Does flexibility increase protein stability?

Since my favorite physicist of the blogosphere put up a great post on the basics of NMR in preparation for a geeky post on the subject, I figure I'll add in a geeky post of my own. As Chad mentions in his post, the dependence of the resonance frequency on local chemical structure allows us to get a great deal of information about covalent bonds and their conformation from NMR spectra. In addition, certain NMR experiments can provide us with detailed information about the motions of atoms with respect to one another. Although the various approaches differ in the level of detail they offer, the sum of NMR dynamics experiments fairly effectively cover the range from picosecond motions to month-long fluctuations. My particular interest in these approaches is in their use to dissect intramolecular signaling in proteins.

The major part of my graduate studies at UNC under Andrew Lee was devoted to understanding the pathways by which changes in dynamics are propagated in eglin c (explore this protein at the PDB). Eglin c is an excellent subject for this kind of study because it has very good spectroscopic properties, and because it has no discernable allosteric properties at all. This means we can do a lot of different experiments on it, and also that our observations about long-range dynamic interactions are likely to be generalizable to any protein that has a hydrophobic core, not just allosteric ones.

My work with eglin c involved making mutations to the protein and determining what dynamic changes resulted. In the course of this I discovered several mutants that caused significant changes in the motions of amino acid side chains on the timescale of picoseconds to nanoseconds (1,2). This is the range covered by the Lipari-Szabo model-free formalism (3). One mutation in particular, V54A (read: valine 54 to alanine), caused almost universal rigidification of the core of eglin c on this timescale. However, the protein was significantly less stable.

This is curious because a lack of stability is known to correlate with an increased frequency of localized unfolding of the backbone. In elements of secondary structure, hydrogens attached to amide nitrogens tend to be caught in hydrogen bonds. When placed in a solution of deuterated water, these hydrogens can only be replaced by deuterium when the hydrogen bond breaks (this is called hydrogen-deuterium exchange or HX). This process can be monitored by NMR (and less specifically by mass spectrometry), and is known to speed up when the structural elements are less stable. So the V54A mutant is more flexible on this slower timescale, and less flexible with respect to some faster processes.

The significant caveat here is that we only looked at some groups in the protein. For reasons that are a bit too complex to discuss in this post, it was only feasible to observe the dynamics of methyl groups. This is a problem because the core of eglin c has a substantial number of aromatic side chains in it. Any picture of dynamic behavior would be incomplete without examining these side chains and quantifying the changes in HX behavior, which I did not do.

In an upcoming article in Biochemistry, Josh Boyer and Andrew Lee address these deficiencies (4). In order to look at the aromatic side chains, they employ a clever biosynthetic labeling scheme devised by Akke's group (5). While this only allowed them to look directly at δ-carbons of these residues, the general rigidity of aromatic rings meant that they could generalize the observations to essentially the whole side chain. They found using this approach that the dynamic response of aromatic side chains to the V54A mutation was more heterogeneous than that of the methyl side chains. As you can see in the figure I have shamelessly stolen from their paper, the central portion of the core appears to have rigidified across all residue types, while the edges of the core and the outward-facing residues of the protein all appear to have become somewhat more flexible. The location of V54 is shown here in black.

This heterogeneity of the dynamic response was also observed when the HX results were taken into account. The distribution of responses, however, is very unexpected. The top portion of the figure at left shows rigidified side chains as a transparent surface, and destabilized backbone amides (as measured by HX) as solid surfaces. Note that the two regions of destabilized amides are linked by contiguous side chains. The lower figure shows side chains that have become more flexible as transparent pink surfaces. The solid backbone surface indicates a region of the protein that has become more stable. You will notice that this stabilized region lies a significant distance from the mutation site (though as the figure above shows it is connected to it by a network of contiguous side chains), and that it corresponds with a bundle of aromatic sidechains that have become more flexible. A significant portion of the protein is clearly destabilized, as was expected from the previous results on V54A. However, the co-localization of side-chain rigidity and backbone instability (and vice-versa) doesn't jive with our expectations.

