Best Poster Awards – In Situ Structural Biology Workshop

The EMBO Workshop: In Situ Structural Biology: From Cryo-EM to Integrative Modelling was our final virtual conference of 2020, but there was no trace of Zoom fatigue amongst the 466 participants who joined us from 6 – 8 December!

80 international researchers presented their posters during the two posters sessions on the following topics:

  • Biophysical analysis in cells
  • COVID-19
  • Imaging across scales
  • Integrative modelling
  • Molecular sociology
  • Structural analysis in situ
  • Structural biology

Each of the participants had the chance to vote for their favourite poster, resulting in two posters winning the Best Poster Award kindly sponsored by EMBO Press.  Here are the winners:

New insights on the catalytic mechanism of arsenite oxidase

PHOTO: Filipa Engrola

Authors: Filipa Engrola, Márcia Correia, Teresa Santos-Silva, Maria Romao, (UCIBIO@FCT-NOVA, Portugal)

Arsenic (As) and antimony (Sb) are two metalloids that, due to anthropogenic and natural causes, pose an environmental  threat, considered as priority pollutants by the World Health Organisation and the United States Environmental Protection Agency. Although the safety guards recommend a maximum of 10 μg/L of As and Sb in drinking water, these values are exceeded in many regions worldwide, with no remediation approach that is simultaneously effective, clean and economically sustainable [1,2]. The ancient bioenergetic enzyme arsenite oxidase (Aio), from microorganisms Rhizobium sp. NT-26 (NT-26 Aio) and Alcaligenes faecalis (A.f. Aio), is currently being studied for its use as a biosensor and in bioremediation processes. Both Aio enzymes contain a large subunit (AioA) that harbours a molybdenum centre and a [3Fe-4S] cluster, and a small subunit (AioB) that possess a Rieske [2Fe-2S] cluster and have demonstrated to oxidise AsIII, as well as SbIII, into the easier to remove and less toxic forms of AsV and SbV, respectively [3,4]. Aiming to elucidate the catalysis mechanism of the enzymes, a combination of expression and purification of the proteins, crystallisation, structural analysis, enzyme kinetics and affinity tests were conducted. X-ray structures of the ligand-free form of the enzyme had been previously determined (PDB: 4AAY, 5NQD and 1G8K [3,5,6]). In our work, Aio crystals in complex with two different forms of the substrate analogue – Sb oxyanions, with a reaction kinetic 6500 times slower than AsIII [6] – diffracted up to ca 1.8 Å resolution. The structures show the reaction intermediates bound at the active site, with a μ-oxo bridge binding Sb to the Mo atom. Analysis of bond lengths and geometry of the ligands at the Mo active site allowed us to revisit the catalytic mechanism of As oxidation [7], contributing to the understanding and future biotechnological application of this family of enzymes in water treatment.

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Allosteric hotspot in the main protease of SARS-CoV-2

PHOTO: Léonie Ströhmich

Authors: Léonie Strömich, Sophia N Yaliraki, (Imperial College London, UK)

Since the beginning of 2020 we have seen the coronavirus SARS-CoV-2 causing a global pandemic with almost 34 million cases and over 1 million deaths worldwide [as of 01.10.2020] [1.] As a result, we have seen a surge in research efforts to develop effective treatments for the underlying disease, COVID-19. One approach is to target the main protease (Mpro) of SARS-CoV-2 as it is essential for virus replication in an early step of the viral life cycle [2.] Most efforts are centred on inhibiting the orthosteric binding site of the enzyme. However, considering allosteric sites on the protein allows for more selective drug design and widens the chemical search space. Here, we report an allosteric hotspot in the SARS-CoV-2 Mpro dimer by using novel atomistic graph theoretical methods: Markov transient analyses follow the propagation of a random walker on a graph and have been shown to successfully identify allosteric communication in catalytic proteins [3.] We further score the so identified allosteric hotspots against random sites in similar distances and thus identify a statistically significant putative allosteric site in the SARS-CoV-2 Mpro. We then simulate a binding event at this hotspot region using data from a recent XChem fragment screen by the Diamond Light Source [4.] which provides a starting point for rational drug design. This study uses highly efficient network theoretical models to shed light on allosteric communication and uncovers putative allosteric sites in the SARS-CoV-2 main protease. This provides a valuable contribution to the ongoing efforts to find a cure against COVID-19 by broadening the horizon for drug discovery efforts.

Image: Léonie Ströhmich

[1.] Official World Health Organization COVID-19
dashboard: (Accessed: 01.10.2020).
[2.] Hilgenfeld, R. (2014). FEBS Journal, 281(18), 4085-4096.
[3.] Amor, B., Yaliraki, S. N., Woscholski, R., & Barahona, M. (2014) Molecular BioSystems, 10(8), 2247-2258.
[4.] Douangamath, A., Fearon, D., Gehrtz, P., Krojer, T., Lukacik, P., Owen, C. D., … Walsh, M. A. (2020) Nature Communications, 11, 5047.

