J.D. Bernal and Crystallography's Beginnings

Last week at Birkbeck we held the annual lecture celebrating the life and work of J.D. Bernal, the founder and first head of the School of Crystallography at Birkbeck. Bernal - "Sage" as many of his contemporaries knew him - was one of the most influential figures in the early development of structural biology. In 1934, while at Cambridge University, he and his student Dorothy Crowfoot obtained the first X-ray diffraction pattern from a protein. That protein was pepsin; its structure was only solved many decades later. (The link is to a structure of porcine pepsin by a group led by another head of Birkbeck Crystallography, Professor Tom Blundell.)

Dorothy Crowfoot - as Dorothy Hodgkin - went on to win the 1964 Nobel Prize for Chemistry, for her work on the structures of vitamin B12 and penicillin. She also solved the structure of insulin. Bernal's mind was no less brilliant than his student's, but the ultimate prize still eluded him. Most commentators agree that he is one of the greatest scientists never to win the Nobel, although several of those he worked alongside and inspired, including Francis Crick, Aaron Klug and Max Perutz, did so.

It is possible that Bernal simply never allowed himself to stay focused on any one area for long enough to win the Nobel. This was essentially the view of Sir Lawrence Bragg, himself a Nobel Laureate (Physics, 1915): "if one traces back almost any fruitful line of crystallographic work, it will be found that Bernal assisted at its conception but left the child to be brought up by foster-parents. This is particularly so in the case of molecular biology and in the analysis of protein crystals. Immediately on seeing the first x-ray differentiation pictures from protein crystals...he assumed that protein structures would sooner or later be solved, and handed out problems to his students and to anyone whom he could persuade to take them up."

Bernal's politics may also have disenchanted him from the Establishment. He was a life-long and well-known Communist (in an era when this was far more common among intellectuals than it is today) and an internationalist, and visited the Soviet Union many times. The painter Picasso was among the giants of the European Left who knew him well; one meeting between the two, after which Picasso left behind a mural on the wall of Bernal's flat in the college, has even been made into a play. (The Wellcome Trust now own the mural, and have recently installed it in their London headquarters.) He was also a pacifist, and chaired the World Peace Council from 1959-1965.

Bernal's contributions to scientific thought go far beyond the practice of what is known as "hard science". He gave much attention to the interaction between science and society, and how science should be managed and funded. One of his many books, The Social Function of Science, published in 1939, is considered the earliest text on the sociology of science. And this year's Bernal Lecture focused on an earlier book still: The World, the Flesh and the Devil (1929): subtitled An Enquiry into the Future of the Three Enemies of the Rational Soul .

There is not enough space in a blog post to do justice to this fascinating man. Those of you who would like to know more about him are referred to a biographical memoir written in 1980 by Dorothy Hodgkin and available online.

Gabriel Waksman on bacterial secretion systems

Gabriel Waksman, head of the School of Crystallography at Birkbeck, has given a seminar in Birkbeck's School of Biological and Chemical Sciences describing his group's work bacterial secretion systems. Gabriel studied at the University of Paris and then spent the first part of his academic career in Washington University in St. Louis, Missouri, before joining Birkbeck in 2003. He now combines his position here with a chair in Biochemistry at University College London and directs the new Institute of Structural Molecular Biology which links the research in the two colleges.

Gabriel's research group works mainly on elucidating the structure and function of bacterial secretion systems, which produce hair-like appendages, or pili, on the surfaces of Gram negative pathogens. These are very important for infection as they allow bacteria to recognise, and then attach to, host cells. Mutant bacteria in which these proteins are not expressed are not pathogenic.

Two of the best understood of these systems are found in variants of E. coli which infect the human urinary tract. The P pilus recognises and binds to kidney cells, causing the kidney infection pyelonephritis, and the type I pilus recognises bladder epithelial cells and causes urinary infections. Both pili can be found on the same bacterium. The structures of both pili are similar, consisting of many protein subunits; it is the tip subunit that recognises its target cell types by binding to different cell surface sugar residues.

