MyD88 and MAL are Toll-like receptor (TLR) adaptors that signal to induce pro-inflammatory cytokine production. We previously observed that the TIR domain of MAL (MAL^TIR) forms filaments in vitro and induces formation of crystalline higher-order assemblies of the MyD88 TIR domain (MyD88^TIR). Due to their crystal size, conventional crystallography proved to be challenging. However, through serial femtosecond crystallography (SFX) we were able to determine the structure of MyD88 crystals. Here, we present the SFX structure of the MyD88^TIR assembly, which revealed a biological relevant two-stranded higher order assembly arrangement of TIR domains analogous to that seen previously for MAL^TIR. Our study provides structural and mechanistic insights into TLR signal transduction¹.

¹Clabbers, M., Holmes, S. et.al. MyD88 TIR domain higher-order assembly interactions revealed by microcrystal electron diffraction and serial femtosecond crystallography. Nature Communications, accepted March 2021, DOI: 10.1038/s41467-021-22590-6

Post-translational modification of proteins regulates many biological processes. Acetyltransferase transfers acetyl groups to lysine residues on target proteins and is a major type of post-translational enzyme. General control nonderepressible5 (GCN5, also known as Kat2a) is one of the histone acetyltransferases that promote transcriptional activity.
Metazoans possess two GCN5 isoforms that arise from alternative splicing. The lower molecular weight isoform (isoform2) is similar in size and function to yeast GCN5, consisting of an acetyltransferase (AT) domain and a bromodomain at the N and C termini, respectively. Another higher molecular weight isoform (isoform 1) has an N-terminal extension. The amino acid sequence of this N-terminal extension is similar to the N-terminal domain of p300/CBP-associated factor (PCAF, also known as Kat2b) and is conserved among vertebrates. This N-terminal domain is called the PCAF_N domain.
Ubiquitination is also a post-translational modification that targets lysine residues. This modification regulates many cellular processes, including cell division and immune responses, among others. Ubiquitination is catalyzed by the sequential reaction of ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3), which is responsible for the ligation of ubiquitin onto a substrate in conjunction with the E2.
A previous study reported that PCAF also harbors ubiquitination activity in addition to acetyltransferase activity. PCAF_N is identified as a domain that contains a ubiquitin E3 ligase activity. Although the longer isoform of GCN5 possesses the PCAF_N domain, it has not been revealed whether GCN5 functions as an E3 enzyme.
We demonstrated that GCN5 exhibited ubiquitination activity and its activity was supported by the ubiquitin-conjugating enzyme UbcH5. Moreover, we determined the crystal structure of the PCAF_N domain at 1.8 Å resolution and found that the PCAF_N domain folds into a helical structure with a characteristic binuclear zinc region, which could not be predicted from sequence analyses. The zinc region is distinct from known E3 ligase structures, suggesting this region may form a new class of E3 ligase. Our biochemical and structural study provides valuable insight into not only the functional significance of GCN5 but also into ubiquitin biology.

Lectins, carbohydrate recognizing proteins, play an important role in various physiological and pathophysiological processes as well as both mutualistic and parasitic interactions between microorganisms and hosts [1]. In connection with the last-mentioned process, lectins from pathogenic bacteria can mediate the first step of infection and they are considered an important virulence factor.

Photorhabdus laumondii is an entomopathogenic bacterium, which is known for its complicated life cycle, including mutualism and pathogenicity towards two different invertebrate hosts [2]. This contribution is focused on the newly described PLL lectin family, which shares a seven-bladed beta-propeller fold. All five members of this family are highly similar to each other in primary, secondary, and tertiary structure. However, the oligomeric state of these lectins differs significantly. Members of the PLL family have been confirmed to bind multiple monosaccharides, including l-fucose and O-methylated saccharides. X-ray structures of PLL family discovered two sets of binding sites with different ligand specificity per monomer, “polar” sites and “hydrophobic” sites. Amino acids involved in the ligand-binding are highly conserved within the lectin molecule. Ligands are bound in both types of binding sites via hydrogen bonds and via CH-π interaction with aromatic residues. Lectin/saccharide interaction is mostly mediated via hydrogen bonds. However, hydrophobic sites are deepened with a hydrophobic pocket. The importance of non-polar interactions, such as CH-π interactions between aromatic amino acids and apolar part of carbohydrate molecules, was shown recently [3].

