Crystallins under the Lens

Written by Jill Faircloth

For generations, anyone who argued against evolutionary theory would point to the human eye and exclaim that nothing so perfectly adapted to its purpose could have evolved in a series of random steps. The well-rehearsed counter argument is that even a very basic recognition of light and shadow via an organic pinhole camera is useful as an aid to survival and that this could provide the first stepping stone towards the sophistication of the vertebrate eye (see references 1 and 2). The theory is supported by a succession of organisms with gradually increasing vision.

On a molecular level, the proof is harder to achieve but Christine Slingsby of Birkbeck's Department of Biological Sciences has used crystallography to do just that. In investigating the structure of the proteins of the vertebrate eye lens, Slingsby has not only greatly increased our understanding of their characteristics and mechanisms but also provided fascinating insights into their evolution.

Professor Slingsby's work is featured in several pages in the PPS course: Greek Key Motif, Beta Sandwiches, Lens Proteins and Cataract and Eukaryotic Genomes. Last year she published a paper (reference 3 here) which summarised the key conclusions of her research during the last ten years. This review is available in the Birkbeck e-library here.

Vertebrate lenses comprise layers of highly elongated fibre cells which give transparency and focus but the refractive power is given by high concentrations of transparent proteins from two superfamilies: the alpha crystallins and the beta-gamma crystallins. These proteins, which are all mainly made up of beta strands, have been co-opted from their original functions to generate a functioning lens.

You don’t need to look far to find the probable origin of α crystallins. They are small heat shock proteins (sHsps), molecular chaperones that are present in most types of cell in most organisms. They are upregulated: that is, produced in greater quantities, by cells under environmental stress as part of the protein homeostasis response.

Despite their name, βγ crystallins are unrelated to α crystallins; all crystallins interact to form a refractive index gradient which can vary as required. Apart from the vertebrate lens where they are very prevalent, and in stark contrast to α crystallins, βγ crystallins are found only in other vertebrate eye tissues (except as a component of a much larger gene/protein known as Aim1) and this makes their origin harder to identify. Beta and gamma crystallins each contain four Greek key motifs organised as two βγ-crystallin domains.

There are several requirements for an eye lens protein. It must be expressed at very high levels, unlike sHsps, so the sHsp gene promoters would have required modification. The proteins must pack tightly and uniformly enough so that there is no irregularity on the scale of the wavelength of light and they must be soluble but must not crystallise or separate into different phases. In addition, lens fibre cells have lost their organelles, which could cause light scattering, and so have no mechanisms for protein repair or disintegration. Accordingly, these proteins need to have a lifespan as long as the vertebrate using them.

One of the main reasons for crystallins having been adopted as lens proteins could be that the two α crystallins are able to dynamically form polymers with highly diverse size and shape. This ability was demonstrated as the first crystal structure of a sHsp revealed a hollow octahedral structure of 24 α crystallin monomers. The next one to be solved showed point group 32 symmetry and was constructed from six dimers arranged in two interlocking rings.

This figure shows the beta-sandwich structure of the alpha-crystallin domain of a monomer, the formation of the dimer with the B6 beta strand exchanging into the partner beta-sandwich, and the oligomer with six dimers forming interconnecting discs. The dimers link using motifs on the C terminal extension which insert into the pocket between the B4 and B8 strands, shown in dark blue, and by interaction of the N terminal helices.

Figure taken from Slingsby, C. et al. (2013. PDB 1GME

In addition to the wide range of alpha-crystallin oligomers, the numerous βγ-crystallin chains can be assembled to create a wide range of polymers which coexist in a polydisperse stable but flexible arrangement of varying density.

Beta-crystallins thus appear to function in a similar way to α-crystallins, forming a diverse range of differently sized hetero-oligomers that adjust the refractive index throughout the lens.

Gamma crystallins are different because they are monomeric and polar. They are present in differing concentrations throughout the lens and their polarity results in distinctive orientations towards other crystallins which may regulate inter-crystallin interactions. There is evidence that disruption of these dipoles results in cataracts.