Our physical intuitions suggest that rigid things are stable and flexible things are not, and in many cases these intuitions have been borne out—in studies of backbone dynamics of proteins from thermophilic organisms, for example. However, the present results can be rationalized if one supposes that conformational entropy makes a significant contribution to stability. Indeed, because the structure of V54A is known to be essentially unchanged from wild-type (2), the enthalpic contributions (hydrogen bonds, charge-charge interactions, etc.) will not be altered. Entropic contributions will therefore dominated the observed changes in stability.

Rigid regions of a protein may have no place to disperse thermal energy other than localized unfolding, while flexible regions may have the option to dump some of that energy into side-chain fluctuations. When mutations increase the available space for those fluctuations, therefore, stability may be expected to increase, and vice versa. Obviously, the benefits of flexibility to ordered backbone structure will diminish as the fluctuations approach a magnitude that permits solvent to permeate the site.

Boyer and Lee's results, though probably expected by those who have followed previous research in eglin c, do not mesh with our macroscopic understanding of the relationship between rigidity and stability. However, they are not so outlandish that they can't be rationalized in terms of what we already know about proteins. An investigation of this anticorrelation in other eglin c mutants (promised in the paper) would be welcome. In addition, it will be interesting to see if this phenomenon is observed in other proteins (especially thermophilic proteins). As new labeling techniques and experimental approaches expand the range of molecules that can be effectively investigated using NMR, we may learn significantly more about the role of conformational entropy in the stabilization of protein folds.

1. Clarkson, M.W., Lee, A. (2004). Long-Range Dynamic Effects of Mutations Propagate Through Side Chains in the Serine Protease Inhibitor Eglin C. Biochemistry, 43(39), 12448-12458. DOI: 10.1021/bi0494424

2. Clarkson, M., Gilmore, S., Edgell, M., Lee, A. (2006). Dynamic Coupling and Allosteric Behavior in a Non-Allosteric Protein. Biochemistry, 45(25), 7693-7699. DOI: 10.1021/bi060652l

3. Lipari, G., Szabo, A. (1982). Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules. 1. Theory and range of validity. Journal of the American Chemical Society, 104(17), 4546-4559. DOI: 10.1021/ja00381a009

4. Boyer, J.A., Lee, A.L. (2008). Monitoring Aromatic Picosecond to Nanosecond Dynamics in Proteins via 13C Relaxation: Expanding Perturbation Mapping of the Rigidifying Core Mutation, V54A, in Eglin c. Biochemistry DOI: 10.1021/bi702330t

5. Teilum, K., Brath, U., Lundstrom, P., Akke, M. (2006). Biosynthetic ¹³C Labeling of Aromatic Side Chains in Proteins for NMR Relaxation Measurements. Journal of the American Chemical Society, 128(8), 2506-2507. DOI: 10.1021/ja055660o

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Allosteric interactions of PDZ domains

Another article entering the preprint stage last week also has some history behind it, although it doesn't have quite the wayback machine mojo of the last articles I discussed. A collaboration between researchers at Radboud University Nijmegen and the University of Pisa produced a really interesting story about the protein tyrosine phosphatase PTP-BL (citation 1 below). Like many phosphatases, PTP-BL is a large, multi-domain protein; it is typical to view these domains as independent functional units and see their conjunction in a protein as just a form of scaffolding. In the case of PTP-BL, however, it appears that the domains interact with each other, and that this has consequences for their binding specificity. In addition, their results agree with previous work identifying allosteric networks in the PDZ domain.