[1] Lis, H. and Sharon, N. (1998) Chem Rev. 98, 637-74.

[2] Clarke, DJ. (2020) Microbiology. 166, 335-348.

[3] Wimmerová, M., Kozmon, S., Nečasová, I., Mishra, S.K., Komárek, J., Koča, J. (2012) Plos One. e46023.

Alzheimer’s disease (AD) is a neurodegenerative disease accompanied by the accumulation of misfolded proteins. AD pathology is characterized by the extracellular amyloid plaques and the neurofibrillary tangles (NFTs). NFTs consist of paired helical filament (PHF) and abnormally phosphorylated tau is known to form the PHF.

Tau protein that exists in the brain neuron cells is important for the stabilization and elongation of the microtubules. Tau contains a microtubule-binding domain (MBD) consisting of three or four repeats of about 30 similar amino acids (R1-R4). The MBD is not only important for the binding of tau to microtubule but also plays a key role for the abnormal self-aggregation of tau. Even though the sequences of repeat peptides of MBD are similar to each other, the ability of self-aggregation of these peptides is quite different. Especially, the short regions of both ²⁷⁵VQIINK²⁸⁰ and ³⁰⁶VQIVYK³¹¹ that are the start sequence of R2 and R3 respectively have an important role for the tau self-aggregation.

Several therapeutic approaches targeting tau aggregation have been proposed, such as a tau aggregation inhibitor. Among tau aggregation inhibitors, a monoclonal antibody may be particularly effective because of its specificity to the target molecule. Thus, in searching for a tau aggregation inhibitor, we made a monoclonal antibody to tau (Tau2r3) using the ²⁷²GGKVQIINKKLD²⁸³ epitope peptide from the MBD in tau and prepared the Fab domain (Fab2r3) from Tau2r3. We analyzed the inhibitory function of Fab2r3 for tau aggregation and determined the tertiary structure of the Fab2r3 complex with VQIINK peptide.

The results of thioflavin S (ThS) fluorescence and TEM (negative-staining electron microscopy) measurement clearly showed that Fab2r3 inhibited the tau aggregation, and the inhibitory function of Fab2r3 seems to occur through specific binding to the VQIINK sequence by the isothermal titration calorimetry (ITC) analysis.

To elucidate this inhibition mechanism, we analyzed the tertiary structure of Fab2r3 and VQIINK peptide complex by X-ray crystallography. The VQIINK peptide makes many hydrogen bonds and two hydrophobic interactions with Fab2r3. Among these interactions, we supposed that the hydrophobic interaction has a key role for the antigen recognition of the Fab2r3.

The fusogenic avian Orthoreovirus (ARV) infection can cause considerable economic losses in the poultry industry, mostly infecting young chickens. The ARV has been associated with various disease conditions in poultry (enteric and respiratory diseases, myocarditis, hepatitis, stunting-malabsorption syndrome, and the most important one viral arthritis/tenosynovitis) [1]. The ARV are non-enveloped icosahedral particles of 85 nm external diameter with 10 dsRNA genomic segments (23.5 kb) encased within two concentric protein shells, forming the outer capsid and the core [2].

Reoviruses' RNA replication and morphogenesis occur exclusively within cytoplasmic inclusion bodies, also known as ‘viroplasms’ or viral factories (VF). VF are globular, dynamic, phase-dense, cytoplasmic inclusions lacking membranes or cellular organelles. VFs are formed by abundant viral non-structural (NS) proteins and structural proteins recruited into VF by interaction with NS proteins. NS proteins are expressed inside the infected cells but are not part of the mature virion. The two most abundant VF proteins are μNS and σNS. μNS is a 70 kDa protein that is forming viroplasm inside infected cells and attracts and associates with other viral proteins including 41 kDa RNA chaperone σNS [3]. σNS is causing specific RNA-RNA interaction between all 10 genomic segments specifically by destabilizing of RNAs helical regions [4]. There is missing information about a fashion of coupling of μNS with σNS protein needed for the understanding of viroplasm formation mechanism.

