists of 10 steps, catalysed by a total of eight enzymes (some of which are bifunctional). The first step in this pathway is synthesised by an enzyme called HisG (or ATP-phosphyribosyltransferase) which catalyses the following reaction:
ATP + PRPP ---> PR-ATP
(PRPP is Phosphoribosyl pyrophosphate; PR-ATP is phosphoribosyl-ATP. The action of the enzyme is, therefore, to transfer a phosphoribose group on to the ATP molecule.
This enzyme is interesting for several reasons, besides the pharmaceutical and biotechnological interest in its inhibition. For one thing, unusually, there are no specific active site residues; the substrate is stabilised in the active site cleft by binding to magnesium ions.
Adrian's talk was subtitled "the long and the short of it", because some bacteria have a "short" form of this enzyme, and others a "long" form with an extra 80-odd residues at its C-terminus. All bacteria with the short form also have an additional enzyme, HisZ, which binds to HisG in an equivalent position to the C-terminal domain of the long form during catalysis. The long form of the enzyme consists of three discrete folded units called domains. There are two similar ones at the N terminus, followed by a long alpha helix and the C terminal domain, which is absent in the short form and has a similar structure to that of the small protein ferredoxin. The active site is between the two similar domains.
You will learn much more about domains and their folds in the next section, Towards Tertiary Structure. For now, look at this structure (PDB 1Q1K) of the long form of HisG (from E.coli) and try to identify the three domains and the active site. You might find it helpful to look at a single chain only.
Histidine is known as an essential amino acid; that is, it cannot be synthesised de novo in humans, but must be supplied from the diet. However, bacteria, fungi and plants all have enzymes that enable them to synthesise histidine from simple chemical precursors. The enzymes in the histidine synthesis pathway are therefore, at least potentially, good targets for novel antibiotics and herbicides as there are no equivalent human enzymes for them to inhibit, so they should be relatively free of side effects.
In bacteria, the histidine synthesis pathway consists of 10 steps, catalysed by a total of eight enzymes (some of which are bifunctional). The first step in this pathway is synthesised by an enzyme called HisG (or ATP-phosphyribosyltransferase) which catalyses the following reaction:
ATP + PRPP ---> PR-ATP
(PRPP is Phosphoribosyl pyrophosphate; PR-ATP is phosphoribosyl-ATP. The action of the enzyme is, therefore, to transfer a phosphoribose group on to the ATP molecule.
This enzyme is interesting for several reasons, besides the pharmaceutical and biotechnological interest in its inhibition. For one thing, unusually, there are no specific active site residues; the substrate is stabilised in the active site cleft by binding to magnesium ions.
Adrian's talk was subtitled "the long and the short of it", because some bacteria have a "short" form of this enzyme, and others a "long" form with an extra 80-odd residues at its C-terminus. All bacteria with the short form also have an additional enzyme, HisZ, which binds to HisG in an equivalent position to the C-terminal domain of the long form during catalysis. The long form of the enzyme consists of three discrete folded units called domains. There are two similar ones at the N terminus, followed by a long alpha helix and the C terminal domain, which is absent in the short form and has a similar structure to that of the small protein ferredoxin. The active site is between the two similar domains.
You will learn much more about domains and their folds in the next section, Towards Tertiary Structure. For now, look at this structure (PDB 1Q1K) of the long form of HisG (from E.coli) and try to identify the three domains and the active site. You might find it helpful to look at a single chain only.
Monday, 8 December 2008
Welcome 2008-9 Students to the PPS Blog
Yes - welcome!
I run this blog to help PPS students understand how what they study throughout the course relates to recent research, mainly in structural biology but also in bioinformatics and some other related subjects. I plan to update it every few weeks (or more frequently if there is enough to say) between the end of the Autumn term and the end of the course, with reports of lectures, conferences and new papers in the areas covered by the PPS course, and link these to the course material and the wider scientific literature. Some other lecturers at Birkbeck may also occasionally add posts.
The first blog post linked to the 2008-9 course will appear before the end of this week, and will describe crystal structures of enzymes presented at one of the talks in our weekly seminar programme here at Birkbeck. In the mean time, do scroll down and look at some of last year's posts, although you won't be able to follow the links to the course material there.
You will be encouraged to comment on blog posts and, if you wish, to make your own - you will all be added as authors in the New Year. Anyone who can view the blog should be able to comment, but only authors can make posts.
Enjoy the blog - and let us know what you think of it!
Clare Sansom
I run this blog to help PPS students understand how what they study throughout the course relates to recent research, mainly in structural biology but also in bioinformatics and some other related subjects. I plan to update it every few weeks (or more frequently if there is enough to say) between the end of the Autumn term and the end of the course, with reports of lectures, conferences and new papers in the areas covered by the PPS course, and link these to the course material and the wider scientific literature. Some other lecturers at Birkbeck may also occasionally add posts.