However, the interesting part of this story starts almost a decade ago with a bioinformatics experiment carried out by Lockless and Ranganathan published in Science (citation 2 below). Standard bioinformatic analysis of homologous proteins involves aligning their sequences based on similarity and looking for residues which are conserved across evolutionary time and space. It is generally believed that invariant residues are essential to either the structural integrity of a given protein fold or its function. Lockless and Ranganathan decided to take this a step further and ask which residues were co-conserved with a particular amino acid position, that is, whether amino acid changes at some position X are correlated with changes at some other position Y. They performed this experiment using the PDZ domain, a very common ligand-binding domain that appears in multiple proteins (and often in multiple copies within the same protein) in nearly all eukaryotes as well as bacteria. As the target of their co-conservation analysis, they chose a histidine in the binding cleft of PDZ.

Naturally, one would expect other residues within the binding site to show co-conservation, and this is indeed the case. The surprise, however, comes from the fact that in addition to these expected residues, an additional patch of residues on the opposite side of the domain also appeared to be co-conserved with the histidine, as shown in their figure at right. The histidine in question is residue 76, and co-conserved residues are rendered with pinkish molecular surfaces. The peptide bound by this particular PDZ domain is shown as yellow sticks. What you can see here is that there appears to be some linkage in an evolutionary sense between residues in the active site and residues in the β-strand structure on the other side of the protein. Lockless and Ranganathan did some binding studies that seemed to support their findings, and attributed this apparent pattern to structural perturbations.

A few years later, NMR virtuoso Ernesto Fuentes performed an NMR dynamics study on the second PDZ domain of human protein tyrosine phosphatase 1e (citation 3 below), in which he compared side-chain dynamics of the free (isolated) domain to those of the protein in a ligand-bound state. The results of that study are remarkably similar to those of the Lockless and Ranganathan work, though not identical. The ligand is shown in green, while side-chains of dynamically-responding residues are shown in red, yellow, and blue. As expected, most of the dynamic changes upon ligand binding occur right next to the binding site. However, two distal surfaces of the domain also appear to feel dynamic effects from the binding of the peptide. This gives even more direct evidence of some kind of allosteric interaction between the binding site of a PDZ domain and parts of the protein that are further away.

The new paper by van den Berk et al. puts a kind of exclamation point on this story. Their effort began, essentially, as a fishing expedition to find what peptides exactly the various PDZ domains of PTP-BL—it has five total—bind. Among others, they found that PDZ2 would bind to peptides from APC (binding site -VTSV) and RIL (binding site -VELV). This was the case when the PDZ2 domain was tested alone. When a construct containing both the PDZ1 and PDZ2 domains was tested, however, the RIL peptide no longer bound to PDZ2. This was true whether or not the 200 amino-acid linker between them was included in the construct, indicating that a bona-fide interaction between PDZ1 and PDZ2 was responsible.

van den Berk et al. then used NMR chemical shift perturbation mapping to identify the binding site of PDZ1 on the PDZ2 domain. They found, in what should hardly be a surprise at this point, that the primary site of interaction is a distal surface of PDZ2. On the basis of modeling studies they suggest that the mobility of Ile 48 is critical to enabling the binding of the bulkier RIL peptide; they attribute the allosteric effect of PDZ1 to a restriction of PDZ2 Ile 48 so that it cannot move out of the way and allow RIL to bind. Thus we come to the model at right. Note that van den Berk et al. did not, as far as I can tell, determine what part of PDZ1 binds to PDZ2. It's possible that the normal binding cleft is used, but the binding site on PDZ2 looks like a broad hydrophobic surface rather than a narrow structure that could easily insert into a binding cleft. This suggests that the binding site on PDZ1 is still available, and thus that peptide binding to PDZ1 could fine tune the behavior of PDZ2.

The functional importance of this change in affinity is not yet clear. RIL is also bound by another PDZ domain in PTP-BL, so the PDZ1-PDZ2 interaction does not abrogate RIL binding. Additionally, even in the absence of PDZ1, APC has a higher affinity for PDZ2 than RIL, so the PDZ1-PDZ2 interaction is not really switching the target of PDZ2 or anything. However, if the local concentration of RIL is significantly higher than APC, improved specificity for APC may be necessary for kinetic reasons. Alternately, the improved APC specificity may be an incidental feature of an interaction that evolved for another reason, perhaps to bring APC into close proximity to some protein bound to PDZ1.