By examining genomes of organisms which predate the development of the camera eye, Slingsby has shed light on the evolutionary pathway of crystallins as lens proteins. PPS students will bave read about the single-domain βγ-crystallin in the urochordate (invertebrate) sea squirt, Ciona intestinalis, that has exactly the same double Greek key structure as a vertebrate crystallin but includes a calcium binding sequence in each Greek key motif. This ancestral link was further demonstrated by the remarkable discovery that the gene promoter for Ciona-crystallin could successfully target reporter gene expression for proteins associated with vision in vertebrates.

Investigation of the genome of a cephalochordate, which is part of the lineage of both vertebrates and urochordates, revealed a less complex ancestor to βγ-crystallins. Signature sequences from the βγ-crystallins have also been found in bacterial and archaeal proteins. The implication of this is that all of the proteins of the vertebrate lens could well have evolved from proteins present in ancient species with no visual function. An interesting twist is introduced by the knowledge that the nonchordates, or animals without a spine, can use quite similar cellular lenses that involve non-crystallin proteins.

This suggests that lenses evolved independently in different animal kingdoms, relatively late on an evolutionary timescale, utilizing different proteins that were available in the respective phyla, that is proteins which already had an established purpose but which had qualities allowing them to form lenses. Since all species seem to have had access to at least a basic form of βγ-crystallin, it is an impressive demonstration that evolution can not only capitalise on the multiple possibilities presented by one family of proteins to develop a functioning visual system, but also repeat the trick from a different starting point.

References

1. Dawkins, R. (1994). The eye in a twinkling. Nature 368, 690-691
2. Nilsson, D.E., Pelger, S. (1994). A pessimistic estimate of the time required for an eye to evolve. Proc. Biol. Sci. 256(1345): 53-8.
3. Slingsby, C., Wistow, G.J. and Clark, A.R. (2013). Evolution of crystallins for a role in the vertebrate eye lens. Protein Sci. 22(4):367-80.

New protein structures presented at the 2013 BCA Winter Meeting

The work of crystallographers in the UK is supported through the British Cryatallographic Association, which has about 700 members based in academia and industry. It is organised into four groups representing different disciplines within crystallography, including one for structural biologists called, not surprisingly, the Biological Structures Group. Every December, this group organises a one day conference to present some of the most recent developments in structural biology. I have blogged these meetings before, and searching this blog for "winter meeting" will find a few of those posts.

The 2013 meeting was billed as both a "final" event in the centenary year of the Braggs' landmark discoveries and part of the build-up to the International Year of Crystallography, but these were not the only anniversaries highlighted there. 2013 also marked the sixtieth anniversary of the publication of the structure of DNA. The 2013 Winter Meeting was held in King's College London, which played a very important part in that discovery: Maurice Wilkins and Rosalind Franklin, who obtained the X-ray diffraction patterns that led to the discovery of the double helix, were based there. (Wilkins shared the Nobel Prize for this discovery with Watson and Crick; Franklin died in 1958, four years before that prize was awarded.) And the first precise physical model of the double helix is still on display in the college.

Maurice Wilkins' original DNA model

The first researcher to speak at the meeting was Birkbeck's own Professor Bonnie Wallace. Her work on the structures of voltage gated sodium channels has been described on this blog before, most recently in April 2013. These proteins are responsible for the transport of ions in and out of cells, an essential signalling mechanism in all multi-cellular organisms. Their structures, however, are among the most intractable of all membrane proteins (PPS section 11, to be released in May, covers this fast moving field). Wallace has used a combination of X-ray crystallography, spectroscopy and molecular dynamics to explore the structure and mechanism of sodium channels in bacteria. The bacterial sodium channel is simpler than the mammalian equivalent, consisting of a tetramer in which helices from each monomer line the pore. The Wallace group's most recent strucure (PDB 3ZJZ) shows the position of the C-terminal domain of these channels for the first time. This domain consists of a coiled coil formed by one helix from each monomer that is linked to the rest of the protein by a flexible region. Moving the coiled coil up and down causes a conformational change that allows the channel to open and close.

