Hello from a recent graduate of the Structural Molecular Biology MSc

Hello everyone.  My name is Jill Faircloth and, as the title suggests, I graduated from the MSc last year.  My plan is to become a freelance science writer and Clare has very kindly allowed me a voice on this blog.  I will be blogging from the Birkbeck Science week next week but first I thought I’d introduce myself.

I read Chemistry as an undergraduate but soon discovered that aspirations to a glorious research career were incompatible with my lab skills.  I therefore put on a suit and became a chartered accountant before moving to ICI and working my way up the finance ladder.  That’s where I was when I had my first child and I would have stayed but an opportunity arose in my husband’s career and we left for San Francisco for a couple of years.  This and the subsequent baby gave me (plenty) of time to realise that I couldn’t go back to the spreadsheets and I decided to follow my interest in structural molecular biology.

This took me to where you are now and, since it is March, and assuming the timetable hasn’t changed too much, you have just navigated symmetry and are now battling through the fascinating but rather dense protein lifecycle.  Since I know that it can be a little overwhelming when you’re in the middle of one of the lengthier sections, I thought I’d give you a quick heads up of what’s in store.  Section 9 is on molecular forces and has quite a different flavour from the rest of PPS.  There’s more physics to contend with but by the end of it the logic behind protein interactions is much clearer.  After that you’re into the home straight and what I found to be the most enjoyable part of the course.  The last 3 sections deal with how the function of a protein is dictated by its detailed molecular structure and by the interactions which are promoted or inhibited by that structure.  For me, this is where I could really get a sense of quite how remarkable the evolution of proteins is.  Various protein types are examined and used as examples of the elegance of mechanisms in which subtle molecular changes can trigger impressive macro consequences.  This is what the rest of the course has been building up to.

After that you will be out of the comparative comfort of working through the sections, with some of you performing the delicate balancing act of simultaneous revision and project work.  This is where I can offer you a few top tips from the previous class.  You should definitely get hold of at least 5 years of past papers.  Even if you don’t have time to work through them all, as you go through you will see that certain topics have a higher probability of featuring than others.  Also, don’t leave all of your work on the project until after the exam, particularly if you are new to HTML.  HTML takes a little time to crack, although a speedy way to do it is to look at the text of previous years’ projects and copy the bits for inserting titles, tables, pictures and links.  Another reason for getting some foundation research in early is that sometimes a key review paper may not be available online and, although the Birkbeck library is extremely helpful, there is a time lag in retrieving hard copies which you may not be able to accommodate in the time between the exam and the project submission.  Please don’t let these comments put you off.  Producing the project is hard work but is extremely satisfying as you get the scope to really engage with a subject in a way that there isn’t often time for during the coursework.  Speaking personally, it was the experience which inspired me to try my hand at science writing, hopefully the beginning of a shiny new career.

The other subject which may be on your minds is the choice of course for the second half of your MSc.  I chose TSMB on the basis that we were told by Prof Nick Keep that it was the best basis for a PhD and at the time I was labouring under the fond delusion that I would be able to attract sponsorship to do a part time PhD.  I have asked an old PPS colleague who chose PX for his opinions so that I could give you a brief student’s view of the choices on offer. 

TSMB is Techniques in Structural Molecular Biology.  It covers a myriad of techniques that are employed to decipher the molecular structure of proteins from the DNA technology employed to code the protein of interest, through the wet lab skills used to identify, isolate and prepare a sample to the physics of different methods of structure solution.  The sections consequently vary greatly in density and difficulty but the course has been thoughtfully structured so that the more demanding sections are interspersed with the lighter ones.  I found the course very interesting and indispensible for understanding structural biology literature.  I can definitely see why Nick said that it would be the best basis for a PhD although I should say that the consensus among the students that I used to chat with regularly was that it was more challenging than PPS.

PX is Protein Crystallography, which is where a crystal of a protein is obtained and x-rays are directed through it to produce a diffraction pattern.  This pattern can be interpreted to give the electron density of each non-hydrogen atom in a molecule and hence its structure.  This is a technique in which Birkbeck has an extremely strong reputation and consequently is a tempting course for anyone wishing to specialise.  My inside source reported that there was a good variety between sections and that on balance he found it slightly less demanding than PPS although I would have to say that his perception probably speaks to his non-biological background.  There is also the opportunity in this module to handle real data and solve a structure as a project. 

Being that I am over my 1000 word limit, I had better sign off.  I hope that one or two of these observations may be helpful and wish you all the best of luck with the rest of the course.

