Promiscuous proteins

Gone are the days when the one protein, one function presumption prevailed. Many proteins are multifunctional and multispecific, that is they have multiple binding partners for carrying out various roles in the cell. Here's a new review by Erijiman et al. in Biochemistry about multispecifity, covering various examples of promiscuous proteins and the different ways in which they achieve their multispecificity.

Proteins can interact with multiple binding partners by having distinct binding interfaces or domains. By this route, it's possible for the protein to optimise each binding site for its specific partner as the interfaces are independent (although there may be some cross-talk). An example of this from the proteins that I'm interested in is the Rel protein of bacteria. This protein has a synthesis domain for producing the alarmone ppGpp, and a hydrolysis domain for degrading it. The interfaces are on different sides of the protein, so are in some sense independent, although binding of a molecule in one site may influence the function of the other site by switching the conformation of the protein.

As an alternative solution, a protein may bind through one interface that is able to interact with multiple partners. An example of this is the archaeal elongation factor EF1A, which delivers aminoacylated tRNA, release factor aRF1 and mRNA decay protein aDom34 to the ribosome, binding all three by overlapping binding sites.

My rather simplistic representation of how a protein's binding interfaces can be distributed. A: independent binding sites eg Rel. B: overlapping binding sites eg aEF1A.

Multispecificity is great for the cell (especially cells with reduced, streamlined genomes) in that from just one gene, you get a lot of functional value. However, it also introduces some compromises for the protein, in terms of optimising its specificity for binding partners (especially true for proteins with overlapping binding sites), and brings about challenges in terms of regulating the different functions. A way to escape these problems is by gene duplication and subfunctionalisation for the different binding functions of the protein. Indeed this has occurred in some organisms for both of my examples above. In proteobacteria, Rel has been duplicated, resulting in RelA and SpoT, specialised for ppGpp synthesis and hydrolysis respectively. Similarly, in eukaryotes, two duplications of EF1A-like proteins have led to eEF1A, eRF3 and Hbs1, specialised for binding aa-tRNA, eRF1 and eDom34 respectively. However, it would be wrong to say that eEF1A now only has one function, as in fact it has many many more functions... but that's another story!

For more info on these proteins, check out my other blog posts:

- Just how hungry am I and what should I do about it (about RelA/Rel/SpoT) 

- Testing predictions from phylogenetics: mRNA decay mechanisms in archaea (about aEF1A)

Refs

Erijman A, Aizner Y, & Shifman JM (2011). Multispecific recognition: mechanism, evolution, and design. Biochemistry, 50 (5), 602-11 PMID: 21229991

Hogg T, Mechold U, Malke H, Cashel M, & Hilgenfeld R (2004). Conformational antagonism between opposing active sites in a bifunctional RelA/SpoT homolog modulates (p)ppGpp metabolism during the stringent response [corrected]. Cell, 117 (1), 57-68 PMID: 15066282

Saito K, Kobayashi K, Wada M, Kikuno I, Takusagawa A, Mochizuki M, Uchiumi T, Ishitani R, Nureki O, & Ito K (2010). Omnipotent role of archaeal elongation factor 1 alpha (EF1α in translational elongation and termination, and quality control of protein synthesis. Proceedings of the National Academy of Sciences of the United States of America, 107 (45), 19242-7 PMID: 20974926

Tsunami and earthquake crisis - Non-Believers Giving Aid

Because praying for Japan doesn't help anyone, but donations save lives. 

 http://givingaid.richarddawkins.net/

"Non-Believers Giving Aid and the Richard Dawkins Foundation for Reason and Science are once more partnering with the International Committee of the Red Cross to bring much needed help to people whose lives have been torn apart by natural disaster. Every cent and penny of money donated via Non-Believers Giving Aid will be forwarded to the International Red Cross – and if you are in the UK and you complete the Gift Aid Declaration along with your donation, we will pass that on in its entirety too."

Well, this is unexpected! Drosophila mitochondrial translation elongation Factor G1 contains a nuclear localization signal.