The first blog post linked to the 2008-9 course will appear before the end of this week, and will describe crystal structures of enzymes presented at one of the talks in our weekly seminar programme here at Birkbeck. In the mean time, do scroll down and look at some of last year's posts, although you won't be able to follow the links to the course material there.
You will be encouraged to comment on blog posts and, if you wish, to make your own - you will all be added as authors in the New Year. Anyone who can view the blog should be able to comment, but only authors can make posts.
Enjoy the blog - and let us know what you think of it!
Clare Sansom
Tuesday, 29 July 2008
London Structural Biology Club meeting
We at Birkbeck have just hosted a meeting of the London Structural Biology Club. This is a network of students and researchers in structural biology based in London and the South-East of England. Members get together for a couple of afternoons a year to hear research presentations, and the talks are followed by further informal discussion over refreshments (usually pizza and beer).
Four talks were given at the Birkbeck meeting, with each presenting not only new structural studies but also novel insights into molecular function and mechanisms derived from those structures. First to talk was Carien Dekker from the Institute of Cancer Research in London. She described the protein interaction network - known as the "interactome" by analogy with "genome" and "proteome" - of a eukaryotic cytosolic chaperonin, CCT. Chaperonins are a sub-class of chaperones, the proteins that assist other proteins in forming their stable three-dimensional structures, and they consist of two ring-like structures that associate back-to-back forming a cavity in which their substrate proteins fold. Dekker and her co-workers have used a number of different proteomics techniques, including the insertion of a long internal tag into a loop of the protein, to discover the range of substrates for this chaperonin. Proteins involved in functions as diverse as protein import into the nucleus, protein degradation, and chromatin remodelling. CCT is also necessary for the formation of the septin ring complex, and thence for cytokinesis (the last stage of cell division).
This work was published very recently in the EMBO Journal. Dekker and her colleagues are now working on the structure of CCT, which they hope will reveal more details of the function of this chaperonin: watch this space.
The second talk was given by David Komander, who has just moved from the Institute of Cancer Research to set up his own lab at the prestigious MRC Laboratory of Molecular Biology in Cambridge. He described some intriguing details of the ubiquitin system, through which proteins can be tagged for degradation. Ubiquitin (mentioned briefly in section 7 of the PPS course material) is a small protein (only 76 amino acids) with an alpha+beta fold (see PDB entry 1UBQ). Its C-terminus can be covalently linked to lysine side chains or N termini of other proteins. As ubiquitin itself has seven lysine residues (as well, of course, as an N-terminus) it can polymerise to form short chains. Proteomics has shown that all possible combinations of ubiquitin linkages can exist, but linkages in which the molecules are connected through lysines K48 and K63 are the most common. Poly-ubiquitin tags composed of different linkages have been linked with different functions; for example, binding a K48-linked ubiquitin chain to a protein will tag it for proteasomal degradation, whereas a K63-linked chain will tag a protein for signalling. The structures of these two forms of poly-ubiquitin have been shown to be very different, with K63-linked poly-ubiquitin forming an extended chain and K48-linked poly-ubiquitin a compact fold.
Ubuquitinlyation is a reversible process, and Komander has been studying the enzymes (deubiquitinases) that catalyse the hydrolysis of the peptide bonds between two ubiquitins, or between ubiquitin and another protein. These DUBs are analogous to the phosphatases that remove phosphate groups from protein side chains; their specificity , however, is more complex than that of phosphatases. Earlier this year, Komander and David Barford published the structure of the N-terminal domain of one such protein, A20 (Komander & Barford (2008), Biochem. J. 409, 771-785l; full text available). This is a cysteine protease domain known as the ovarian tumour (OTU) domain. These structural studies suggest both a novel architecture for the protein's catalytic triad and a novel mechanism - reversible oxidation - for the regulation of protein ubiquitinylation.
The "home team" at Birkbeck contributed a talk from Han Renaut, in Professor Gabriel Waksman's group. Waksman's own account of this work, on the structure of bacterial secretion systems, was blogged back in May and will not be described in more detail now.
Lastly, we heard from Erhard Hohenester who described an unpublished structure of SPARC, a protein that binds collagen. About 30% of the dry weight of the human body is composed of fibrils of this structural protein. It has a unique structure, being composed of three strands wound round each other in a triple helix. Every third residue of each strand must be a glycine, and the protein also contains a high percentage of proline. Some proline residues are post-translationally modified with the addition of an -OH group to form hydroxyproline. Besides being the major structural component of animal tissue, collagen binds to and forms complexes with many proteins including integrins and some tyrosine kinases. However, until now the only structure of a complex of collagen with another protein was with integrin (see PDB entry 1DZI).
SPARC, or osteonectin, is secreted by osteoblasts during bone formation, and binds calcium as well as collagen. Its structure as an isolated protein has been known for over ten years; it has two domains, one alpha-helical and the other containing many disulphide bonds (PDB 1BMO). Details of the new crystal structure of the collagen-SPARC complex, solved by Hohenester's group, must wait until the paper is published, but it is possible to say that it binds the hydrophobic sequence GVMGFO (which is a rare sequence in collagens, although one often involved in protein-protein interactions) into a hydrophobic pocket on the PARC molecule. [Note that that "O" is not a mistake; it is the single letter amino acid code for hydroxyproline.]
