Activated caspases stick together

In a post last week I mentioned a technique for obtaining the high-resolution structure of a protein inside a living cell, but I also pointed out that this technique was difficult and expensive, and might not be applicable to large proteins. Techniques improve and become more powerful, of course, but you might not want to wait for NMR to catch up to your question. Fortunately, high-resolution in vivo structures may not be necessary if you already have relevant dilute-solution structures of your protein and merely want to distinguish between different known conformational states. In a recent paper in PNAS, researchers from San Francisco used conformation-specific antibodies to locate activated caspase-1 in cultured cells.

Caspase-1 is a cysteine protease that plays a role in the immune response, as well as being released during apoptosis. From crystal structures we know that this protein can adopt two different structures, of which only one represents a catalytically competent state of the enzyme (the on-form). Caspase-1 also possesses an allosteric site where an inhibitor can bind, locking the enzyme in an inactive conformation (the off-form). When it's not bound to anything (the apo-form) caspase-1 is presumed to have a conformation similar to the off-form. Like many proteases, caspase-1 has a large, inactivating tail when it is made (the pro-form) that must be cleaved off before activation is possible. The structure of the caspase-1 proenzyme is not known. Current models of inflammatory response propose that after processing, the on-form binds to scaffolding proteins in an "inflammasome". In order to confirm this proposition, the authors decided to generate antibodies that would bind specifically to the on-form or the off-form of caspase-1.

The key to this experiment was combining irreversible inhibitors that could essentially lock the caspase into one conformation with the phage-display technique for optimizing antibody recognition. The authors had the advantage that both the active site and the allosteric site have cysteines in them. In an oxidizing environment, small molecules can covalently bind to the protein via disulfide bonds, thus locking the enzyme into the on-form or off-form. The authors immobilized these "locked" forms of caspase-1 and used them to screen antibody fragments (Fabs) using phage display. In addition to the typical selection approach, the authors performed anti-selection at one point using the "wrong" conformation to increase the specificity. After several rounds of selection, and some controls to ensure that the antibodies were binding to caspase and not the inhibitors, Gao et al. had several candidates for further optimization and screening. After they completed that process, they had two antibodies, Fab_on and Fab_off, specific for the two conformations. Each antibody bound to its intended target with a K_D of less than 5 nM. The authors also made full antibodies (IgG_on and IgG_off) from these Fabs for expression in mammalian cells.

The authors took these new antibodies for a spin with the apo-form of caspase-1. One might naively expect that only Fab_off would bind to this protein, but in fact Fab_on bound as well, albeit with substantially reduced affinity relative to the on-form. One possible interpretation of this finding is that the apo-form is equivalent to the off-form, but that Fab_on can convert it to the on-form via an induced-fit mechanism. If this is the case, then we would expect Fab_off to have the same affinity for the apo-form as it has for the off-form. However, the authors find that Fab_off has reduced affinity for the apo-form relative to the off-form. This indicates that the apo-form is an ensemble of conformational states, most of which more closely resemble the off-form than the on-form. Consistent with this view, the authors found that they can activate or inhibit apo-form activity by adding Fab_on or Fab_off, respectively.

By contrast, IgG_on did not bind detectably to a model of the pro-form, suggesting that this form's conformational ensemble contains no members that are close in structure to the active form. The weak affinity of IgG_off for the pro-form suggests that there are substantial differences between this conformation and the off-form as well.

At this point we know that IgG_on will bind tightly to the on-form of caspase-1, weakly to the apo-form, but not to the pro-caspase. This means it will likely be an effective probe of active caspase-1 in cells. The authors performed this experiment in THP-1 cells that they differentiated into macrophages. While IgG_off produced diffuse fluorescence in these cells, IgG_on stained small, concentrated bodies in a fraction of the cells. This suggests that active caspase-1 is localized to supramolecular structures in these cells, which the authors argue are identical to a structure previously identified as the "pyroptosome".

Although this particular experiment took advantage of binding-site cysteines that are particular to caspase-1, it should be possible to extend this approach to other proteins. Even non-covalent inhibitors or activators should be useful in this approach as long as the concentration is held high enough to saturate the target site during the selection step. Of course, the conformational change must alter the structure enough that the antibodies have something to grasp — it may not be possible to get specific antibodies if the shift is too subtle. If this requirement is met, however, it should be possible to determine the distribution of specific conformational states in cells, or even (as the authors suggest) to use antibodies as activators or inhibitors in vivo.