The possibility that allosteric communication pathways might exist in small protein modules like the PDZ domain was initially met with a great deal of resistance. The classic descriptions of allosteric and cooperative interactions all involved very large protein oligomers. Dynamics experiments like those carried out by Fuentes et al. and functional studies such as this one, however, have borne out the predictions of the bioinformatics studies. Allostery, or the potential for allostery, is a feature of small, isolated domains just as it for large protein assemblies.

Another point to keep in mind out of this paper is that it is a mistake to assume that domains within a protein are completely independent. It turns out that this is often the case, that, for instance, a binding domain and a catalytic domain exist together without really interacting, and when that happens it's not a problem to analyze each domain separately. However, domains within a protein can and do interact with one another, even when they are separated by sizable linking regions (the linker here is >200 amino acids). Often the easiest (and sometimes the only) way to investigate the structure of large proteins is to look at their domains individually. This paper, among others, is a reminder that this strategy is not always appropriate and may fail to capture important features of the domains' functions.

(1) van den Berk, LCJ, Landi, E, Walma, T, Vuister, GW, Dente, L, and Hendriks, WJAJ. "An Allosteric Intramolecular PDZ-PDZ Interaction Modulates PTP-BL PDZ2 Binding Specificity." Biochemistry ASAP (2007)

(2) Lockless, SW and Ranganathan, R. "Evolutionarily Conserved Pathways of Energetic Connectivity in Proteins." Science 286 (1999) p. 295-299.

(3) Fuentes E.J., Der C.J., and Lee, A.L. "Ligand-Dependent Dynamics and intramolecular signaling in a PDZ domain." J. Mol. Biol. 335 (2004) pp. 1105-1115.

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Cryptochrome: Not Just for Circadian Rhythm Anymore?

The annual movements of birds are frequently cited as one of the great wonders of nature, and rightly so. This widespread behavior's initial evolution and subsequent refinement into the amazingly specific systems we observe in the modern world are testaments to the enormous power of natural selection to shape not only the physiological systems that enable long-range directional flight, but also the social mechanisms that specify destination and mode of travel. In general terms, we understand what biological components enable migratory behaviors, but the specifics of the system are not yet completely understood. We know that birds have a magnetic compass, and that their choice of direction is also light-dependent to some extent. Today in PLoS ONE, Miriam Liedvogel and colleagues attempt to eplain this linkage with biophysical experiments.

The target of their studies is cryptochrome, a eukaryotic protein related to the bacterial DNA repair protein photolyase. Cryptochrome is a blue-light photoreceptor, known to play a role in light-detection in plants, and important for circadian rhythm in mammals. Eukaryotic cryptochromes have a photolyase-like domain that contains a flavin chromophore, and a large, intrinsically unstructured C-terminal domain that is important for signaling. The lovely and talented Carrie Partch showed that photoreception in the photolyase homology region disrupts stable structure in the C-terminal domain of arabidopsis cryptochrome, and that this is important for signaling (shameless promotion). Liedvogel et al. show in their paper that the flavin nucleotide reacts to the incidence of blue light by forming pairs of radicals. This is important because the electronic behavior of these radicals can be affected by external static or oscillating magnetic fields. Moreover, cryptochromes may be active in the eye during magnetic orientation, as Mouritsen's lab showed previously.

Liedvogel et al. demonstrate the existence of radical pairs using a transient absorbance experiment following irradiation of a 20 µM cryptochrome solution with a blue laser. Their Figure 1 (to see it, follow the link above—PLoS is free) shows absorbance maxima characteristic of flavin radicals that persist for a few milliseconds following excitation. These peaks are not present in absorbance spectra averaged over a longer period of time (Figure 3). Liedvogel et al. propose that these radicals, though persisting for only a few milliseconds, nonetheless last long enough to plausibly interact with the earth's magnetic field.

