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.
The Nobel Prize in Chemistry in 2021 has been awarded to German Benjamin List and British David MacMillan. Prof Stefan Howorka from the ISMB at UCL Chemistry explains: ‘The two researchers have developed a new class of catalysts that are inspired by Nature. Enzymes are widely used in biology as they initiate and specifically control biochemical reactions to achieve the desired stereochemistry while limiting the creation of undesirable by-products. Reconstructing these catalytic functions with smaller and cheaper synthetic units is of considerable scientific and industrial interest. Ideally, synthetic catalysts should also avoid precious metals such as platinum which are not environmentally friendly. List and MacMillan succeeded independently of each other in developing efficient biomimetic and “green” catalysts. In the late 1990s, List wondered whether amino acids found in the enzymes’ active site would also be able to achieve part of the same catalytic role if added in isolation. As proof-of-principle, List tested the catalytic properties of proline and related compounds in an aldol reaction. The specific question was whether the use of a chiral proline would control the stereochemical outcome of the reaction. Indeed, the chirality of the catalyst controlled which enantiomer of the aldol products was formed. MacMillan was working in the same field. MacMillan was motivated to develop new catalysts that avoid the widely used metals. Rather, he focused on environmentally harmless and inexpensive organic frameworks that contain -in addition to carbon- oxygen, nitrogen, sulphur or phosphorous. Similar to List, MacMillan also tested chiral versions of his organic catalysts but with a different reaction, the Diels-Alder cycloaddition. The reaction was successful as enantiopure products formed depending on the chirality of the catalysts. Reflecting the catalysts’ composition and enantioselective control, MacMillan coined the term ‘asymmetric organocatalysis’ This new field has grown dramatically and develops simple, easy-to-manufacture and environmentally friendly catalyst. This has a huge impact in science and industry to produce new pharmaceuticals or molecules that can capture light in solar cells. This year’s Nobel prize and the Nobel prize given in 2018 for ‘the directed evolution of enzymes’ underscore the importance of developing new catalytic tools, Prof Howorka concludes.
References: J. Am. Chem. Soc. 2000, 122, 2395-2396; J. Am. Chem. Soc. 2000, 122, 4243-4244
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.
The Wallace Group has recently received a 3-year grant from the Rosetrees
Trust for work on: ‘“Seeing” General Anaesthetics Bound to Sodium
Channels: Using Novel Structure/Function Information for Molecular
Understanding and Design, Enabling Improved Function and Safety’ – a
collaboration with Professor Hugh Hemmings’ Lab at the Weill Cornell Medical
Centre in New York.
Amandine Marechal gave the Keynote Lecture, ‘Respiratory supercomplexes: what can we learn from yeast?’, in the Biological Structures group Membrane Proteins Session (chaired by Professor Bonnie Wallace), and Altin Sula gave an invited talk on ‘Sodium Channel/Drug Interactions’.
Dec 2020: Postdoctoral EMBO fellowships awarded to ISMB postdoc Sander Van der Verren and former ISMB PhD student Joshua Hutchings.
ISMB’s Sander Van der Verren has been awarded a prestigious and highly competitive EMBO fellowship to work at ISMB in Giulia Zanetti’s laboratory. The fellowship will support structural studies of the human COPII coat complex and its regulatory factors. COPII mediates vesicular transport of proteins out of the Endoplasmic Reticulum, and its regulation in humans is important for secretion of a wide range of cargoes, including the most abundant secretory proteins: collagens. As part of the Birkbeck cryo-EM lab, Sander will use cryo-tomography and single particle cryo-EM techniques to understand how regulatory factors TANGO1 and cTAGE5 assemble and interact with the coat to promote collagen secretion.
Sander says: “I am honoured to have been awarded an EMBO postdoctoral fellowship and look forward to tackle outstanding questions in the membrane trafficking field.”
A Zanetti lab recent alumnus, Josh Hutchings, was also successful in securing an EMBO fellowship. His postdoctoral studies in Elizabeth Villa’s lab at UC San Diego will focus on in situ characterisation of the LNK complex.
The Microbiology Society Outreach Prize 2020 was awarded for the charity project ‘Joi Hok’, which uses educational intervention as a means of spreading awareness of tuberculosis.
Professor Sanjib Bhakta, Professor of Molecular Microbiology and Biochemistry at the ISMB and ‘Joi Hok!’ founder Sreyashi Basu have been awarded the 2020 Microbiology Outreach Prize by the Microbiology Society, for effectively bridging the gap between laboratory science and public health research with their project ‘Joi Hok!’, a community tuberculosis (TB) awareness programme.
The programme’s aim is to alter the perception of TB among the local community in Kolkata, India, through a network of local artists, musicians and health professionals. A series of routine interdisciplinary workshops were introduced in semi urban and rural schools around the city over a span of 6 months.
Sanjib Bhakta commented: “TB has progressively worsened with the advent of antibiotic resistance, hence there is urgent need to review and deal with the disease not only as a medical concern or even public health problem alone, but also as a socio-economic challenge.”
Sreyashi Basu commented: “we used traditional folk-art and music as a creative tool to engage and educate underprivileged youth on the influence of local stigmas and antibiotic resistance in the context of TB. We have been fortunate to see a discernible impact amongst the masses whereby the transfer of knowledge from children to household members has resulted in an increased awareness of TB in the community and encouraged patients to adhere to anti-TB treatment regimens .”
Professor Mark Harris, General Secretary of the Microbiology Society, said: “The Microbiology Society Prizes panel felt that the ‘Joi Hok!’ tuberculosis awareness programme demonstrated an inventive and exciting approach to informing the public about an important disease. The novelty of the programme was its innovative use of traditional art and music to raise awareness of tuberculosis in rural India, with potential to positively influence treatment and control in a country that has the highest global incidence of this devastating infection – a worthy winner of the Microbiology Society Outreach Prize 2020.”
The Microbiology Society is a membership charity for scientists interested in microbes, their effects and their practical uses, and is one of the largest microbiology societies in Europe. The Microbiology Outreach Prize is awarded for an outstanding outreach initiative, that shows innovation, originality and a sustained impact over time.
The Joi Hok programme uses traditional art and music to raise awareness of Tuberculosis in rural India.
Congratulations to Prof. John Christodoulou (jointly affiliated between UCL and Birkbeck in the ISMB), on being awarded the British Biophysical Society’s 2020 Sosei Heptares Prize for Biophysics. The prize is in recognition of Prof. Christodoulou’s original and influential contributions to the advancement of biomolecular NMR and the biophysical analysis of protein folding on the ribosome.
John is serving as the director of the NMR facility at the ISMB and has been a driving force behind high-calibre structural biology at the ISMB for more than twelve years.