Beyond the Central Dogma

Biological information may be represented in different ways. The famous central dogma of molecular biology, as described by Francis Crick and others in the 1970s, states that "DNA makes RNA makes protein" (see this page in PPS section 6: Bioinformatics).. Nowadays, some exceptions to this simple statement of the single-direction flow of information are known, such as the synthesis of DNA molecules from a RNA template by the retroviral reverse transcriptases or the synthesis of telomeres by telomerase. However, it is very likely that early biology used a simpler molecular architecture. The "RNA World" hypothesis states that early life was based on RNA alone - the only molecule that is capable of both storing information and catalysing chemical reactions - and was only later superseded by  the DNA-RNA-protein world of today.

Such an RNA world would have relied on an ability of RNA molecules, or "ribozymes" to synthesise other RNAs following a template: another way of saying that in this world, each of the components of the "central dogma" would have been represented by a type of RNA. Phillip Holliger, a group leader in the Protein and Nucleic Acid Chemistry division of the MRC Laboratory of Molecular Biology, Cambridge, gave an interesting seminar to the Institute of Structural and Molecular Biology at UCL in early February in which he described his recent work exploring how the chemistry of this "RNA World" might have worked.

Holliger began by introducing the concept of a RNA replicase: an RNA sequence that can catalyse the extension of a RNA primer. No natural RNA replicases are known: if there was once such a primordial molecule, it has been lost in time. In order to understand the RNA replicase, therefore, it is first necessary to synthesise one.  In 2001, David Bartel and his group at MIT in Boston, Massachusetts, published a paper (Johnston et al. (2001), Science 292, 1319-25) describing a synthetic ribozyme that was able to catalyse the addition of over a complete turn of a RNA primer strand on a template sequence to a "reasonable" level of accuracy.  Holliger's group is one of several that have, since then, been making improvements to the basic replicase.

The work in the Holliger group has involved developing a technique known as compartmentalized bead-tagging (CBT)  for directing the "evolution" of a synthetic RNA polymerase. Put very simply, this involves using water-in-oil emulsions to select and isolate ribozymes from a library that had specific RNA primer extension properties. Observing that their original ribozymes had poor ribozyme-template-primer interactions, they generated a library of ribozymes with additional random 5' domains. Three rounds of CBT were needed to isolate a ribozyme named C19 that had improved RNA polymerase activity. Secondary structure prediction suggested that the new 5' domain of this ribozyme consisted of a short sequence complementary to the 5' end of the RNA template used in the experiments, followed by a hairpin domain. This sequence complementarity promotes the formation of a stable ternary complex between the ribozyme and the template and primer RNA strands thereby allowing processive synthesis of long RNA molecules. Further directed mutations have yielded a ribozyme (tC19Z) that can catalyse the synthesis of a RNA that is itself catalytic: a mini-version of a hammerhead ribozyme (link to PDB structure 1MME).. Holliger's group may still be quite a long way from generating a truly self-replicating molecule (which would have been necessary in a primordial RNA world) but they are making progress towards this goal.

This work can essentially be seen as "restricting" or "shrinking" the central dogma to one type of  information-containing macromolecule from three. Holliger and his group are now using similar techniques to try to "expand" the dogma by adding an extra branch, developing polymerases that can synthesise and reverse transcribe artificial poly-nucleotides based on unnatural building blocks not found in nature.  


Holliger's work on the ribozymes was published last year in the journal Science: Wochner et al. (2011), Science 332, 209-212. Click here to access this paper (login to Birkbeck e-library required).


.

BCA Winter Meeting: Structures of Supramolecular Assemblies

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

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

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

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

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

Nature cover illustrating the structure of respiratory complex I

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

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

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

Structural Secrets of an Ancient Viral Plague

Research in Biological Sciences at Birkbeck, and several related departments at neighbouring University College London, is combined into the Institute of Structural Molecular Biology. The Institute holds a regular seminar programme - every Wednesday lunchtime during termtime - in which it invites excellent scientists, many with links to the colleges, to present their research. A few weeks ago, the seminar speaker was an electron microscopist, Sarah Butcher, who is based at the University of Helsinki in Finland. Her group has been investigating the structure of a virus that causes a very well-known disease: measles.

Measles has been known of for millennia. The disease (although of course not its cause) was first described in ancient Egypt. It is one of the most infectious viruses known, but people who encounter measles (if at all) as an unpleasant childhood affliction are often surprised to learn that it is a killer. About 164,000 people lost their lives as a result of measles infection in 2008, most from lingering immunosuppression rather than the acute infection. Most deaths occur in Africa and south Asia; a smaller epidemics have recently arisen in the UK when the MMR vaccination lost popularity over the MMR autism scare.