The technique of rational or structure-based drug design, which involve modelling the interactions between a library of potential ligands and a protein binding site, has proved particularly successful in the design of anti-viral drugs. Several inhibitors of HIV protease and of influenza virus neuraminidase that were designed in this way have become very successful drugs. David Stuart from the University of Oxford and the Diamond synchrotron gave a talk illustrating how structure-based in silico techniques are now being applied to design drugs against another virus family: the Picornaviruses. Members of this large family are responsible for a diverse range of diseases, ranging in humans from polio to the common cold. The foot-and-mouth virus, which affects livestock and which devastated parts of the UK countryside in 2001, is also a member of this family.

One of the viruses studied in Stuart's goup is a human picornavirus that causes similar symptoms to the foot-and-mouth virus and that represents a serious threat to public health in East Asia. The disease is known as hand foot and mouth virus, and the virus as CAV16: like all picornaviruses, it consiss of a single strand of RNA enclosed within an icosahedral (20-sided) protein capsid. The intact virus particles are very fragile and diffraction patterns must be captured before the particles disintegrate in the X-ray beam. Stuart and his Chinese collaborators have used one of the microfocus beamlines at Diamond to take snapshots of the virus structure at several points during its life cycle. One of these is of an "uncoating intermediate" that shows one of the viral proteins (VP1) emerging from the capsid so that it can be embedded in the membrane of a host cell Ren et al., 2013). Stuart and his co-workers are now designing compounds to bind to these intermediate structures and prevent the virus from entering its human host cells.

All cells, whether prokaryotic or eukaryotic, contain long molecules of DNA that must be packaged in order to fit into the confined space available. Fortunately for developers of anti-bacterial drugs (and users of antibiotics) bacterial cells package DNA using a different mechanism from mammalian ones. In bacteria, enzymes called topoisomerases bind to, cut and re-join double-stranded DNA so that it can be unwound or untangled ahead of replication. Ivan Laponogov, a postdoctoral research assistant at King's College, described recent work in his group on the structure of one of these enzymes. Bacterial topoisomerase II ia a target for an important class of antibiotics, the fluoroquinolones, but resistance to these drugs is increasing.

These enzymes are powered by ATP and act as "clamps", capturing one double-helical strand of DNA and passing it through a break in another to remove supercoils and knots in the nucleic acid structure. The structure presented at this meeting was the first of a complete topoisomerase dimer bound to DNA in the "open clamp" position. This structure was solved with and without a fluoroquinolone drug (levofloxacin) bound. The structure with drug bound showed that molecule intercalating between DNA bases at the point where the nucleic acid would be cleaved, preventing that cleavage. The structure without the drug showed the DNA in a different position; the position of a functionally important magnesium ion also changed between the structures.

Many essential cellular processes involve a post-translational modification in which poly-(ADP ribose) or PAR is added to amino acid side chains, and the processing of this molecule involves a wide variety of enzymes. Inhibitors of one of these, poly-(ATP ribose) polymerase or PARP, have recently been developed as drugs against cancer. David Leys from the University of Manchester described his work on the structure of another enzyme in the PAR life cycle: poly-ADP-ribose glycohydrolase (PARG), which catalyses the removal of PAR from proteins.

Mammalian PARG enzymes have three domains, a N-terminal regulatory region and two C-terminal domains forming the catalytic region; the equivalent bacterial enzymes lack the N-terminus. Leys and his groups first solved structures of a bacterial PARG bound to ADP-ribose (PDB 3SIG) and to a known inhibitor with a similar structure. They found that a C-terminal helix in the protein was clamped around the terminal ribose of PAR, enabling the release of a single ADP-ribose from the polymer. This basic mechanism is similar in the mammalian enzyme. More recently, the Leys group has solved the structure of PARG bound to an intact PAR substrate (PDB 4L2H); modelling studies based on this structure suggest that the enzyme acts predominantly as an exo-glycohydrolase, that is, it catalyses the removal of one residue at the end of the polymer chain. Understanding the structure and mechanism of these enzymes should enable us to develop small-molecule inhibitors of PARG, and these may one day rival the PARP inhibitors as anti-cancer drugs.