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Best Poster Awards – EMBO Workshop: Tools for Structural Biology of Membrane Proteins

183 researchers convened at the Centre for Structural Systems Biology (CSSB) in Hamburg, Germany, for the recent EMBO Workshop: Tools for Structural Biology of Membrane Proteins (7 – 9 October 2019) to present and discuss new technologies and approaches applied in studying membrane protein structure, dynamics and functions.

Out of the 82 posters presented, 6 were awarded a poster prize based on popular vote. Here we present the poster abstracts of four of the winners.

Structural insights into the role of the conserved ATPase component, EccC, in the mycobacterial T7SS

Katherine Beckham is a postdoctoral fellow in Matthias Wilmanns’ group at EMBL Hamburg. PHOTO: Katherine Beckham

Authors: Katherine Beckham (1), Luciano Ciccarelli (1), Mandy Rettel (2), Mikhail Savitski (2), Jan Kosinski (1), Annabel
Parret (1), Matthias Wilmanns (1)

Mycobacteria have a unique membrane structure with a complex hydrophobic outer-membrane rich in mycolic acids. To transport substances across this impermeable barrier, mycobacteria rely on a highly specialised translocation machinery – the Type VII secretion system (T7SS). Pathogenic mycobacteria encode up to five distinct T7SSs ESX-1 to 5 [1]. Our previous work characterised the structure of the of the inner-membrane complex of the ESX-5 T7SS from Mycobacterium xenopi using negative stain electron microscopy, revealing a hexameric 1.8 MDa complex comprising the four conserved core components: EccB5, EccC5, EccD5 and EccE5 [2]. The large cytosolic domain of EccC5, an FtsK/SpoE-like ATPase, is absent in our current EM map due to its conformational flexibility, which may be required to accommodate a range of protein substrates. Our current work aims to understand the role of EccC5 in secretion. In isolation this component can oligomerise into a hexameric ring-like conformation, as observed for other ATPases in this family. In addition, chemical cross-linking of the ESX-5 complex coupled with mass spectrometry (XL-MS) supports the oligomerisation of EccC5 in the secretion complex, suggesting that it may form a channel or ‘translocation tunnel’. Thus, we propose that EccC5 may exist in two conformational states: an extended, flexible monomeric state and a more compact hexameric state. Using an integrative structural biology approach, we are combining structures of isolated proteins derived from X-ray crystallography and electron microscopy studies with XL-MS data. Together these data aim to further elucidate the secretion pathway across the mycobacterial cell envelope.

[1] Houben, E. N. G., et al. Take five — Type VII secretion systems of Mycobacteria. Biochim. Biophys. Acta – Mol. Cell Res.1843, 1707–1716 (2014).
[2] Beckham, K. S. H. et al. Structure of the mycobacterial ESX-5 type VII secretion system membrane complex by single-particle analysis. Nat. Microbiol.2, 17047 (2017).

(1) EMBL Hamburg, Germany, (2) EMBL Heidelberg, Germany

Poster currently not available

Dissection of protonation sites for antibacterial recognition and transport in QacA, a multidrug efflux transporter

Puja Majumder is a Ph.D student at the Indian Institute of Science. PHOTO: Puja Majumder

Authors: Puja Majumder (1), Shashank Khare (1), Arunabh Athreya (1), Nazia Hussain (1), Ashutosh Gulati (2), Aravind Penmatsa (1)

Emergence of multidrug-resistance poses serious threat to the society. One of the effective way by which bacteria gain drug resistance is through active efflux of antibiotics and other antibacterial compounds using multidrug efflux transporters. Among the battery of efflux pumps present in pathogenic bacteria, our work is focused on QacA, a drug-proton anitiporter (DHA) with 14-transmembrane helices that provide resistance to methicillin resistant Staphylococcus aureus (MRSA) strain, with homologs present in other pathogenic organisms. QacA is a highly promiscuous transporter, capable of effluxing diverse array of monovalent and divalent cationic antibacterial compounds and dyes. This study using a homology model, dissects the role of six protonatable residues present in the transport vestibule of QacA. Systematic mutagenesis resulted in identification of D34 (TM1) and E407 (TM13) as crucial residues and D323 (TM10) and D411 (TM13) as conditional residues needed for transport process of QacA. Whole cells, inside-out vesicles, substrate-induced proton release and microscale thermophoresis based assays were used to investigate the transport and binding properties of the transporter and its mutants. The activity of purified protein was checked with reconstituted QacA in a proteoliposome using substrate-induced proton transport assay. We identify two sites, D34 and D411 playing vital role in recognition of most of the substrates tested while E407 facilitates substrate efflux as a protonation site. It was also observed that E407 has an additional role as a recognition site for the transport of dequalinium, a divalent quaternary ammonium compound. These observations rationalize the promiscuity at the residue level of QacA for diverse substrates. The study identifies the role of acidic residues in QacA with implications for substrate recognition, promiscuity and processive transport in multidrug efflux transporters, related to QacA.