So... problem solved? Not exactly—Liedvogel et al. do a pretty good job explaining the limitations of this research and the attendant models. Although this work opens up some intriguing possibilities it certainly leaves us with more questions than answers. Specifically, it would be nice to know whether this radical-pair formation actually causes some kind of signal; given the relatively short lifetime of these species this is not at all certain. Also it would be important to determine whether the presence of magnetic fields in any way modulates cryptochrome signaling. This is also a case where the nature of the experiment may be misleading us—a dilute solution excited by a laser may not really resemble a cellular milieu stimulated by ambient light. To be fair, this cuts both ways; cofactors present in the cell could inhibit or enhance radical formation.

In this regard, it will be important to perform in vivo experiments, at least at the level of cultured cells. Experiments of that nature are likely to be very difficult, however, given the highly specific context in which the signaling occurs. Given that the experiment entailed the creation of a cDNA library from bird retinal cells under the appropriate signaling conditions, a yeast two-hybrid screen under conditions of blue-light irradiation may be a possibility.

The subsequent challenge will be to test whether and how a magnetic field alters signaling. The problem there is one of controls—the earth's magnetic field is pretty difficult to escape. However, once signaling partners are isolated, one could set up experiments involving NMR spectrometers... not as direct devices for investigation, but rather as part of the experimental setup. The magnetic field inside (and thus, with an unshielded instrument, outside) an NMR spectrometer has a directionality, and not all of them are oriented the same way. Some point away from the core of the earth, others towards it. The magnetic field of a typical MRI machine is oriented parallel to the ground. In addition, the field of almost any superconducting magnet is vastly greater than that of the earth. Growth experiments performed in the leaking field outside of these spectrometers could be compared to experiments performed in the ambient magnetic field in order to gain insight into the effects of external polarization on cryptochrome signaling. Dilute-solution experiments performed within an NMR spectrometer may also be of value.

Liedvogel et al. argue that the cryptochrome would have to be tethered or immobilized in some way in order for its interactions with the magnetic field to encode information. This is certainly the simplest possibility, but may not go far enough. Simply tethering the molecule is unlikely to prevent isotropic or near-isotropic tumbling, especially considering the unstructured nature of the c-terminal domain that seems to govern signaling. However, tethering the protein at a single point might work if the actively signaling species is short-lived, experiences primarily local interactions, and is dispersed along a line or plane of tissue. In this case, the averaging due to tumbling might be countered by a gradient imposed by the arrangement. This is especially attractive because this form of activation appears to be low-yield: increased average activity of a tethered ensemble rather than a rare specific signal incidence might be the mechanism.

This is an interesting paper that amounts to a feasibility study. Liedvogel et al. demonstrate that blue-light stimulation can induce the formation of radical pairs, and it is known that the behavior of these species are affected by magnetic fields. This shows that it is possible for cryptochromes to play a direct role in migratory magnetic navigation. Additional evidence suggests that they play some role, but this may be coincidence; for instance, magnetic compass calibration may be a system controlled by the circadian rhythm function of cryptochromes. Actually proving the existence of a direct role for cryptochromes in orientation control will require substantially more work.

Liedvogel M, Maeda K, Henbest K, Schleicher E, Simon T, et al. (2007) Chemical Magnetoreception: Bird Cryptochrome 1a Is Excited by Blue Light and Forms Long-Lived Radical-Pairs. PLoS ONE 2(10): e1106. doi:10.1371/journal.pone.0001106

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Allosteric inhibition in medicine

If you wanted to keep someone from driving off in their car, how would you do it? There are some obvious answers: you could put some kind of blockage in front of and behind it, or you could slash the tires, or you could damage the engine, or you could empty out the gas tank. All of these approaches directly interfere with the mechanisms that allow the car to move. Alternately, you could take a more indirect approach: you could break the doors so they don't open, or remove the steering wheel, or take out the brake and accelerator pedals, or remove the stickshift. None of these latter approaches make it impossible for the car to move — the engine and wheels still function — but by removing the means that allow human beings to control the car these approaches still manage to ensure that the vehicle cannot be driven.