A hundred years on from the "invention" of crystallography and sixty years on from the structure of DNA, these elegant, fascinating and complex structures presented at one meeting give a snapshot of recent progress in structural biology. Furthermore, each of these structures has already provided insights into human disease that may yet lead to the development of useful drugs.

A Very Short History of Crystallography

You might possibly have been intrigued to read in my last post that 2014 has been designated as the International Year of Crystallography. This year was chosen to celebrate the fact that this discipline - the study of atomic and molecular structure through their crystal forms - is now almost exactly a hundred years old. Admittedly, the first paper in the discipline, rather charmingly titled just "The diffraction of short electro-magnetic waves by a crystal" was published in 1913, and the Nobel Prize awarded two years later, but 2014 is at least a good compromise.

It can be said, perhaps simplistically, that crystallography was invented by the father-and-son team of William Henry and William Lawrence (known as Lawrence) Bragg, at the Universities of Leeds and Cambridge in the UK. The Braggs, however, did not aim to found a new discipline or even to investigate the atomic properties of matter. They were more interested in solving a problem that had been puzzling the cleverest physicists in the world for almost two decades. X-rays had been discovered by Wilhelm Rőntgen in Germany in 1895, but their very name (the unknown X) suggests reveals their controversial nature. Were they particles or waves?

The older Bragg, William, was convinced that X-rays were particles, and set out to prove this to his son (who favoured the wave theory) by exploiting the discovery of another German physicist, von Laue, that X-rays shone at a crystal were scattered and could produce a pattern on a film. Lawrence was the first to realise that these patterns could be explained by the theory that the X-rays were reflected from planes of atoms in the crystal and interfered with each other.

Lawrence presented these results to the Cambridge Philosophical Society late in 1912 and published them in the paper mentioned above the following year. This paper also included the first formulation of one of the best known of all laws of physics: Bragg's Law. This relates the wavelength of incoming X-rays and the angles that they are scattered (diffracted) to the spacing between planes of atoms in a crystal, enabling scientists to determine the geometry of atomic crystal lattices.

The Braggs worked together in Leeds and published their first structures, including that of sodium chloride (common salt) before Lawrence was sent to France to fight in the First World War. He was in the trenches when he heard that he and his father had been awarded the 1915 Nobel Prize for Physics; that news reached him shortly after that of the death of his brother Robert. At only 25, he was (and still remains) the youngest ever recipient of a Nobel Prize.

Technical advances between the wars enabled scientists working in this new discipline to solve the structures of rather more complex molecules. Kathleen Lansdale, one many women who began their research careers as the Braggs' students, solved the structures of benzene derivatives and was the first to see that aromatic rings were flat. And two later developments paved the way for the explosion in structural science that characterised the later twentieth century. In 1934, John Desmond (J.D.) Bernal, who later became the first head of the School of Crystallography at Birkbeck (the predecessor department of our Biological Sciences) and his student Dorothy Crowfoot (later Hodgkin) obtained the first X-ray diffraction patterns from protein crystals. And in the following year Lindo Patterson developed a function that greatly simplified the mathematics involved in structure determination.

Even fifty years ago however, solving crystal structures was a long and at times tedious business. A typical crystallogaphy PhD thesis of the 1960s or 1970s would contain the structures of maybe three small or medium-sized molecules. It is now possible to generate as many in a few hours, so it is possible to see clearly how structures of molecules respond to changes in conditios such as temperature and pressure.

All these discoveries have been made possible by advances in technology, and particularly by the development of synchrotron radiation as a source of powerful beams of X-rays. Synchrotron radiation is produced when charged particles are accelerated radially, and synchrotrons built primarily as X-ray sources were first built in the 1980s. The UK's synchrotron, Diamond at Harwell in Oxfordshire, is currently the fifth largest in the world. It has 23 separate "beamlines", each providing a beam of X-rays with properties that have been optimised for a particular experimental technique.

Synchrotrons provide facilities for solving structures from single crystals of large and small molecules, including, of course, proteins, and from micro-crystalline samples (the latter technique is known as powder diffraction). Although structural biology attracts much of the attention (see almost all the other posts on this blog) structures of smaller molecules can still provide important insights. Sandy Blake, a crystallographer at the University of Nottingham, is using Diamond beamlines to solve the structures of novel materials called metal-organic frameworks or MOFs that are able to store gases including hydrogen (which is a potential fuel source) and greenhouse gases.