All subunits associated with a particular pilus are encoded by genes within a gene cluster and named accordingly: the P pilus genes are Pap genes and the type I genes Fim genes. Structures of at least one representative of each type of subunit have now been solved, many by Gabriel's group and its collaborators. Knowing these structures has enabled the group to understand the mechanism through which the pili are formed.

The pilus subunits that polymerise to form the main part of the fibrous structure all have similar structures. They are immunoglobulin-like, mainly-beta structures in which one sheet is lacking a central beta strand, and they are therefore unstable independently unless they are bound to a chaperone protein. A strand from the chaperone fits into the gap, forming regular hydrogen bonds with the neighbouring strands, and this stabilises the chaperone-subunit complex. This is then transported to the growing pilus, where the N terminal peptide from a subunit already in the structure replaces the chaperone strand in the new subunit, adding it to the polymer via a mechanism called "donor strand exchange". The resulting fibre therefore consists of a string of similar subunits, with the N terminal peptide of one subunit forming a strand in the central beta sheet of the previous subunit in the assembly.

The Waksman group's most recent structural studies concern the protein through which the pilus is assembled, known as the usher. This is a mainly beta membrane protein (link is to material in PPS section 11, which will be released next week) which is embedded in the E. coli outer membrane. The structure of the E. coli P pilus usher, solved by X-ray crystallography, shows the beta barrel and a middle or plug domain which interrupts the main beta sheet of the barrel. With 24 strands, it is the largest outer membrane beta barrel protein structure to be elucidated so far. In its inactive form, the plug domain fits inside the barrel, completely blocking it. They also used cryo-electron microscopy to isolate the structure of a type I pilus complex during pilus assembly. The usher forms a dimer within the cell membrane but, interestingly, the EM studies show that a pilus is secreted through only one monomer of the dimer.

This is very complex work which can only be touched on in a blog post. If you would like to know more, have a look at a few of these papers (links to abstracts in PubMed):

• Sauer et al. (1999), Science 285, 1058-61
• Sauer et al. (2002), Cell 111, 549-51
• Remaut et al. (2006) Molecular Cell 22, 831-42
• Remaut et al. (2008), Cell 133, 1-13 (to be published May 2008)

Predicting RNA binding from protein sequences

The first Birkbeck seminar of the new term was given by Sue Jones, from the University of Sussex. Sue is no stranger to Birkbeck as she did her Ph.D. with Janet Thornton at University College, and later worked with her at the European Bioinformatics Institute and the biotech company Inpharmatica. Today she described a piece of software that she and her colleagues have developed for predicting motifs in protein sequences that are likely to bind to RNA.

Proteins function largely by interacting with other molecules - they are "social" molecules. Protein interaction partners include other proteins, carbohydrates, "small" molecules and ions, and the focus of today's talk: nucleic acids. The structures and functions of RNA molecules are diverse and include protein coding (mRNA), protein synthesis (tRNA and ribosomal RNA), splicing, hydrolysis of nucleic acid bonds (in RNA enzymes or "ribozymes") and control of gene expression (the so-called "micro-RNAs or miRNAs). RNA-binding domains in proteins include RNP domains, dSRNA binding domains, and K homology (KH) domains - all these are mixed (alpha and beta) structures.

Jones and her colleagues surveyed known structures of protein-RNA complexes and marked residues that were in close contact (through van der Waals or hydrogen bonding) with the RNA. They described each amino acid in terms of predicted accessible surface area, conservation within the family of homologous proteins, and chemical properties. Not surprisingly, positively charged and polar amino acids were favoured in binding to the negatively charged nucleic acid over negatively charged and hydrophobic ones; glycine, which is flexible, and tryptophan, which can form base stacking interactions were also favoured.

Jones then built these features, averaged over a "window" of 5-25 amino acids, into a support vector machine to predict RNA binding features in proteins of unknown function. (This technique is a form of "machine learning"; you don't need to know about it for this course, but if you're interested in knowing more and can cope with maths at a relatively high level, see the Wikipedia entry.) This was found to be at least as reliable as any similar tools that are publicly available.

There will be more about protein-nucleic acid binding in the next section of course material, Protein Interactions and Function, which is due to be released at the end of April.