The measles virus is a paramyxovirus; an enveloped virus with a single strand of RNA as its genome, and closely related to the viruses that cause mumps, respiratory syncytial virus (RSV) infection and para-influenza in infants and children. It has two surface proteins and iis thought to attach directly to the membranes of the cells it infects via one of these.

Until recently, structural studies of the measles virus have been fairly limited. Many groups have studied it using an electron microscopy technique called negative staining, but that can only see the virus' surface. Structures of one intact measles virus protein and domains of three others have been deposited in the Protein Data Bank; the haemagglutinin (e.g. PDB code 2RKC); two separate domains of the phosphoprotein (1OKS and 2K9D) and a structure of fragments of two proteins simply called P and N bound together (1T60).

Sarah Butcher and her group used a technique called cryo-electron microscopy, which allows the interior of viruses to be visualised, to study the measles virus. Their results led them to focus on the matrix protein, which is thought to be important for the assemby of the virus (the protein coloured cyan in the images below). All previous models had placed the matrix protein covering the inner part of the viral membrane. What the Butcher group saw, however, was completely different. They could see a protein surrounding parts of nucleocapsid - the viral RNA and its associated, protective protein - and further analysis identified this as the matrix protein. The matrix binds tightly to parts of the nucleocapsid to make rod-like structures, and these fold into anti-parallel units that are somewhat remniscent of antiparallel beta sheets in proteins. This model suggests that the process of virus replication will be more complex and yield more potential drug targets than has previously been thought.

Two models for the organisation of proteins and RNA in the measles virus. Top: the old model, with the matrix protein (cyan) surrounding the virus coat. Bottom: the Butcher group model, with the matrix protein surrounding parts of the nucleocapsid. Figure credit: Proc. Nat. Acad. Sci. USA (2011)

Structures of proteins from other viruses, particularly HIV and influenza, will be covered quite extensively later in the PPS course. We don't study the technique used in this study, cryo-electron microscopy, in PPS but it is covered in one of the options for the second year of the PPS course, Techniques in Structural Molecular Biolog

Welcome to new students!

A warm welcome to the Principles of Protein Structure blog to all students who have just started studying Birkbeck's Principles of Protein Structure course!

I run this blog to link the material that you will be studying in the course to new research developments in the areas of protein structure and function and related aspects of biotechnology and medicine. Throughout the taught course (but more often in the later part of the course) I will post reports of recent developments. I might, example, report on talks given in the ISMB seminar series run jointly by the Department of Biological Sciences at Birkbeck and research departments in neighbouring University College London. The overall title of the programme for Autumn 2011 is "“Proteins of the Future: Mechanism, Evolution and Design” which is closely connected to the content of the PPS course. Other posts may be reports from conferences or summaries of recently published papers in protein structure, protein bioinformatics and allied areas.

Do, if you get a chance, look through some of the earlier posts on the blog to see the kind of topics that we will be discussing. However, don't be discouraged if at this stage of the course you find the science presented there difficult to understand. I can assure you that it will get easier!

And the best of luck for the 2011-12 PPS course and for your studies at Birkbeck! We hope that many of you will go on to complete our MSc in Structural Molecular Biology.

Best wishes,

Dr Clare Sansom

Senior Associate Lecturer, Biological Sciences, Birkbeck and Tutor, Principles of Protein Structure

Waking Up to Structural Biology

Professor Nicholas Keep (known to generations of Birkbeck students as Nick) was appointed to a chair in biomolecular structure in 2009. It was June 2011, however, before he gave his inaugural lecture at the college. In this lecture he gave an overview of the techniques he uses as an experimental structural biologist and some of the discoveries he has made through them.

Nick’s career so far has been a glittering one. He took both his degrees at Cambridge and did postdoctoral work at UCL and back in Cambridge at the prestigious Laboratory of Molecular Biology (LMB) before being appointed as a lecturer at Birkbeck in the mid-90s. Since then he has risen steadily through the ranks and is now not only Professor but Executive Dean, with academic and financial oversight of the whole Faculty of Science. It is perhaps not surprising that it took him two years to fit in his inaugural lecture.

He began his lecture with a whistle-stop tour of the history of structural biology, beginning at Birkbeck and with the first Professor of Crystallography here, J.D. Bernal (see June 2008 post). Before he even arrived in London, however, Bernal had published (with Dorothy Hodgkin) the first ever diffraction pattern to be obtained from a protein crystal. Very much later, Nick as an undergraduate Cambridge student was inspired by two Nobel laureates, Max Perutz (who published the first-ever three-dimensional protein structure, that of myoglobin, in 1958) and Tim Hunt, to specialise in this aspect of molecular science. His PhD research was on the structure of methylmalonyl-coA mutase , an enzyme that binds to vitamin B12. This provides another link back to Dorothy Hodgkin, whose Nobel Prize in 1964 was awarded partly for determining the structure of this important vitamin.

