New research, published in Cell, illuminates the molecular “trains” that transport cargoes essential for human health and development.
Virtually every cell in the human body grows an antenna-like structure on its surface, which is used to receive vital signals from the body and the outside world. Perturbations in this process cause a wide range of human disorders spanning loss of eyesight, cystic kidneys, breathing problems, and infertility, among other conditions.
New research from the Institute of Structural and Molecular Biology has shed new light on the molecular “trains” that underpin this process, and how they misfunction in disease. Using cryo-election microscopy, a powerful technique for determining the three-dimensional shape of biological molecules, the team was able to see the structure of the proteins that make up the trains and how they carry their vital cargoes. Cell biology experiments showed that the range of cargoes transported by the trains is even wider than anticipated. The findings will help researchers to interpret patient mutations in the proteins that cause disease and design new experiments.
This research, funded by the Wellcome Trust and BBSRC with co-first authors Dr. Sophie Hesketh and Dr. Aakash Mukhopadhyay and co-senior authors Dr. Katerina Toropova and Dr. Anthony Roberts, was published in Cell on 2 December 2022: https://www.cell.com/cell/fulltext/S0092-8674(22)01422-2
Researchers in Biological Sciences at Birkbeck, in collaboration with a group at the Peter MacCallum Cancer Centre in Melbourne, have determined the structure of a protein assembly used by the immune system to kill unwanted cells. The immune system uses cytotoxic T lymphocytes and natural killer cells to act as executioners when it detects the presence of virally infected or cancerous cells. The cytotoxic and killer cells contain small membrane parcels filled with the protein perforin, which can punch holes through cell membranes, along with the toxic granzyme enzymes. When an infected cell is detected, the killer cell latches onto it and ejects some of the membrane parcels with their toxic contents so that the perforin protein punches holes in the target cell membrane, through which the toxic granzymes enter, rapidly causing the target cell to die (Figure 1). The cytotoxic and killer cells are professional assassins that can kill many victims in rapid succession, briefly attaching, ejecting their lethal cargo, and then moving on to the next victim. Perforin is an essential protein for survival, and unfortunate individuals who lack functional perforin usually die of infection or cancer in early childhood. On the other hand, over active killer cells can also cause serious damage, by triggering inflammation and killing healthy cells.
A former postdoctoral researcher, Marina Ivanova (now at Imperial College), determined the perforin structure in the group of Professor Helen Saibil, and the paper has been published in Science Advances. Perforin is made as single protein molecules that are stored inside their membrane compartment until they are needed, but when they are released, they join up into rings of around 22 molecules and undergo a dramatic shape change in order to punch the hole through the target membrane. Two parts of the protein that are at first coiled up in the molecule extend and join up into the ring to punch through the membrane. This shape change is shown in Figure 2, with the part of the protein that makes the big change highlighted in pink.
Figure 3 shows two views of a perforin ring (multicoloured molecules) enclosing a hole in a cell membrane (shown as a pale blue slab). Now that we know the details of the pore structure, it will be possible to think about designing drugs to either enhance or prevent its activity. This could eventually lead to new therapeutics for certain autoimmune diseases and the condition familial hemophagocytic lymphohistiocytosis.
Transcription is carried out by evolutionary conserved RNA polymerases and subject to regulation by different strategies. The control of individual genes is enabled by a plethora of DNA-binding transcription factors that respond to changes in the environment and enable the up- or downregulation gene expression; the structural basis and mechanisms of genespecific regulation has been characterised in great detail. Much less is known about the global regulation of transcription that is enabled by RNAP-binding factors. Two research articles from the ISMB RNAP laboratory published back-to-back in Nature Communications explore and characterise the global control of the archaeal RNAP in two different scenarios. Firstly, a multidisciplinary study combining functional genomics with in vitro transcription assays hints at a paradigm shift for the regulation of transcription in Archaea (Blombach et al., 2021), and secondly, a cryo-EM analysis elucidates, for the first time, the structural basis of RNAP inhibition by repressors involved in the archaeal virus-host arms race (Pilotto et al., 2021). Dr Fabian Blombach, the lead author of the first study explains: ‘The current paradigm in the field dictates that transcription regulators positively or negatively interfere with the recruitment of RNA polymerase to the promoter at the stage of initiation. We challenged this hypothesis using a functional genomics approach by mapping the dynamic (re-)distribution of RNAP correlating it with the cellular RNA levels, genome-wide and at single nucleotide resolution. We could show that it is not only simple ‘access’ of RNAP to the promoter that determines the expression level of a gene, but sophisticated mechanisms that occur during early elongation (Fig. 1). The factors Spt4/5 and Elf1 are successively recruited to the transcription elongation complex before the RNA polymerase makes a transition into productive transcription. Quite surprisingly, we also found that RNA polymerases in the early elongation phase recruit a ribonuclease, the transcription termination factor aCPSF1, that negatively regulates transcription likely by RNA cleavage-induced premature termination mechanism, a mode of regulation that is evolutionary conserved in bacteria and eukaryotes.’
