Author Archives: Ditlev

About Ditlev

I lead a structural biology research lab at the Department of Molecular Biology, Aarhus University, Denmark, focusing on the molecular mechanisms underlying RNA metabolism and decay in eukaryotic organisms. In the lab we use molecular biology, biochemistry, and x-ray crystallography to study the structure and function of a number of important protein and RNA macromolecules involved in regulation and decay of RNA in both the nuclear and cytoplasmic compartments of the eukaryotic cell.

Characterisation of a novel NADase toxin-antitoxin system

In a paper published today in Molecular Microbiology, we provide a thorough functional and structural characterisation of a novel toxin-antitoxin module of the RES-Xre class. Based on the presence of three conserved residues (R, E, and S), the RES toxins were proposed to constitute a new family of metal-dependent RNases. However, our work demonstrates for the first time that the toxin fold is reminiscent of the ADP-ribosyl transferase enzymes such as the cholera and diphtheria toxins. However, rather than transferring an ADP-ribosyl moiety to a target protein in a eukaryotic cell, the toxin acts intracellularly in the bacterium itself, by degrading NAD+ into nicotinamide and ADP-ribose. We show that this has the effect of stopping all major gene expression functions, starting with transcription, followed by translation, and finally replication. In this sense, the end result of RES toxin activation is thus the same as many of the RNase (RNA interferase) type toxins, which function by degrading translational RNAs (mRNA, tRNA, or rRNA).

For more information, see the paper in Mol. Microbiol:

Skjerning, R. B., Senissar, M., Winther, K. S., Gerdes, K., and Brodersen, D. E. (2018) “The RES domain toxins of RES-Xre toxin-antitoxin modules induce cell stasis by degrading NAD+”, Mol Microbiol, 111(1): 221-236.

New review on ABC cassettes in phosphonate breakdown

Phn4 with outline

Crystal structure of the E. coli carbon-phosphorus (C-P) lyase core complex.

In a new review, just published online today, we delve into the possible functional roles of the ATP-binding cassettes (ABC cassettes) encoded in the E. coli phn operon, which is required for uptake and degradation of phosphonate compounds during phosphate limited growth. ABC cassette proteins are most well known for their function in ABC transporters and it looks like the phn operon also encodes a protein with this role (PhnC). Curiously, however, the operon also encodes two other ABC modules for which the function is less well known. One of them, PhnK, we know binds to a large protein complex known as the C-P lyase core complex (shown in the figure to the right) but the reason for this is not clear. It is thought that ATP is required for the reaction, so this could suggest a possible role for the ABC cassette.

Phn operon

Overview of the 14-cistron phn operon of E. coli

The other protein, PhnL, is even more mysterious as we neither know if it binds to the enzymatic core complex nor what its role might be. If any of this has caught your interest, head over to BioEssays and have a look at the paper:

Manav, M. C., Sofos, N., Hove-Jensen, B., and Brodersen, D. E. (2018) “The ABC of phosphonate breakdown: A mechanism for bacterial survival”, BioEssays, 40(11): e1800091.

 

Ditlev Brodersen awarded NNF HM Ascending Investigator grant

Today, Ditlev Brodersen was awarded the 2018 Hallas-Møller Ascending Investigator grant from the Novo Nordisk Foundation (10 m DKK) to study microbial defence mechanisms by structural methods. The grant will allow the lab to pursue a range of new projects within the main goal of understanding how microbes (bacteria and archaea) defend themselves against nutritional stress, antibiotics, other organisms etc.

Link to news story on the Department’s web site.

Link to news story on the Novo Nordisk Foundation’s web site.

Link to popular news story about the research project on ScienceNews.dk.

 

First archaeal anti-CRISPR protein revealed

Protein fibres formed by the AcrID1 anti-CRISPR protein

Protein fibres formed by the AcrID1 anti-CRISPR protein

Today, in collaboration with the labs of Xu Peng (University of Copenhagen) and Eugene Koonin (National Institutes of Health, USA), we describe the first example of an archaeal anti-CRISPR protein. We show that the anti-CRISPR protein, which is expressed by a special type of hyperthermophilic DNA viruses, block the CRISPR-Cas system of their host, Sulfolobus islandicus, by directly interacting with one of the Cas proteins and likely forming an oligomer.

We go on to determine the crystal structure of the AcrID1 anti-CRISPR protein and show that it forms a tight dimer with a tendency to oligomerise. These features (along with the extreme pH and temperature stability) are likely to be important for the ability of such a small protein to act as a “stick in the wheel” against the archaeal CRISPR-Cas system.

For more information, read our paper in Nature Microbiology.

He, F., Bhoobalan-Chitty, Y., Van, L. B., Kjeldsen, A. L., Dedola, M., Makarova, K., Koonin, E. V., Brodersen, D. E., Peng, X. (2018) “Anti-CRISPR proteins encoded by archaeal lytic viruses inhibit subtype I-D immunity”, Nat Microbiol, e-pub ahead of print.

New review on toxin-antitoxin systems published in Mol. Cell

TA review Mol Cell

Overview of the four most significant types of toxin-antitoxin systems in bacteria and archaea.

Today, our lab has published a comprehensive new review on toxin-antitoxin systems in collaboration with the labs of Kenn Gerdes and Namiko Mitarai at the University of Copenhagen. In this paper, we critically review the current literature in the field of toxin-antitoxin research and and highlight how multiple levels of regulation shape the conditions of toxin activation to achieve the different biological functions of TA modules

For more information, have a look at our review in Molecular Cell.

Harms, A., Brodersen, D. E., Mitarai, N., Gerdes, K. (2018) “Toxins, targets, and triggers: An overview of toxin-antitoxin biology”, Mol Cell, e-pub ahead of print.