He then spent a few minutes going through the essential principles of protein crystallography. This involves purifying and crystallising a protein and then exposing the crystals to a parallel beam of X-rays. These X-rays are deflected from the atoms in the crystal in a regular way to produce a diffraction pattern, and this can be interpreted to give first a map showing the density of electrons in the molecule, and then a model of the positions in space of all atoms in the molecule (missing out, in most cases, the lightest atom, hydrogen). In an ideal case, this whole process can now take a month or so, but this is rare: many structures still take years to solve. Birkbeck now has excellent facilities for purifying proteins and growing crystals. We can collect the X-ray data on site but often go to more powerful machines – synchrotrons – located in the UK and beyond. Britain’s first synchrotron is still in use, but in Jordan; it was replaced about five years ago by a state-of-the-art facility, Diamond , near Harwell in Oxfordshire. Birkbeck’s scientists also use synchrotrons elsewhere in Europe; Nick’s favourite is in Grenoble.

Nick then went on to describe some of the research projects that he has led or contributed to at Birkbeck. Working with Lin Field and Jing-Jiang Zhou at Rothamsted Research in Hertfordshire, he solved the structure of an insect protein that binds odorant molecules, which has led to some useful insights into the mechanism behind insects’ extremely sensitive sense of smell. Much of his work, however, is and has been in proteins that are involved in one way or another with human disease. Duchenne Muscular Dystrophy is a progressive, muscle-wasting genetic disorder that almost always affects boys and that is caused by mutations in a large muscle protein called dystrophin. Nick and his colleagues have solved the structures of a part of this protein that binds to another muscle protein, actin, and of related proteins.

It was only when he reached some of his final examples that the pun in his title, “Waking up to Structural Biology”, became clear. The bacterium Mycobacterium tuberculosis (see also April 2011 post ) infects the lungs of about a third of the world’s population. In most people, however, it remains in a wholly benign, dormant condition. In about 5-10% of cases, however, the dormant bacteria will “wake up” when an infected individual is under stress (for example, by exposure to another infection) and overt tuberculosis (TB) develops. Bacteria in the dormant stage are untreatable by any current TB drugs. Nick and his group first studied the structure and function of a protein known as resuscitation promoting factor that is involved in this “waking up” process,. He observed similarities between its sequence and that of a very well-known protein, lysozyme , which breaks down bacterial cell walls, and later, when the structure was solved, it was seen to have a lysozyme-like fold. This led to his identification of a glutamate residue as key to this enzyme’s activity. He is now looking at other proteins involved in M. tuberculosis resuscitation including a small heat shock protein (a protein that helps keep other protein structures stable under stresses such as raised temperatures), Acr1. This is the most abundant protein in dormant TB. He ended with a glimpse of a new unpublished TB protein structure.

Nick concluded a fascinating lecture by thanking his lengthy list of co-authors and particularly his research group, stressing the collaborative nature of science, and Gabriel Waksman, head of the Department of Biological Sciences, closed the proceedings by praising his achievements in teaching and administration as well as research.

Both second-year options in the MSc Structural Molecular Biology programme are concerned with the techniques used to study the structures of biomolecules. Techniques in Structural Molecular Biology (TSMB) is a general course covering crystallography, NMR, electron microscopy and some of the molecular biology and bioinformatics techniques associated with them, whereas Protein Crystallography (PX) is, as its name implies, a more specialist course.

The Structural Biology of Pilus Biosynthesis: Or, How Bacteria Man the Pumps

Science Week at Birkbeck College was celebrated for the second time in early May 2011 with presentations from each of the college’s three science departments. The two lectures from researchers in Biological Sciences were linked by the common theme of nano-machines in biology. Just as a car engine, for example, is built up from many interacting parts, so some proteins work together in large complexes to do particular jobs within cells. Professor Helen Saibil, whose ground-breaking work in electron microscopy has featured earlier in this blog (see November 2010 post) presented some of her research into chaperones, protein machines that carry out “quality control” work enabling other proteins to form into and stay in the precise three-dimensional shapes they need to function.