This London Structural Biology Club meeting was sponsored by Alpha Laboratories Ltd.
Four talks were given at the Birkbeck meeting, with each presenting not only new structural studies but also novel insights into molecular function and mechanisms derived from those structures. First to talk was Carien Dekker from the Institute of Cancer Research in London. She described the protein interaction network - known as the "interactome" by analogy with "genome" and "proteome" - of a eukaryotic cytosolic chaperonin, CCT. Chaperonins are a sub-class of chaperones, the proteins that assist other proteins in forming their stable three-dimensional structures, and they consist of two ring-like structures that associate back-to-back forming a cavity in which their substrate proteins fold. Dekker and her co-workers have used a number of different proteomics techniques, including the insertion of a long internal tag into a loop of the protein, to discover the range of substrates for this chaperonin. Proteins involved in functions as diverse as protein import into the nucleus, protein degradation, and chromatin remodelling. CCT is also necessary for the formation of the septin ring complex, and thence for cytokinesis (the last stage of cell division).
This work was published very recently in the EMBO Journal. Dekker and her colleagues are now working on the structure of CCT, which they hope will reveal more details of the function of this chaperonin: watch this space.
The second talk was given by David Komander, who has just moved from the Institute of Cancer Research to set up his own lab at the prestigious MRC Laboratory of Molecular Biology in Cambridge. He described some intriguing details of the ubiquitin system, through which proteins can be tagged for degradation. Ubiquitin (mentioned briefly in section 7 of the PPS course material) is a small protein (only 76 amino acids) with an alpha+beta fold (see PDB entry 1UBQ). Its C-terminus can be covalently linked to lysine side chains or N termini of other proteins. As ubiquitin itself has seven lysine residues (as well, of course, as an N-terminus) it can polymerise to form short chains. Proteomics has shown that all possible combinations of ubiquitin linkages can exist, but linkages in which the molecules are connected through lysines K48 and K63 are the most common. Poly-ubiquitin tags composed of different linkages have been linked with different functions; for example, binding a K48-linked ubiquitin chain to a protein will tag it for proteasomal degradation, whereas a K63-linked chain will tag a protein for signalling. The structures of these two forms of poly-ubiquitin have been shown to be very different, with K63-linked poly-ubiquitin forming an extended chain and K48-linked poly-ubiquitin a compact fold.
Ubuquitinlyation is a reversible process, and Komander has been studying the enzymes (deubiquitinases) that catalyse the hydrolysis of the peptide bonds between two ubiquitins, or between ubiquitin and another protein. These DUBs are analogous to the phosphatases that remove phosphate groups from protein side chains; their specificity , however, is more complex than that of phosphatases. Earlier this year, Komander and David Barford published the structure of the N-terminal domain of one such protein, A20 (Komander & Barford (2008), Biochem. J. 409, 771-785l; full text available). This is a cysteine protease domain known as the ovarian tumour (OTU) domain. These structural studies suggest both a novel architecture for the protein's catalytic triad and a novel mechanism - reversible oxidation - for the regulation of protein ubiquitinylation.
The "home team" at Birkbeck contributed a talk from Han Renaut, in Professor Gabriel Waksman's group. Waksman's own account of this work, on the structure of bacterial secretion systems, was blogged back in May and will not be described in more detail now.
Lastly, we heard from Erhard Hohenester who described an unpublished structure of SPARC, a protein that binds collagen. About 30% of the dry weight of the human body is composed of fibrils of this structural protein. It has a unique structure, being composed of three strands wound round each other in a triple helix. Every third residue of each strand must be a glycine, and the protein also contains a high percentage of proline. Some proline residues are post-translationally modified with the addition of an -OH group to form hydroxyproline. Besides being the major structural component of animal tissue, collagen binds to and forms complexes with many proteins including integrins and some tyrosine kinases. However, until now the only structure of a complex of collagen with another protein was with integrin (see PDB entry 1DZI).
SPARC, or osteonectin, is secreted by osteoblasts during bone formation, and binds calcium as well as collagen. Its structure as an isolated protein has been known for over ten years; it has two domains, one alpha-helical and the other containing many disulphide bonds (PDB 1BMO). Details of the new crystal structure of the collagen-SPARC complex, solved by Hohenester's group, must wait until the paper is published, but it is possible to say that it binds the hydrophobic sequence GVMGFO (which is a rare sequence in collagens, although one often involved in protein-protein interactions) into a hydrophobic pocket on the PARC molecule. [Note that that "O" is not a mistake; it is the single letter amino acid code for hydroxyproline.]
This London Structural Biology Club meeting was sponsored by Alpha Laboratories Ltd.
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