The traditional approach in structure-based drug design is to attempt to block the active site of a protein or important interacting sites, analogous to the "direct" approaches I outlined above. By adding a lump of stuff that obstructs enzymatic activity, ion transport, or (more recently) binding interactions these drugs attempt to interfere directly with the biochemistry of the target protein. This approach has been pretty successful, and certainly no reasonable person would want to depart from it, but there are some weaknesses here. For one thing, many diseases don't originate from enzymes or ion channels, and developing drugs that obstruct binding interactions can be pretty tricky.

Imagine your disease arises in the following system. You have some kind of receptor (R in my little cartoon over to the side there) that can bind a ligand (L). Binding of L activates R in some way so that it can bind to a partner (P) and this causes some gene to be activated. This cartoon roughly represents the way in which hormone receptors (like for estrogen or testosterone) function. Now, suppose something has occurred to derange this system: perhaps too much of L is being made, or R and P have been aberrantly expressed in some tissue where the gene they activate is toxic or causes inappropriate cell proliferation.

If we come at this by the conventional approach of targeting the L-binding pocket, we have a problem, because any drug that has an affinity for R high enough to displace the natural ligand is likely to produce the same or similar conformational changes that cause P to bind. We could, of course, try to develop a drug that interferes with P's binding directly, but this approach has its own problems. For one thing, these binding surfaces are often quite large and highly structured, features that are often difficult to replicate with a small molecule. You can create a drug that inserts itself into the site somehow and gets in the way of P binding, but it's not always possible to design such an agent that specifically hits only your protein. A peptide mimic of R might work, but these are also difficult to deliver.

But there's another strategy that might work, too. Rather than try to block R from binding L or P directly, what if we could cause some other change in R that prevents P binding whether or not L is around? Just like removing the accelerator or stickshift from the car of my example, this doesn't directly block either function of the molecule. R can still bind L, and the binding surface P interacts with is still present. However, our drug (D) has caused a new change in R's conformation that prevents the binding of P. A simplified cartoon is at left.

This is precisely what seems to have happened in a study on the androgen receptor (AR) appearing the October 9 issue of PNAS and reported by Eva Estébanez-Perpiña and coworkers from UCSF and St. Jude's. Interestingly, this group of researchers was initially seeking to produce a direct inhibitor — in this case, a drug that would bind to the part of AR that interacts with its coregulators. This interaction is therapeutically interesting because aberrant AR activity plays a role in prostate cancer, among other things. Several promising compounds were found that produced substantial inhibition of AR activity at relatively low concentrations. The surprise was that only some of these preferred to bind the targeted site (AF-2). Instead, a number of them were found to locate preferentially to a previously unsuspected site at the top of the molecule, now called binding-function 3 (BF-3), which you can see in the figure I shamelessly stole on the right here.

The reason that this appears to work is that binding to BF-3 changes the structure of AF-2, via an allosteric interaction. Rather than directly getting in the way of the essential reaction, the drug acts at a distance to disrupt the binding surface. Although much still needs to be done to refine these lead compounds into usable drugs, this is a significant demonstration that such an approach has therapeutic potential. In this case, the discovery of allosteric inhibitors was made by accident, but as our understanding of protein structure and allostery improves, expect to see more efforts to approach drug design in this way.