Most eukaryote genomes encode two mitochondrial translation elongation factor Gs. I recently had a paper in MBE about the origin and evolution of these factors, and I've blogged about it previously. I spotted a very surprising article in PloS One today: "The Drosophila Mitochondrial Translation Elongation Factor G1 Contains a Nuclear Localization Signal and Inhibits Growth and DPP Signaling." For some reason, mtEFG1 is dual targeted to the nucleus as well as the mitochondrion. The localisation signal is proposed to be found at the C terminus, unlike the mitochondrial transit peptide, which is found at the N terminus. The authors suggest a model in which "if mitochondrial ATP synthesis is low or EF-G1 is overexpressed and import of EF-G1 proteins into mitochondria is a limiting step, some EF-G1 proteins can accumulate outside of mitochondria and translocate into the nucleus, where they inhibit cellular growth and proliferation."

The authors carry out mutagenesis and subcellular localization analysis of mtEFG1, and find that although the Drosophila mtEFG1 gene is essential, it's not required in every tissue. This leads them to suggest that in some tissues, mtEFG2 and not mtEFG1 is the primary translocation factor. This would be very unexpected as neither spirochete or human spd/mtEFG2 can not promote translocation, and instead spd/mtEFG2 is proposed to be specialised for EF-G's role in ribosome recycling. Additionally, the alignment in my paper shows mtEFG2s don't have the conserved amino acids involved in translocation functions, such as interaction with peptidyl-tRNA. However intramolecular and ribosome interaction sites are well conserved in mtEFG2, suggesting it maintains EF-G-like structural integrity and ribosome binding abilities. Maybe this is sufficient to promote translocation in some conditions? In fact, even the more distantly related EF-G2 of Thermus, which belongs to a whole other ancient subfamily is capable of translocation, hinting that although classical EF-G is very well conserved at the primary sequence level, at least in some conditions the ribosome can accommodate and translocate with more divergent homologs that maintain an EF-G-like structure.

The model of mtEFG1 subcellular location being related to mitochondrial ATP synthesis proposed by Trivigno and Haerry presents a paradox, which they acknowledge: "If EF-G2 functioned as an elongation factors in tissues like the heart, mitochondrial translation and ATP synthesis would occur at normal levels, and EF-G1 would be imported into mitochondria and not accumulate in the nucleus. On the other hand, in tissues like the liver, where EF-G2 cannot function as an elongation factor, mitochondrial translation would decrease, ATP levels would drop, EF-G1 import into mitochondria would decrease and accumulation in the nucleus increase, which would further exacerbate the problem."


So, in conclusion, it's all rather surprising and the model just doesn't seem quite right... it's all very well for a bioinformatician to say this I know, but more experiments needed!

Refs

Atkinson GC, & Baldauf SL (2011). Evolution of elongation factor g and the origins of mitochondrial and chloroplast forms. Molecular biology and evolution, 28 (3), 1281-92 PMID: 21097998

Trivigno C, & Haerry TE (2011). The Drosophila Mitochondrial Translation Elongation Factor G1 Contains a Nuclear Localization Signal and Inhibits Growth and DPP Signaling. PloS one, 6 (2) PMID: 21364917

Tsuboi, M., Morita, H., Nozaki, Y., Akama, K., Ueda, T., Ito, K., Nierhaus, K., & Takeuchi, N. (2009). EF-G2mt Is an Exclusive Recycling Factor in Mammalian Mitochondrial Protein Synthesis Molecular Cell, 35 (4), 502-510 DOI: 10.1016/j.molcel.2009.06.028

Connell, S., Takemoto, C., Wilson, D., Wang, H., Murayama, K., Terada, T., Shirouzu, M., Rost, M., Schüler, M., & Giesebrecht, J. (2007). Structural Basis for Interaction of the Ribosome with the Switch Regions of GTP-Bound Elongation Factors Molecular Cell, 25 (5), 751-764 DOI: 10.1016/j.molcel.2007.01.027

"Just how hungry am I? And what should I do about it?" How bacteria decide.