At 100, crystallography is still a young discipline but it has radically transformed many other areas of science and, through them, the world we inhabit today. This has been reflected in decisions made by the Nobel committees over the decades. The International Union of Crystallography maintains a list of Nobels awarded for ‘achievements directly related to, or involving the use of, crystallography’. There are now 29 of these, and the latest year with no crystallography-related Nobel was 2008. Even the 2013 Chemistry prize, awarded to Martin Karplus, Michael Levitt and Arieh Warshel, appears on the list: their discipline of computational chemistry would be impossible without structural knowledge obtained through crystallography.

And it almost goes without saying that protein structure, and structural biology more generally - the disciplines taught in this course and its associated MSc - owe their existence to the development of X-ray crystallography.

This blog post is based on an article I wrote for the Royal Society of Chemistry's membership journal, Chemistry World. It will be published in the January 2014 issue of the journal.

Welcome to PPS Students 2013-14!

This post is very like one I wrote at exactly this time last year. This is because what I have to say now is very, very similar...

I would like to offer 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 2013 is Molecular Mechanisms of Intracellular Trafficking: an important topic that relates quite closely to some of the material we cover in the later sections of the course. Other posts may be reports from conferences or summaries of recently published papers in protein structure, protein bioinformatics and allied areas.

And one topic that you are bound to hear more of on this blog, particularly after the New Year, is the history of structural science, particularly X-ray crystallography. Crystallography was the first method to be developed for solving the structure of biological macromolecules, and it is still the most important. The United Nations has designated 2014 as the International Year of Crystallography - the date is between the centenaries of the publication of the first papers on X-ray diffraction and the award of the 1915 Nobel Prize for Physics to the father-and-son team of William and Lawrence Bragg who made the principal discoveries.

Some of the posts on this blog are written by "guest blogger" Jill Faircloth, who took the MSc in Structural Molecular Biology a few years ago and is now working as a freelance science communicator. She introduces herself in this post written in March 2012, in which she also describes how she found the later part of the PPS course and her thoughts on the two choices available for the second year of the MSc.

Do, if you get a chance, look through some of the earlier blog posts 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 2013-14 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

GroEL, giving misfolded polypeptides a second chance

Saibil, H.R., Fenton, W.A., Clare, D.K., Horwich, A.L. (2013) Structure and Allostery of the Chaperonin GroEL. J Mol Biol. 2013 May 13;425(9):1476-87

A recent paper, by Professor Helen Saibil’s team at Birkbeck, reviews the current understanding of chaperonin GroEL.  Chaperonins attract unfortunate proteins which are incompletely or incorrectly folded and provide them with an isolated chamber in which to bind and release until they achieve their native functional state.  GroEL and its partner GroES are profiled in PPS in Section 7 (symmetry) and Section 8 (action as a chaperone).  

GroEL is remarkable in its construction.  It consists of 14 identical protomers arranged in two back-to back rings, each of the two rings with seven subunits.  This forms a barrel with a 7-fold rotational symmetry axis through its centre and, perpendicular to this, seven 2-fold axes of symmetry, giving an overall symmetry of 72.

Each subunit comprises two main domains linked by an intermediate domain (see figure (c)).  The largest domain is equatorial at the centre of the barrel.  This contains the ATP binding site and is in contact with its two neighbours in the ring as well as the equatorial domains of its partner ring.  These domains form a stable platform from which the other two domains undergo large movements orchestrated by the cycle of ATP binding, hydrolysis and release.  

The apical domains are exposed at the outer ends of the GroEL barrel.  They are smaller and include a hydrophobic surface which is the binding site for many different nonnative polypeptides.  The intermediate domain has a hinge at the junction with each of the two main domains, such that it can mediate large movements of the domains as rigid bodies.  This can be seen by comparing figures (c) and (f).