The other speaker from Biological Sciences was Professor Gabriel Waksman, a distinguished structural biologist who combines a very successful research career with running both Birkbeck’s Biological Sciences department and the Research Department of Structural and Molecular Biology at UCL. Professor Waksman’s work for many years has focused on the complex structures through which bacteria interact with the outside world. Pathogenic bacteria cause problems for their hosts only when they interact with them, by secreting toxic substances into their environment or attaching to host cells. Now, when bacteria are rapidly developing resistance to many traditional antibiotics and more antibiotics, particularly with novel mechanisms of action, are desperately needed, some of these mechanisms are at last becoming understood.

Gram negative bacteria, which have double cell walls, often carry hair-like fibres or filaments known as pili on their surfaces. Some bacteria use these to bind to receptors on the surfaces of host cells, a process that can trigger the host cell surrounding and engulfing the bacteria in infection. Different forms of bacteria even from the same species carry different pili that bind to different cell receptors: for example, some E. coli bind to and infect bladder cells causing cystitis, while others infect kidney cells causing pyelonephritis. E. coli can also use pili to bind to each other, forming colonies around the bladder that are particularly difficult to treat.

The type of E. coli that infects the kidney carries a large number of so-called P pili on its outer membrane. These consist of a relatively thick rod near the cell wall and a thin filamentous tip. The whole pilus is made up of thousands of similar protein subunits encoded by genes within the Pap gene cluster. Almost all are identical PapA subunits, and these form the rod: the tip consists of just a few homologous PapE subunits, tipped by the sensor, PapG, which recognises and binds to kidney cells.

Pilus subunits, like all proteins, are synthesised in the cytoplasm; they need to be transported to the outer membrane and polymerise to form the pilus structure, and this complex task is achieved by other Pap proteins. When a pilus subunit is synthesised it is first translocated across the inner membrane into the periplasm, where it will be immediately degraded unless it can bind to a PapD protein. This acts as a chaperone, moving the subunit to the outer membrane where it docks with the membrane-bound PapC. This latter, or “usher” protein, is the core of the pilus biosynthesis molecular machine, and PapC and PapD together give the process its name: the chaperone-usher pathway. PapC has a large central pore through which the intact pilus is secreted.

Over many years, Gabriel Waksman and his group have solved the structure of many of these Pap proteins, and they have now built up an accurate picture at atomic resolution of how the chaperone-usher pathway works. The first structure to be solved was that of a binary complex of one pilus subunit, PapK, bound to the chaperone PapD (PDB 1PDK). PapK – and, subsequently, each of the pilus subunits – was found to have an immunoglobulin type fold, but with one beta-strand missing. This structure can only be stabilised when another protein, either the chaperone or a second pilus subunit, completes this fold with a strand of its own. One by one, starting with the tip subunit PapG, chaperone-subunit complexes migrate to the PapC usher, where the chaperone strand is replaced by a strand from another subunit in what has been termed a “donor strand exchange” model of polymerisation. The pilus fibre therefore forms from a series of “typical” immuno-globulin-like subunits in which each subunit is completed with a single strand from the next nearest subunit. The pilus biogenesis process only stops when a PapH, or “terminator” subunit is incorporated.

The usher forms a wide pore in the outer membrane (PDB 2VQI) and acts both to synthesise and to secrete the pilus polymer. It is a long, multi-domain protein. Using X-ray crystallography, Waksman’s group first determined that the pore comprises a very large beta barrel derived from the central domain of the usher, with a small sub-domain embedded within this domain forming a “plug” that blocks the pore when it is not being used. The usher also has a short N-terminal domain that dangles down into the periplasm and grabs on to chaperone-subunit complexes. The function of the C-terminal domain, however, remained unknown until the group solved the structure of an intact usher-chaperone-subunit complex.

It was only when this intact structure was solved that Waksman’s group really began to understand the mechanism of this complex “molecular machine”. For this, the group used a homologous system in which the usher is a protein known as FimD, the chaperone is FimC and the bound subunit FimH. This structure was a “first” in several ways, not least because it was the first time that an intact, folded protein was observed inside the pore of another protein structure. In this structure, the C-terminal domain of the FimD usher was seen to bind to the chaperone-subunit complex. It appears that, once the N-terminal usher domain has grabbed on to a chaperone-subunit complex and moved it into the usher, that complex will move up the usher structure to the binding site on the C-terminal domain, freeing the N-terminal domain to capture the next subunit.

This work, the culmination of fifteen years’ study of this secretion system, has just appeared in Nature (published online ahead of print 1 June 2011). More importantly, however, this elegant piece of structural biology may be exploited in the war against bacterial infection. A drug that bound to the usher and prevented pilus biosynthesis – a “pilicide” – would not kill the bacteria, but it would prevent them from binding to their target cells and also from forming the antibiotic-resistant colonies that can remain in the urinary tract for years and that lead to persistent infection.