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.

Science Week 2013: Structures of Sodium Channels

Since 2010, Birkbeck College has held a week of lectures, most often in the spring, to highlight some of the research carried out in the School of Science. This year’s speakers included Professor Bonnie Wallace from the Department of Biological Sciences, who presented a fascinating and accessible lecture on the structures of sodium channels, and what these new structures are already teaching us human health, and particularly about some rare neurological diseases.

Professor Nicholas Keep, Dean of the Faculty of Science (and director of the MSc in Structural Molecular Biology and the second-year option TSMB) introduced Professor Wallace. She has been at Birkbeck for about twenty years and now directs the department’s impressive research work on the structural biology of membrane ion channels. You will learn a lot about membrane proteins in general in section 11 of PPS; they are ubiquitous, are responsible for the transport of both chemicals and signals into and out of cells, and form some of the most important drug targets. They are also, as Wallace made very clear in her talk, some of the most challenging of all proteins for structural biologists to work with.

All cell membranes are semi-permeable, which means that some substances can pass across them easily while others are excluded. Ions, which are charged, are generally excluded by the hydrophobic (“water hating”) membranes. This could be something of a problem, as ion transport into and out of cells is an essential physiological process. Ion channels are evolution’s solution to this problem: proteins embedded in membranes that allow ions to selectively enter and leave cells.

Much of Wallace’ work over the last ten years has focused on the structures of voltage gated sodium channels. These open to allow sodium ions to enter cells, and close to prevent them from doing so, in response to changes in potential across the membrane, and they are found throughout nature. Small molecules can bind to these channels, holding them either open or closed; some of these are severely toxic, but others are important drugs for cardiac arrhythmias, epilepsy, and pain.

Human voltage gated sodium channels are composed of a single protein chain, divided into four similar domains. Each of these domains has six transmembrane helices, four of which (labelled S1-S4) act as a voltage sensor while the other two (S5 and S6) fold together to form an eight-helix pore. This protein has so far proved impossible to crystallise, and the breakthrough involved a bacterial protein. Similar proteins are found in the membranes of some species of bacteria, enabling them to live in “extreme” environments that are rich in salt. Their structures are similar to those of the human protein, but in this case the channel is built up from a complex of four identical proteins, each of which is homologous to a single domain of the human channel.

Although this simpler bacterial protein proved easier to work with than the human protein, it was still not at all easy. It took over ten years for Professor Wallace and her group to isolate the gene, clone and purify the protein, obtain crystals and finally solve the structure of the pore. The structure was finally solved using the powerful X-rays generated at Diamond, the UK’s only synchrotron radiation source located near Harwell in Oxfordshire.

These channels exist in three different structural forms: “open”, “closed” and “inactivated”. Many years before the detailed structures were solved Wallace and her group had used a biophysical technique, circular dichroism (CD) spectroscopy, to examine the conformational changes that occurred when mammalian and bacterial channels switched from one state to the other. As always, however, the full atomic-crystal structures yielded very much more information.

The first of these structures to be solved was a slightly strange one: the pore was held in the “closed” conformation that prevents sodium ions from entering the cell, although the voltage sensor was in the structure associated with the “active” state (PDB code 3RVY). The “top” part of this structure, towards the extracellular membrane surface, has a hydrophobic surface, and the pore in this part of the membrane acts as a selectivity filter to allow sodium ions in while keeping others, including potassium and calcium ions, out. Wallace and her group were the first to solve the structure of a fully open channel and showed that the upper portion of the channel containing the selectivity filter was virtually unchanged. The conformational change associated with opening and closing the channel occurs at the internal or cytoplasmic side of the protein (PDB 4F4L). When the pore closes, a small turning motion of the “bottom” part of the helical bundle causes the diameter of the pore to shrink, in a motion rather like the closure of a camera lens; the resulting channel is too small for sodium ions to pass through, so any inside the pore become trapped there.

Two subunits of the bacterial sodium channel pore in the “open” conformation, shown as a ribbon structure

All voltage gated sodium channels have a domain at the C-terminal end of the molecule that is necessary for channel activity but that was not visible in any of the crystal structures. Wallace and her group looked at this part of the molecule in the bacterial protein using a particularly powerful form of CD spectroscopy called synchrotron radiation CD spectroscopy that she had pioneered, and showed that each subunit had an extremely flexible protein chain separating the pore from a C-terminal helix. Using this information, the group have proposed a novel mechanism for channel opening in which the conformational change in the pore is enabled by these helices oscillating up and down.

Two subunits of the bacterial sodium channel pore in the “open” conformation, shown as a ribbon structure

The final part of Wallace’ talk was devoted to the role of sodium channels in health and disease, and as a drug target. A few unfortunate individuals have mutations in a type of channel that is involved in the response to painful stimuli. If this channel is jammed open, patients experience a constant, burning pain termed erythromelalgia, most commonly in their hands and feet. Wallace showed that an equivalent mutation from phenylalanine to valine at the base of one of the bacterial protein subunits caused the channel to open just enough for ions to pass through. There are also people in whom these channels are jammed in the closed position, and they feel no pain, even if they walk on hot coals. It may one day be possible for drugs based on our knowledge of these structures to be designed to ease both these conditions.

E. coli, turbocharging the workhorse

Vincentelli, R., Romier, C. (2013) Expression in Escherichia coli: becoming faster and more complex.  Current Opinion in Structural Biology.  In press, corrected proof.