 

X-ray crystal structures of GroEL and GroEL-GroES complexes.  (a) Longitudinal cross-section of GroEL (PDB 1OEL). (b) Top view of the GroEL barrel. (c) A protomer of GroEL, aligned approximately as the top left protomer in (a).  (d-f) Show the same set of views with GroES (d-e) and ATP (f) bound (PDB 1SVT).  Example helices have been coloured to demonstrate the extent of the rotation angles.  The red and orange helices of the apical domains can be seen to undergo a significant rotation.  Compare this with the relatively minor movements of the green helices in the intermediate domains and the violet helices of the equatorial domains.

The operations of the GroEL chaperone are initiated by rapid binding of ATP to the equatorial domain of one of the rings.  This is followed by the binding of the unstructured, partly folded or misfolded polypeptide.

Natively folded proteins have their hydrophobic residues buried in the stabilising core whilst those which have lost their way have exposed hydrophobic patches.  These patches bind to the hydrophobic surfaces of the apical domains.

The final ligand is GroES, a ring of seven homo-oligomers, which forms a lid for the GroEL barrel.  Each GroES monomer has a flexible hydrophobic loop which binds to the hydrophobic regions of the apical domain alongside the substrate polypeptide.  This loop is visible in figure (d). 

The apical domains undergo significant concerted rotations together as one movement, with the domains being held as rigid bodies (compare figures (a) and (d)).   These rotations replace the hydrophobic polypeptide binding surface with hydrophilic residues, so propelling the nonnative protein into the central lidded cavity, where it is isolated to refold.

As ATP binding stimulates positive cooperative movements within the cis ring, that is the ring binding the nucleotide, it is also responsible for negative cooperation between the rings.  This means that while the movements are coordinated to bind GroES and promote protein folding in the ATP bound cis ring, in the trans or partner ring the opposite rotation prompts the release of GroES, more than 100Å away, and the expulsion of the now native protein.  

Mutation studies have revealed that salt bridges, which are studied in PPS Section 9, hold the rings steady until full ATP occupancy is achieved and are probably involved in the positive cooperativity whilst the negative cooperativity is thought to be triggered by a pivoting of the equatorial domains.  This interferes with the staggered contacts between each equatorial domain and two of its partner equatorial domains on the opposite ring.

Recent work using single particle cryo-electron microscopy techniques, which is studied in the TSMB course, has captured images of the intermediate states between ATP binding and the active chaperone state where GroES is fully bound.  

Once ATP binds, the intermediate and apical domains tilt 35˚ sideways from the lower hinge.  This brings the intermediate domain towards the ATP binding pocket where the residue ASP398 forms several hydrogen bonds.  This action causes the breakage of salt bridges between the intermediate and apical domains of neighbouring protomers and between neighbouring apical domains with new salt bridges forming which support the new tilted architecture.

Following this, the apical domains lift and separate, to use an old advertising slogan, causing further breakage of salt bridges between apical domains.  This separation could help to unfold the misfolded polypeptide before it is released into the chaperone chamber and also positions the hydrophobic binding areas for docking of the GroES binding loops.

Once GroES is bound, the apical domains lift even further outwards and undergo a 100˚ twist to create the active folding chaperone with the GroES lid in a domed position and the polypeptide is released into the cavity to complete its folding.

The next stage is hydrolysis of the ATP, which triggers the release of the ligands on the cis ring, and the acceptance of ligands on the trans ring.   

The mechanism is believed to involve separation of a β sheet contact between equatorial domains of the trans ring.   The equatorial domains are primarily responsible for holding the rings together so that the ADP complex has reduced stability. Hydrolysis is followed by ATP binding to the equatorial domains of the trans ring.  This promotes the pivoting of the equatorial domains that defines negative cooperativity and the discharge of GroES, the native protein and ADP from the cis ring, although the exact movements which lead to the discharge are unknown.

It seems likely that the release is mediated through a reversal of the twist in the cis apical domains.  This is speculation, however, as this good Samaritan of nanomachines has not yet given up all of its trade secrets.  The progress made to date, however, in large part by Professor Saibil’s team, is a striking demonstration of the power of this recently developed method in structural biology.