British Crystallographic Association Spring Meeting

I spent Tuesday - Thursday of last week at the British Crystallographic Association Spring Meeting. The meeting has 4 strands Biological, Chemical, Industrial and Physical Crystallography.
Each contribute a plenary and then have their own separate sessions. The Biological Plenary was the Bragg Lecture where one famous crystallographer speaks about another usually older crystallographer and their work. This year Tony Crowther from the MRC Cambridge talked about his work and that of Michael Rossmann from Purdue University. Both made seminal contributions to the method of molecular replacement in protein crystallography. More on that in TSMB. Michael Rossmann when a postdoc for Max Perutz at the LMB in Cambridge was the first person to realise that the chains of hemoglobin looked like the chain of myoglobin and hence that you could solve structures of related proteins by molecular replacement. Michael then developed the mathematics and early software for molecular replacement. Tony Crowther did a Ph.D. with David Blow at the LMB and developed an improved form of the translation function. While working on natural language processing inEdinburgh, Tony also realised how to give a much faster and more acccurate version of the rotation function, which was the basis of the molecular replacement method for a long time. Tony's career was actually mainly in electron microscopy, he returned to the LMB from Edinburgh to work for Aaron Klug and became a group leader in his own right. Both he and Michael Rossmann have done most of their work on viruses and he talked about Michael's work on bacteriophage and his work on Hepatitis.
The Biological Group sessions were on Membrane proteins. Chris Tate from LMB in Cambridge described work they have been doing to stabilise membrane proteins by mutation. They search for alanine mutations that increase the stability of the protein in detergent and then carry out mutations in combination until the protein is stable for half an hour at a temperature 15-20 degrees hotter than the original. They have succeeded in crystallising beta-1 androgenic receptor, which will give important comparison to the beta-2 published just before Christmas.
The most interesting talk for me was from Thomas Sorenson, now at Diamond, on the work he and colleagues had done in the group of Poul Nissen in Aarhus. The group have published structures in several states of eukaryotic ATPase transproters (Calcium, sodium, proton). Interestingly these proteins were discovered by a Dane, Jens Skou http://www.pumpkin.au.dk/en and the group used proteins provided by various groups in Denmark that have worked on the systems for many years. They used natural sources and did not purify the proteins down columns, but just used differential extraction. This means that they isolated the membrane fraction that contained most of the protein and then extracted with detergents and this material was pure enough to crystallise in the presence of the right combination of detergents and lipids. The other biological sessions were on neutron diffraction, probing fast biological reactions, complementary methods, and ligand binding and drug design. Neutron diffraction gives the position of hydrogen atoms as both hydrogen and even more so deuterium diffract neutrons much more relatively than they do X-rays and you get density for hydrogen atoms. The catch is that neutron fluxes are much weaker and you need crystals that are 0.1- 1 mm3 compared to 0.0001 mm3 for a protein crystal. Studying reactions in crystals often means trapping intermediates by freezing out. Arwen Pearson from Leeds gave a good talk about a redox system that she had worked on in Minnesota where the reaction cycle can be carried out in the crystal, even changing space group between states. The catch, and this is common, is that X-rays themselves generate free radicals which can reduce redox centres so by collecting the data the redox state is altered. This meant that they had to collect data from several crystals before they became too damaged. The highlight for me of the Ligand and Drug session was a talk from Chris Phillips at Pfizer about their new non-nucleoside HIV Reverse Transcriptase inhibitor. These target a hydrophobic pocket in the protein, and tend to be rather 'greasy'. The Pfizer group had carefully designed a ligand that was both smaller and more hydrophilic and hence a better drug in terms of bioavailability.
There were many more great talks, but I hope this gives you a flavour of the meeting
Nick

Greetings from Poznan

This is just by way of an apology for my relative silence on PPS blogs and forums lately.

I am half way through two weeks' teaching at Adam Mickiewicz University, Poznan, Poland, funded by a grant to Birkbeck through the EU Erasmus programme (formerly known as Socrates) which funds student and lecturer exchanges between EU countries (and some others). I am teaching a two-week course on bioinformatics mostly, this year, to postgraduate Physics students but I have also taught in other departments.