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(1) Indian Institute of Science, India, (2) Stockholm University, Sweden

Biophysical analysis of circularized MSP nanodiscs for structural studies

Melina Daniilidis is a Ph.D student in Prof. Dr. Franz Hagn’s group at the Bavarian NMR Center (BNMRZ) of the Technical University of Munich. PHOTO: Melina Daniilidis

Authors: Melina Daniilidis (1), Ralf Stehle (1), Franz Hagn (1,2)

Structure and dynamics of membrane proteins are crucial aspects for understanding functional properties of this protein class. Unfortunately, stabilizing them in their isolated form is still difficult. By incorporating membrane proteins into nanodiscs, they can be studied in a native-like
environment using biochemical and structural methods. However, thermal and long-term stability of small nanodiscs limit these studies and make it difficult to carry out nuclear magnetic resonance spectroscopy (NMR) measurements at elevated temperatures. Circularized membrane scaffold proteins (MSPs) produced via split-inteins have been shown to be more stable and homogenous than their linear counterparts. However, their biophysical properties, as well as suitability for membrane protein insertion and structural studies have not yet been assessed in a systematic manner. Thus, we examined circular and linear nanodiscs of varying size using several biophysical methods. An important issue for NMR structural studies is that the size and shape of circular nanodiscs do not expand above the phase transition temperature, increasing their homogeneity and reducing their size as compared to linear nanodiscs at high temperatures. 1H,15N-TROSY experiments could demonstrate that circular MSP1D1 nanodiscs with incorporated VDAC-1 are stable at higher temperatures, making it possible to obtain high-resolution NMR spectra of superior quality. Furthermore, NMR relaxation experiments were carried out to compare rotational correlation times of VDAC-1 in circular and linear nanodiscs, respectively. Despite the higher molecular weight, the circular nanodiscs showed lower rotational correlation times, which corroborated the biophysical results on the temperature-dependecy of the nanodisc diameter and homogeneity. The presented data demonstrate that these very stable circularized MSPs are well applicable to the study of membrane proteins in a lipid environment by NMR, but also other structural methods like electron microscopy.

(1) Technical University of Munich, Germany, (2) Helmholtz Zentrum München, Germany

Poster currently not available

Reconstitution of the activity of RND efflux pumps into proteoliposomes

Dhenesh Puvanendran is a Ph.D student at the Institute of Physical and Chemical Biology in Paris, France. PHOTO: Dhenesh Puvanendran

Authors: Dhenesh Puvanendran, Quentin Cece, Martin Picard, IBPC, France

Efflux pumps are the major systems in bacterial resistance against antibiotics. They are classified by the energy needed to be active (ATP hydrolysis or ion counter-transport). Efflux pumps from the RND (Resistance, Nodulation, and cell Division) family use a proton gradient to be active and are composed of three proteins: a membrane fusion protein (MFP) and a transporter (RND) in the inner membrane, and an Outer Membrane Factor (OMF) localized in the outer membrane. We focus on the MexA-MexB-OprM efflux pump from Pseudomonas aeruginosa.The overall goal of my research is to measure in vitro the velocity of transport by efflux pumps. To that end, we reconstitute MexA and MexB as one population of proteoliposome, and OprM as another population of proteoliposome. The whole tripartite pump forms upon association of the respective populations of liposomes. The proof of concept of this method has already been described, leading to a qualitative monitoring of transport. We now work at defining a reconstitution procedure amenable to now quantify the rate of transport. To do so we take extreme care to precisely determine the efficiency of protein reconstitution and the type of lipids component used to perform liposomes. I will present the roadmap towards the rational, step-by-step, reconstitution of the MexA-MexB-OprM efflux pump as well as the methodologies that are undertaken to measure the velocity of transport, and possible perspectives regarding the screening of efflux pump inhibitors.

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The other award-winning posters are:

Novel antigen-binding chimeric proteins as tolls in crystallography and cryo-EM of membrane proteins presented by Thomasz Uchanski, Vrije Universiteit Brussel, Belgium

Cryo-Electron tomography of synaptic vesicle fusion junctions presented by Lucy Ginger, MRC Laboratory of Molecular Biology, United Kingdom

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