Escherichia coli bacteria may lack rumbling stomachs, but they know they're hungry when their ribosomes are empty. In starvation conditions, the lack of available amino acids leads to an increase in the cellular concentration of tRNAs that are uncharged with amino acids. The presence of uncharged tRNAs on the ribosome is sensed by the protein RelA in E. coli and other gamma- and beta-proteobacteria, and by close homologs called Rel or RSH in other bacteria. In such starved conditions, RelA-like proteins (from here-on I'll call them RSHs (RelA/SpoT Homologs) produce the small 'alarmone' molecule ppGpp. This onsets the so-called stringent response, where ppGpp induces changes in cell physiology, down-regulating the translation machinery and up-regulating amino acid biosynthesis machinery. It's also involved in responses to other environmental inputs such as glucose and fatty acid and iron availability. Here's a link to great review of ppGpp synthesis, hydrolysis and effects.

It's becoming clear that there is tight and complex regulation by ppGpp, and there are a couple of recent papers to this effect, which I'll briefly discuss. Using microarrays, Traxler et al. have carried out transcriptional profiling of the stringent response in E. coli, the organism where most research on bacterial stress and starvation has been carried out. They studied how ppGpp effects two transcriptional regulons, Lrp (leucine responsive protein that regulates amino acid biosynthesis and transport) and the σ-factor RpoS that regulates general stress response. They found that the Lrp regulon requires only a low level of ppGpp for its induction, while the RpoS regulon is induced only in high concentrations of ppGpp (also see comment by Carlos Balsalobre). Thus, the RpoS-dependent response is only triggered as a kind of emergency measure if the induction of the Lrp-dependent response has been induced and failed to save bacterial population growth.

It is also becoming evident that the production of ppGpp and its effects differ among different bacteria.  The bacterium Caulobacter crescentus lives in environments where amino acid concentrations are generally low. Boutte and Crosson have found that in this organism, ppGpp is synthesised in response to glucose and ammonium starvation, but not amino acid starvation. They find the RSH of this bacterium binds the ribosome, and exhibits "AND-type signaling logic", in which detection of an uncharged tRNA on the ribosome is a necessary but alone, insufficient signal for activation of ppGpp synthesis. This hints at synergistic control, where multiple starvation signals contribute to the accumulation of ppGpp at a high levels. This is different to the "OR-type" logic of E. coli, suggesting the environmental niche of the bug affects how starvation is perceived and dealt with.

The circuitry for ppGpp synthesis and its downstream effects are clearly intricate, involving feedback loops and inter-molecular cross-talk. I'm writing up some bioinformatic work now on the distribution and evolution of RSHs that synthesise or hydrolyse ppGpp, or do both. My results, like those of Boute and Crosson suggest the circuitry is dynamic across bacterial taxa, being often rewired during evolution with the help of gene duplications, domain gain and loss and horizontal gene transfer. This will be blogged of course!

For more posts about the stringent response, check out Vasya's blog

Refs

Traxler MF, Zacharia VM, Marquardt S, Summers SM, Nguyen HT, Stark SE, & Conway T (2011). Discretely calibrated regulatory loops controlled by ppGpp partition gene induction across the 'feast to famine' gradient in Escherichia coli. Molecular microbiology, 79 (4), 830-45 PMID: 21299642

Balsalobre C (2011). Concentration matters!! ppGpp, from a whispering to a strident alarmone. Molecular microbiology, 79 (4), 827-9 PMID: 21299641

Boutte CC, & Crosson S (2011). The complex logic of stringent response regulation in Caulobacter crescentus: starvation signaling in an oligotrophic environment. Molecular microbiology PMID: 21338423

Pets in Tartu in need of good homes

We're traveling too much at the moment to get a pet, but we're looking forward to getting a cat as soon as we're settled enough. We're flying to Sweden tomorrow for a few weeks... and then, maybe when we're back in Tartu we could ask the landlady, just hypothetically of course, whether we'd be allowed to have a cat. I have a feeling she'll say yes, in which case, maaaybe we'll head over to the Tartu animal shelter just to have a look... They have so many lovely animals there who are in need of good homes.

They list their cats online here:
http://www.loomadevarjupaik.eu/kassid-koduootel/

And dogs here:
http://www.loomadevarjupaik.eu/koerad-koduootel/

If you're in Tartu and are thinking about getting a pet, please consider the shelter first. It's run by volunteers who do a great job of caring for the animals, with very little money available to them.