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Characterization of the mimivirus cyclophilin

An interesting article showed up today on the Articles in Press page at the Journal of Molecular Biology. Vu Thai, the most talented young crystallographer I have never wanted to punch in the face, and Elan Eisenmesser, a man of sharp wits and sharper dress, along with several collaborators, have performed a really extensive characterization of the cyclophilin encoded in the mimivirus genome. This is an intriguing study because the role of cyclophilins in viral life cycles is still very poorly understood, even though they are known to aid the infectivity of HIV and SARS.

Typically, these viruses do not encode their own cyclophilins -- this would be a pretty costly thing for such a small and limited organism -- but rather hijack them from the local cellular machinery. What still hasn't been cleared up is exactly why that happens. The catalytic activity of cyclophilins is to convert proline residues from a cis conformation, in which the C alpha atoms of sequential residues lie on the same side of the C'-N bond, to a trans conformation in which they are on the opposite side. It's been demonstrated that human cyclophilin A (hCypA) catalyzes this conversion on the HIV capsid. However, it's not known whether this is the key role and reason for its incorporation. hCypA is also known to mediate some cell-binding events for HIV, and it's hypothesized that this is the only reason for inclusion, and the cis-trans isomerization is incidental.

The mimivirus cyclophilin (mimicyp) has the potential to answer some of these questions. One of the keys here is that it has been carried along in the viral genome. Because viruses are such efficient parasites operating under strong selective pressure, it stands to reason that anything the virus considers important enough to carry along with it is critical to its survival. Granted, mimivirus is a bit of an odd duck, one of the largest viruses known. Additionally, its primary targets are amoebae, though mimivirus has also been known to cause pneumonia by direct attack of human cells. And indeed, Thai et al. find that mimicyp most closely resembles an amoeboid cyclophilin.

The results start to get progressively weirder from there. Firstly, mimicyp has a vanishingly low affinity for cyclosporin, the molecule that gave cyclophilins their name. Moreover, it doesn't appear to have catalytic activity towards a standard proline isomerase substrate at all. The sequence of the protein is missing key residues that typically interact with isomerization substrates and contribute to catalysis, and its putative active site is uncharacteristic of active cyclophilins. Mimicyp crystallizes with an unusual trimeric arrangement, and Elan has demonstrated that it also forms multimers in solution that can be dissociated by adding arginine. Nonetheless, mimicyp localizes to the outer capsid of the mimivirus and is thought to be critical to infectivity, though Thai et al. were unable to demonstrate this for infection of Acanthamoebae polyphaga.

So what we have here is a cyclophilin that is catalytically dead, forms strange multimer arrays, and can't yet be shown to be important for infectivity, that nonetheless is always carried along by mature virions, and is so critical to the success of the virus that it is carried along in the genome and expressed despite significant selective pressure in favor of exclusion, mutation, or silenced expression. Granted some of these pressures are alleviated by the sheer size of the genome and virus, but others (particularly expression) are not. Thai et al. suggest a number of reasons why mimicyp might be important, including charge neutralization of the capsid, mediation of entry and viral disassembly, or capsid-masking similar to that performed by hCypA for HIV. And, of course, the possibility exists that mimicyp has some other, as-yet-unidentified catalytic activity that is essential for mimivirus infectivity, or is only critical for infecting certain amoebae or cells.

One might also surmise that the ability of mimivirus to infect vastly different hosts to some degree relies on its carrying along this particular cyclophilin, which is probably quite dissimilar to any protein abundantly found in human cells. This only brings us back to the question of why mimicyp is so important in the first place.

Ultimately this paper does not reveal the precise reason why cyclophilins are used by viruses, and indeed it will probably do more to spur debate than end it. Nonetheless, Thai et al.'s research can be taken as adding weight to the proposition that the catalytic activity of cyclophilins on virions is incidental, or at least that its importance is an idiosyncratic feature of particular viruses. Mimicyp may also be our first introduction to a new class of cyclophilins, catalytically inactive, cyclosporin-free (cyclophobins? cyclomehs?), and multimeric. What it is that these cyclophilins do and why they are important will be an intriguing inquiry to follow.

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