J. Gao, S. S. Sidhu, J. A. Wells (2009). Two-state selection of conformation-specific antibodies Proceedings of the National Academy of Sciences, 106 (9), 3071-3076 DOI: 10.1073/pnas.0812952106

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It came from the Protein Society! (Part 2)

Another feature of the protein society was a continued emphasis on trying to understand natively-disordered proteins, and by extension, the denatured state of natively ordered proteins. Because these two fields are highly related and use the same techniques, it seems to me best to lump them together for now. A couple of interesting points came up that I wanted to get down here for my own memory's sake.

One point, and one that became a recurring theme in several talks at the symposium, was averaging bias. The first real discussion of this came from a really good talk by Michele Vendruscolo on the study of the natively-disordered 131-deletion mutant of staphylococcal nuclease. Some models that Dave Shortle had produced of the disordered state on the basis of paramagnetic relaxation enhancement had predicted ensembles that were too small with respect to the known radius of gyration. Michele pointed out that the PRE is an ensemble measurement, and many different ensembles can give rise to the same PRE. Additionally, the PRE is biased because below a certain threshold the effect is invisible. This means that the measurement ends up being biased towards closer approaches. Essentially his point was that the normal distribution cannot be assumed for the ensemble average of distance measurements in the denatured state (and it's probably a questionable assumption in the native state as well).

Kevin Plaxco gave a talk later on that really hammered this point home. He did a series of SAXS experiments to determine the radius of gyration for a ton of proteins, including several that had shown residual structure in NMR experiments. His results indicated that the experimentally determined radius of gyration matched that predicted for a random coil for all these proteins. As he pointed out, though, the Rg is totally insensitive to local structure, whereas because of anomalous averaging much of the NMR data is hypersensitive to local structure. This means that both results can be right -- any given protein can have some percentage of its structure intact and as long as it's a different piece for each protein and not too much, the ensemble can retain a random-coil-like Rg. If tertiary interactions are preserved this becomes a slightly more difficult proposition to swallow, though. Still, his work, and several other talks and posters presented during the symposium, made an excellent point. We simply cannot rely on the assumption of a normal distribution when we are analyzing NMR data from systems with so many degrees of freedom.

Another thread that showed up repeatedly was the ongoing attempt to understand exactly how disordered states interact and are regulated, especially by post-translational modifications such as phosphorylation. Most disordered regions have multiple binding partners, with affinity enhanced for a particular partner by a particular modification. In the simplest model for these interactions, the modification itself and some of the surrounding primary sequence is recognized. However, there's an increasing amount of data, including a nice talk by a postdoc from Julie Forman-Kay's group, that the post-translational modifications alter the structural characteristics of the disordered state itself. The Forman-Kay talk suggested that phosphorylation induced a condensation of the protein by attenuating a surplus of positive charge.

This could conceivably be taken further. Consider a bit of sequence like DKRSDKA, which could conceivably take the form of a β-strand if it weren't for that concentration of positive charge on one side. A phosphate group on the serine could conceivably stabilize this structure and preorganize it for binding to a ligand, thus increasing affinity by reducing the energetic cost of binding.

It might even be possible to tune things more specifically. Take a sequence like GRDSSKAKSR. If you put this on a helix wheel you'll see a huge blast of positive charge on one side, but also a pair of serines. Phosphorylate S5 and S9 and you could stabilize the helix. At the same time, this would make a β-strand conformation less likely because such a strand would have negative charges on one side and positive charges on the other. By contrast, if you phosphorylate S4 you'd do nothing to stabilize the unfavorable charge concentration on the helix, but the positive charge concentration on the strand would be attenuated (see cartoon). In this way phosphorylation might be used as a kind of conformational switch to preorganize the same sequence in different ways and thus reach different downstream effectors. We know that conformational rearrangements of the kind that lymphotactin undergoes give rise to different signals and protein behaviors. The role of differential preorganization in disordered proteins hasn't been extensively studied yet, but may be equally important.

It's increasingly clear that disordered regions are a major factor in cellular signaling. I'm not having much luck with the one I'm working on now, but I'm excited to see where the next few years lead this field.

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