Escherichia coli, or E. coli, has long been the trusty workhorse of the structural biologist.  It is by far the most popular expression host, that is an organism which is used to translate introduced DNA into target proteins, with an astonishing 90% of structures deposited in the Protein Data Bank (PDB) having at least one subunit if not the entire protein produced by this bacterium.  Targets are becoming more ambitious, however, as the importance of larger protein complexes in biological pathways becomes increasingly apparent.  A recent review (Vincentelli, R., Romier, C. (2013)) examines whether E.coli is fit to tackle these new challenges.  Can the workhorse be taught to jump fences?

E. coli as host for single expression

The virtual monopoly held by E. coli as a host is due to a number of factors, not least its ease of use and low cost.  It is amenable to several different methods of genetic engineering, has rapid growth and benefits from an ever expanding range of host specific tools.

There are mature methodologies available for the production of a single protein.  Reliable and straightforward screening processes, which can discover the conditions required for optimised protein solubility, are accessible for even challenging targets.  Since the solubility of a protein has a direct impact on the size and quality of crystal that can be grown, this is a critical aim.

Genomics laboratories tend to use robotic platforms which can work with thousands of cultures side by side in order to optimise the culture conditions for their long shopping list of proteins.  This wealth of experience now allows biologists to make a rational selection of a smaller set of expression parameters when targeting proteins which are more difficult to produce.  If this restricted method fails then the team can revert to the broader set of conditions.

Supporting this trend towards more efficient screening processes are studies that have identified the parameters with the greatest impact on expression and solubility, a key example being specific fusion protein tags which enhance solubility.

Another recent development which has increased the complexity of proteins open to expression by E. coli, is the ability to co-express post-translational modifying factors.  These factors can be critical in protein folding, complex assembly and catalysis, for example by promoting glycosylations and disulphide bridges.

Streamlining for maximum solubility and quality

The traditional approach to protein production was to perform small scale expression tests designed to discover the conditions for the highest soluble yield.  These conditions would be scaled up and only at this stage would consideration be given to the quality of the sample, specifically protein aggregation, oligomeric states, protein stability and correct folding.

By using growth media with high cell density, particular E.coli strains and protein fusions designed to enhance solubility, initial yields have been increased.  This coupled with the improvement in biophysical characterization, such that assays to examine protein quality can now be performed on just micrograms of a sample, has meant that culture conditions can be optimised for both solubility and quality at the first stage thereby streamlining the expression protocols.

E. coli and the expression of macromolecular complexes

Most biological processes involve macromolecular complexes alongside single proteins and there is an increasing desire to understand the structural and biochemical basis of these larger structures.  The co-expression of partner proteins has been demonstrated to be advantageous for complex formation as protein-protein interactions are often the platform which allows co-folding and co-stabilization.

One example is the fimbrial tip complex of E. coli.  Many Gram-negative and some Gram-positive bacteria are covered in a fringe of short, thin fimbriae which are used to attach both to eukaryotic cells and to each other although the mechanism was previously unknown.  Co-expression of the FIM proteins of the fimbrial tip established that each subunit inserts a β strand into its neighbouring subunit such that allosteric changes in the tip protein trigger signals which can be passed down the fimbria.

Image adapted from (Le Trong, I. et al., (2010)).  A view of a fimbrial tip complex of E. coli.  (PDB 3JWN)

Birkbeck’s head of biological sciences, Prof Gabriel Waksman, has also used this technique to elucidate the interaction of FimH with its transmembrane translocation channel.  His work has been the subject of previous blogs in June ’11 and May ’08.

Another impressive example which illustrates the size of complex that can be achieved through co-expression is the 1.8MDa baseplate of the lactococcal phage TP901-1.  The baseplate is responsible for adhesion of the phage to the host and for delivery of the genome at infection and this particular version consists of 6 subunits of DIT, 18 of BppU and 54 of RBP proteins.

 

Image adapted from (Veesler, D. et al. (2012)).  The baseplate of the lactococcal phage TP901-1.  (PDB 4DIW)

Despite the advantages of co-expression, only a small percentage of large complexes listed in the PDB have been fully produced this way.  Often, subunits are produced using co-expression but then labour intensive in vitro reconstitution strategies are employed to form the complete complex.

In some cases, this is because the protein complexes have critical interactions with nucleic acids but this is not always the case. This raises the question of what the barriers are that are discouraging complex formation via co-expression from E.coli.

Approaching the jumps

Studies have found various parameters that influence the quality and yield of co-expression using E.coli.  Results can vary depending on whether a single vector is used to introduce the target genes rather than multiple vectors, whether multiple genes are used rather than cis and trans copies of the same gene or on the precise location of the affinity tag.

The best approach to tackle this quantity of possible sets of conditions is to use the high-throughput technologies which have been refined so effectively for single expression protein production.  This will need to be combined with the tools in development for miniaturization of biophysical characterization so that the sample quality can be considered during the initial stages rather than creating a further bottleneck as a second round of tests are performed with greater quantities.

As techniques for performing characterization assays on minute samples improve further and, hopefully, a co-expression system can be evolved which allows the production of protein/RNA and protein/DNA complexes, there is optimism that E. coli can extend its hosting duties into ever larger and more intricate protein complexes.

Protein-protein interactions and protein expression for structural biology is covered in detail in the TSMB course.