I have known my host here, Professor Mariusz Jaskolski, since we were both working in the same lab in the States, NCI Frederick, in the early 90's. Mariusz was involved in some of the early work on the structure of HIV protease which is covered extensively in the PPS course. Since then he has gone on to found the first X-ray crystallography group in central-eastern Europe and to solve the structures of viral integrases, asparaginases and others... and many of his students, and others at AMU, have taken PPS and/or other distance learning courses from Birkbeck.

Normal service will be resumed next Monday.

Diamond Beamlines

Today's seminar was by Dr Liz Duke from the Diamond Synchrotron. Liz was the first scientist employed to develop the protein crystallography beam lines over 5 years ago and now the first 3 beamlines are taking users. She outlined the design and some of the highlights (the first time she went into the experimental hall, the first beam and the first diffraction) and the ongoing issues that they want to improve. For example the sample robot takes 4 1/2 minutes to mount a new sample and be ready to collect data, which people find slow (although in tests the quickest a person could do it was 6 1/2 minutes). By really understanding the steps where corners can be cut they hope to take another minute or so off the time. They are now finding the balance between giving users access and finding enough time for themselves to implement the improvements that people want to see. However Diamond like all large projects is under some threat from the STFC (Science and Technology Facilities Council) review of its spending commitments. They do not have enough money in the next Comprehensive Spending Review to continue all the projects currently being funded and have launched a consultation http://www.scitech.ac.uk/STFCConsultation/comment.aspx?ci=1 which closes at the end of this week. Although Diamond is a High Priority there may not be as much money for the ongoing running and development costs as are needed.

Regulation of the EGF receptor

The epidermal growth factor (EGF) receptor is (as its name implies!) a receptor that sits at the surface of cell membranes. Like many other such receptors, its intracellular region contains a tyrosine kinase; the extracellular part binds to a small, soluble protein, epidermal growth factor (EGF). When EGF binds it stimulates a conformational change that leads to a dimerisation of two EGFR molecules. This activates the tyrosine kinases so they pass a signal - essentially saying "EGF has bound here" - through the cell in a cascade of phosphorylation reactions.

EGFR is one member of a family of four similar receptors, known as the ErbB family; it can also be known as ErbB1. You may have heard, indirectly, of another member of this family, ErbB2; this is over-expressed on the surface of breast cancer cells in about a quarter of breast cancers. It is the target of the drug herceptin, which has transformed the lives for many women with so-called "herceptin receptor positive" breast cancer.

This week, Professor Mark Lemmon from the University of Pennsylvania gave a seminar at University College, London, about the structural basis for the regulation of the EGF receptor. This was one of the regular seminars organised through the Institute of Structural Molecular Biology, which brings together researchers at Birkbeck and UCL working in structural biology, chemical biology, biophysics, proteomics and bioinformatics.

Lemmon's research is concerned with the structure of the extracellular, EGF-binding regions of these receptors. These are made up of four domains - two "L-domains" and two cysteine-rich domains, arranged in the order L-C-L-C starting from the N terminus of the protein. EGF binds between the two L-domains, and the two C-domains form the interface between the monomers in the dimer. In the absence of ligand, the extracellular region adopts a "tethered" conformation in which the dimerisation domain is occluded. However, this inactive, auto-inhibited conformation can also exist in the presence of EGF; Lemmon and colleagues solved the structure of the entire extracellular region, with ligand bound and in an inactive conformation (PDB file 1nql).

Lemmon and his colleagues have now studied the transition between the inactive, tethered state and the active, extended one using the technique of small-angle X-ray scattering (SAXS), which is used for observing large conformational changes in molecules. They found, importantly, that introducing mutations into the "tether" region of the protein cannot drive the transition to the active conformation. Rather, in this protein (but not in the apparently ligand-less herceptin receptor) it is only EGF binding that can cause the transition to the active form.

A useful (if a few years old) review of tyrosine kinase structure and function is Hubbard & Till (2000), Annu. Rev. Biochem. 69, 373-398. This is accessible from the Birkbeck e-library with your username and password.