Category Archives: Hot off the press

News about research done in our lab.

Structure of the Qbeta replicase holo complex

Structure of the Qbeta holo complex

Crystal structure of the Qbeta holo complex bound to ribosomal protein S1

Today, our lab in collaboration with the labs of Charlotte Knudsen and Frans Mulder, published a paper describing crystal and NMR structural studies of the Qbeta replicase holo complex bound to ribosomal protein S1. In the paper, we show how S1 interacts with the viral replicase along with the two other host proteins, elongation factors EF-Tu and EF-Ts, to generate the fully functional, viral replicase enzyme. Using NMR, we further show how  two of the OB domains of S1 are involved in binding viral RNA during replication.

For more information, have a look at the paper in Nucleic Acids Research.

Gytz, H., Mohr, D., Seweryn, P., Yoshimura, Y., Kutlubaeva, Z., Dolman, F., Chelchessa, B., Chetverin, A. B., Mulder, F. A. A., Brodersen, D. E. and Knudsen, C. R. (2015) ” Structural basis for RNA-genome recognition during bacteriophage Qβ replication”, Nucleic Acids Res, 43(22):10893-906.

Computational studies of NRPS substrate specificity

Active site of NRPS A domains

Regions involved in conferring substrate specificity among fungal NRPS A domains

Today, our lab in collaboration with the lab of Christian Storm Pedersen at the BiRC bioinformatics centre, published a paper in Bioinformatics describing a novel computational analysis of the substrate conferring code of fungal non-ribosomal peptide synthetase (NRPS) A domains, which are responsible for recognising and activating the individual amino acid substrates during peptide synthesis. We can show that a much larger part of the A domain than hitherto imagined is involved in conferring substrate specificity, and could map this to the structure of the A domain.

For more information, have a look at the paper in Bioinformatics.

Knudsen, M., Søndergaard, D., Tofting-Olesen, C., Hansen, F.,T., Brodersen, D. E., Pedersen, C. N. (2015) “Computational discovery of specificity-conferring sites in non-ribosomal peptide synthetases”, Bioinformatics, 32(3):325-9.

Structure of the 240 kDa C-P lyase

Crystal structure of the C-P lyase core complex

Crystal structure of the C-P lyase core complex determined by x-ray crystallography

Today, our lab published a full article in Nature describing the crystal structure of a 240 kDa core complex of the E. coli C-P lyase, an enzyme required for utilisation of phosphonate compounds in bacteria. Phosphonate are characterised by a direct  carbon-phosphorus (C-P) bond, which is stable to  hydrolysis and therefore requires a totally different enzymatic machinery. In C-P lyase, cleavage of the bond is achieved using radical chemistry in the subunit PhnJ, which is one of four proteins in the hetero-octameric core complex. We also show, using negative stain electron microscopy, that a fifth subunit, PhnK, an ABC cassette protein, binds as a monomer to the core complex via a conserved loop in PhnJ and breaks the two-fold symmetry of the core.

For more information, have a look at the paper in Nature.

Seweryn, P., Van, L. B., Kjeldgaard, M., Russo, C. J., Passmore, L. A., Hove-Jensen, B., Jochimsen, B., Brodersen, D. E. (2015) “Structural insights into the bacterial carbon-phosphorus lyase machinery”, Nature, 525(7567): 68-72.

Structural analysis of the THO complex by SAXS

Structures of the THO complex components Mft1p and Thp2p

Structures of the THO complex components Mft1p and Thp2p

Today, our lab published a comprehensive analysis of the yeast THO complex by small-angle x-ray scattering in the open access journal, PLoS One. The THO complex is intimately involved in coupling transcription to downstream mRNP processing and export from the nucleus. In the paper, we dissect the architecture of the complex through purification of sub complexes and structure determination of these by SAXS. Putting the pieces together reveals the position of each of the 5 proteins in the core complex and opens up new possibilities for a functional understanding of the complex.

For more information, have a look at the paper in PLoS One.

Poulsen J. B., Sanderson L. E., Agerschou E. D., Dedic E., Boesen T., Brodersen D. E. (2014), “Structural Characterization of the Saccharomyces cerevisiae THO Complex by Small-Angle X-Ray Scattering”, PLoS One, 9(7):e103470.