Structural basis for (p)ppGpp synthesis in Gram positive bacteria revealed

ATP and GTP inside RelP

Electron density showing the presence of both ATP (as an ADPNP analogue) and GTP inside the active site of S. aureus RelP.

Today, we have published a comprehensive structural and functional analysis of a (p)ppGpp synthesizing enzyme (a so-called small alarmone synthetase) from S. aureus in the Journal of Biological Chemistry. These enzymes are only found in Gram positive bacteria and thought to respond to cellular stress by producing the alarmone molecule, (p)ppGpp.

Using substrate analogues and product molecules, we were able to trap the enzyme in both the pre-catalytic state (bound to ATP and GTP) as well as in the post-catalytic state (bound to (p)ppGpp). Together, these structures complete our view of the catalytic cycle of this group of enzymes. We also show, using biochemistry, that this enzyme (S. aureus SAS2, RelP) is not regulated allosterically by its product as the other homologous enzyme found in many of these organisms (SAS1, RelQ). Instead, the enzyme appears to be regulated by binding of metal ions, which means that it could respons to minute alterations of the cellular homeostasis.

For more information, read our paper in the Journal of Biological Chemistry.

The article was featured on the cover of the 1 March 2018 issue of the journal.

Manav, M. C., Beljantseva, J., Bojer, M. S., Tenson, T., Ingmer, H., Hauryliuk, V., Brodersen, D. E. (2018) “Structural basis for (p)ppGpp synthesis by the Staphylococcus aureus small alarmone synthetase RelP”, J Biol Chem, 293(9): 3254-64.

New review on structural conservation of the PIN domain

Structural conservation of the PIN domain

Structural conservation of the PIN domain across all domains of life.

Today, our lab has published a comprehensive review on the PIN domain, which is a ubiquitous protein domain found in all domains of life. Our analysis demonstrates that we are far from knowing everything we need to know about PIN domains, which in many cases (but not always) function as small RNA nucleases.

Firstly, there is still no structure of a PIN domain bound to its RNA target, which leaves open question both as to how RNA is specifically bound as well as how cleavage occurs. The active site, which consists of a number of acidic side chains, is conserved in many cases, but the details of catalysis are far from understood. Even more intriguingly, some PIN domains (such as those found among bacterial VapC toxins) appear to cleave very specific RNA species including tRNA and ribosomal RNA. Our review also highlights the need to understand how this specificity arises.

For more information, see the paper in Protein Science.

Senissar, M., Manav, M. C., Brodersen, D. E. (2017) “Structural conservation of the PIN domain active site across all domains of life”, Protein Sci, 26(8):1474-1492.

Protein palindromes discovered in bacterial antitoxins

C. crescentus VapBC apo-DNA morph

Crystal structure of the C. crescentus VapBC complex bound to DNA with disordered tails modelled.

Today, we have published a paper in Nucleic Acids Research describing crystal structures of the Caulobacter crescentus VapBC toxin-antitoxin complex both in the apo form and bound to operator DNA.

For the first time, we have detailed structures of the same TA complex both in the apo form and bound to DNA. Unexpectedly, the “tails” on the antitoxin molecules bind two toxin molecules each. We show that this is possible due to a “protein palindrome” sequence located near the C termini of the antitoxins. Even more surprising, the tails swap position upon binding DNA, suggesting that they might serve to interlock the complex when bound to the operator region. Sequence analysis of more than 4,000 VapB sequences finally reveal that this phenomenon is widespread among bacterial antitoxins.

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

Bendtsen, K. L., Xu, K., Luckmann, M., Winther, K. S., Shah, S. A., Pedersen, C. N. S., and Brodersen, D. E. (2017) “Toxin inhibition in C. crescentus VapBC1 is mediated by a flexible pseudo- palindromic protein motif and modulated by DNA binding”, Nucleic Acids Res, 45(5):2875-2886.

Crystal structure of human RBM7 revealed

The crystal structure of the RRM domain of human RBM7 from the nuclear exosome targeting (NEXT) complex). Sofos et al. (2016)

The crystal structure of the RRM domain of human RBM7 from the nuclear exosome targeting (NEXT) complex). Sofos et al. (2016)

Today, we have published a paper in Acta Cryst. Section F presenting the crystal structure of the RNA recognition motif (RRM) domain of human RBM7, a central component of the human nuclear exosome targeting (NEXT) complex.

The crystal structure reveals an unexpected pentameric assembly of the RRM domain, which has been highlighed on the cover of the journal.

For more information, have a look at the paper in Acta Crystallographica Section F.

Sofos, N., Winkler, M. B. L., and Brodersen, D. E. (2016) “RRM domain of human RBM7: purification, crystallization and structure determination”, Acta Cryst., F72:397-402.

NRPS responsible for sansalvamide discovered

Proposed non-ribosomal peptide synthesis pathway required for formation of the bioactive compound, sansalvamide

Proposed non-ribosomal peptide synthesis pathway required for formation of the bioactive compound, sansalvamide

Today, our lab published a joint paper with the fungal genetics group at Aalborg University headed by Prof. Henriette Giese. In the paper, we demonstrate using genetic knock-out strains, which non-ribosomal peptide synthetase is responsible for production of the potential anti-cancer drug, sansalvamide.

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

Romans-Fuertes, P., Søndergaard, T. E., Sandmann, M. I., Wollenberg, R. D., Nielsen, K. F., Hansen, F. T., Giese, H., Brodersen, D. E., and Sørensen, J. L. (2016) “Identification of the non-ribosomal peptide synthetase responsible for biosynthesis of the potential anti-cancer drug sansalvamide in Fusarium solani”, Curr Genet, 62(4):799-807.