Dr Simona Pilotto, the lead author of the second study elaborates on her work: The inhibition of RNAP and the resulting attenuation of the transcriptome plays a crucial role in the interaction between viruses and their hosts during infection. My aim is to understand the structural basis of ‘switching off’ RNAP, as this is highly relevant for the design of novel antibiotics and antiviral drugs. I have solved the cryo-EM structures of the complexes formed between the Sulfolobus acidocaldarius RNAP and two distinct regulators (RIP and TFS4), and my structures provide insights into the detailed mechanisms underlying the very potent inhibition (Fig. 2). RIP is encoded by the Acidianus two-tailed virus (ATV) and sterically interferes with the interaction of template DNA and transcription factors by molecular mimicry of initiation factors. TFS4 is encoded by the Sulfolobus host and is expressed in response to infection with the Sulfolobus Turreted Icosahedral Virus (STIV). TFS4 is related to elongation factors including TFIIS and targets the RNAP NTP entry pore through which it inactivates RNAP in an allosteric fashion by inducing a widening of the DNA-binding channel disrupting the bridge helix and trigger loop active site motifs. The most intriguing conclusion of my work is that the inhibitory strategies and mechanisms reveal the underlying functional conservation of RNAPs: unrelated inhibitors have evolved to exploit factor- and nucleic acid binding sites, and conformational flexibilities that are intrinsic to all RNAPs to effectively repress its activity.
Blombach, F., Fouqueau, T., Matelska, D., Smollett, K., and Werner, F. (2021). Promoter-proximal elongation regulates transcription in archaea. Nat Commun 12, 5524. Pilotto, S., Fouqueau, T., Lukoyanova, N., Sheppard, C., Lucas-Staat, S., Diaz-Santin, L.M., Matelska, D., Prangishvili, D., Cheung, A.C.M., and Werner, F. (2021). Structural basis of RNA polymerase inhibition by viral and host factors. Nat Commun 12, 5523.
Professor Steven Perkins (Professor of Structural Biochemistry, UCL) made the front cover of October’s Biochemical Journal (vol. 476, issue 19) with his latest paper:
A joint project with UCL Medicine and the University of Bedfordshire, it is a combination of work done on Professor Perkins’ new analytical ultracentrifuge, protein crystallography and recombinant proteins.
Citation: Lau, A. M., Zahid, H., Gor, J., Perkins, S. J., Coker, A. R. & McDermott, L. C. (2019) Crystal structure of zinc-α2-glycoprotein in complex with a fatty acid reveals multiple different modes of lipid binding. Biochem. J. 476, 2815-2834.
A paper by Professor Neil McDonald’s research group, in collaboration with Sir Richard Treisman, has been published in Nature Cell Biology, identifying a new RPEL-family of rhoGAPs that link Rac/Cdc42 GTP loading to G-actin availability.
Professor Helen Saibil’s research group have published a paper titled ‘Two-Step Activation Mechanism of the ClpB Disaggregase for Sequential Substrate Threading by the Main ATPase Motor’ in Cell Reports in June 2019.
Professor Bart Hoogenboom’s research group published a paper titled ‘Quantification of Biomolecular Dynamics inside Real and Synthetic Nuclear Pore Complexes using Time-Resolved Atomic Force Microscopy’ in ACS Nano in June 2019.
We are the first group to publish a paper on how a new cyclic ion-mobility mass-spectrometry (cIMMS) device, manufactured by Waters, can be used to probe protein structure and dynamics. In particular, the tandem ion mobility capabilities of the instrument allow us to probe in very fine detail protein unfolding pathways and for the first time to do so for co-existing and interconverting conformers. We are now using this technology to study proteins involved in protein misfolding diseases such as human amyloid islet polypeptide.
The paper is:
Eldrid, C.; Ujma, J.; Kalfas, S.; Tomczyk, N.; Giles, K.; Morris, M.; Thalassinos, K. Gas Phase Stability of Protein Ions in a Cyclic Ion Mobility Spectrometry Traveling Wave Device. Anal. Chem.2019, 91 (12), 7554–7561 https://doi.org/10.1021/acs.analchem.8b05641
Professor Maya Topf’s research group published a paper titled ‘Protein interactions and consensus clustering analysis uncover insights into herpesvirus virion structure and function relationships’ in Plos Biology on 14th June.
Professor Frances Brodsky’s research group published a paper titled ‘Genetic diversity of CHC22 clathrin impacts its function in glucose metabolism’ in eLife on 4th June.