Small-angle x-ray scattering structures of the Rrp6p-Rrp47p complex

Structure of the Rrp6p-Rrp47p complex by SAXS

Structure of the Rrp6p-Rrp47p complex by SAXS

Today, our lab has published structural and functional studies of the yeast exosome Rrp6p-Rrp47p complex in Biochem. Biophys. Res. Commun. In this paper, we show how the two proteins, despite the fact that they both multimerise, form a stable 1:1 complex with Rrp47p situated on the “top” of the Rrp6p exonuclease domain. RNA degradation experiments with and without Rrp47p present further show that the co-factor has a very limited effect on the activity and specificity of the nuclease. Together, our data suggest a role for Rrp47p in higher-order organisation of the exosome complex, perhaps linked to interactions with other protein factors.

For more information, have a look at the paper in Biochem. Biophys. Res. Commun.

Dedic E., Seweryn P., Jonstrup A. T., Flygaard R. K., Fedosova N. U., Hoffmann S. V., Boesen T.,Brodersen D. E. (2014), “Structural analysis of the yeast exosome Rrp6p-Rrp47p complex by small-angle X-ray scattering”, Biochem Biophys Res Commun, 450(1):634-40.

VapC20 of M. tuberculosis cleaves the SRL of 23S rRNA

tRNA(fMet) and the SRL of 23S rRNAToday, a joint publication with the Gerdes group appeared online in Nature Communications. In this paper, we describe how the VapC20 toxin (VapBC-type) of Mycobacterium tuberculosis specifically cleaves the sarcin-ricin loop (SRL) of 23S ribosomal RNA when activated. We further show that the structure of the SRL is surprisingly similar to that of a tRNA anticodon stem loop (ASL) suggesting that VapC-type toxins bind and cleave RNA targets with similar structure.

For more information, have a look at the paper in Nature Communications.

Winther, K. S., Brodersen, D. E., Brown, A. K., Gerdes, K. (2013), “VapC of Mycobacterium tuberculosis Inhibits Translation by Endonucleolytic Cleavage of the Sarcin-Ricin Loop of 23S rRNA”, Nature Commun, 4:2796.

Yeast Sub2p has a conserved N-terminal domain

Sub2p NTM motifToday, we published a paper in RNA together with the Torben Heick Jensen and Eckhard Jankowsky labs that shows, both experimentally and by using bioinformatics, that the S. cerevisiae helices Sub2p contains a hitherto uncharacterised N-terminal domain with a sequence signature reminiscent of the ubiquitin… We also show, using biochemical assays, that the N-terminal domain does not affect the main helices functions, ATPase activity, RNA binding, and RNA unwinding.

For more information, have a look at the paper in RNA.

Saguez, C., Gonzales, F. A., Schmid, M., Bøggild, A., Latrick, C. M., Malagon, F., Putnam, A., Sanderson, L., Jankowsky, E., Brodersen, D. E., Jensen, T. J. (2013) “Mutational analysis of the yeast RNA helicase Sub2p reveals conserved domains required for growth, mRNA export and genomic stability”, RNA, 19(10):1363-71.

New software for crystallisation made public

Mimer screenshotToday, we published a paper describing a new piece of software useful for designing and setting up macromolecular crystallisation experiments using a simple spreadsheet-based approach. The software, called Mimer, can not only calculate the correct volumes to pipette given stock and desired final concentrations of ingredients, it can also perform incomplete factorial and grid screen designs.

For more information, read the paper in Acta Crystallographica Section F.

Brodersen, D. E., Andersen, G. R., and Andersen, C. B. F. (2013), ” MIMER: An Automated Spreadsheet-Based Crystallization Screening System”, Acta Cryst. F, 69(Pt 7):815-20.

Crystallisation of the isolated Shigella VapC toxin

S. flexneri VapC crystalsToday, we published a brief communication reporting the successful crystallisation of the isolated VapC toxin from Shigella flexneri. Together with our previously published structure of the Shigella VapBC complex, this structure will complete our picture of what happens with VapC upon inhibition by VapB.

For more information, read the paper in Acta Crystallographica Section F.

Xu, K., Dedic, E., Cob-Cantal, P., Dienemann, C., Bøggild, A., Winther, K. S., Gerdes, K., and Brodersen, D. E. (2013), “Protein expression, crystallization, and preliminary x-ray crystallographic analysis of the isolated Shigella flexneri VapC toxin”, Acta Cryst. F, 69(Pt 7):762-5.