Only a low-resolution structure of σNS has been established while no structural information is available for μNS. This is chiefly due to the poor solubility of μNS and polydispersity of σNS which forms oligomers ranging dimers to octamers and RNA containing filaments [4]. To tackle these problems and obtain structural information we have generated various fusion constructs for expression, purification, and further structural study by X-ray crystallography and electron cryo-microscopy. Due to their multi-facet role in virus biology, the detailed knowledge of their structure is necessary for a better understanding of their functions and could provide the rationale for the development of new antiviral drugs.

[1] Jones R. C. (2000). Revue scientifique et technique (International Office of Epizootics), 19(2), 614–625. https://doi.org/10.20506/rst.19.2.1237.

[2] Zhang, X., Tang, J., Walker, S. B., O'Hara, D., Nibert, M. L., Duncan, R., & Baker, T. S. (2005). Virology, 343(1), 25–35. https://doi.org/10.1016/j.virol.2005.08.002.

[3] Benavente, J., & Martínez-Costas, J. (2007). Virus research, 123(2), 105–119. https://doi.org/10.1016/j.virusres.2006.09.005.

[4] Borodavka, A., Ault, J., Stockley, P. G., & Tuma, R. (2015). Nucleic acids research, 43(14), 7044–7057. https://doi.org/10.1093/nar/gkv639.

Surface layer proteins (Slp) assemble into highly regular 2D crystalline arrays and represent the outermost cell envelope in many bacteria and archaea. The Surface layer (S-layer) is composed mostly of a single (glycol)protein species and is in close contact with their surrounding. Therefore, these arrays fulfill various functions like bacterial adherence to other cells or substrates, protection against life-threatening conditions, and maintenance of the cell shape.

The S-layer of L. acidophilus consists of two proteins. SlpA is mainly expressed under normal physiological conditions, whereas SlpX expression is increased under osmotic stress. S-layer proteins have two functional regions in common: a region that is important for the attachment to the cell wall and a region responsible for the self-assembly of the S-layer array.

Our goal is to structurally characterize the S-layer proteins SlpA and SlpX of L. acidophilus and to further understand the mechanism of the self-assembly, how the two proteins interact with each other and how the attachment to the cell wall interact occurs. Since full length S-layers form insoluble 2D crystals we designed three functional protein fragments of both proteins and we obtained diffracting crystals of all. In a joint effort and in combination with various different approaches like Hg-SAD, ARCIMBOLDO at a resolution of 1.4Å, ab initio prediction with RoseTTAfold of an only beta-strand protein and molecular replacement we were able to obtain the crystal structures of all protein domains. The structures of the self-assembly regions of SlpA and SlpX show an interesting domain switch and together they suggest the mode of action how the self-assembly of the S-layer occurs.

Acknowledgments:

The X-ray experiments were performed at synchrotron-radiation facilities ESRF (ID23-1, ID30B, ID30A, ID23-2 and ID29, Grenoble, France), DESY (P11, PETRAIII, Hamburg, Germany), EMBL (P13, PETRAIII, Hamburg, Germany), Elettra (XRD1 and XRD2, Trieste, Italy) and SLS (PXI, Villigen, Switzerland). We are grateful to local scientists for providing assistance in using the beamlines. This work has been supported by the Austrian Science Fund (FWF, P29432)

PTG (protein targeting to glycogen) is a scaffolding protein that is involved in the activation of glycogen synthesis by bringing PP1 (type 1 protein phosphatase) to its substrates. It is proposed as a target for the treatment of Lafora disease (LD), a genetic disorder manifested by catastrophic teenage onset of progressive myoclonus epilepsy. In healthy neurons, PTG is downregulated by the laforin-malin complex resulting in very low glycogen production. Mutations in malin or laforin causes accumulation of PTG, which promotes glycogen synthesis by directing PP1 to glycogen synthase and glycogen phosphorylase. This results in the appearance of neurotoxic inclusion bodies formed by insoluble polyglucosans called Lafora bodies (LB), which are ultimately responsible for Lafora disease (LD). In LD mice models knocking out PTG resulted in a nearly complete disappearance of LB and resolution of neurodegeneration and myoclonic epilepsy, indicating that small molecules interfering with the PTG/PP1 interaction emerge as an excellent therapeutic strategy for LD. Up to date, there was no structural data of PTG and PTG/PP1 complex allowing for identification of potential druggable pockets. We were able to set up expression, purification and crystallization protocols of different constructs of PTG and PTG/PP1 complex for structural studies. Here we present preliminary crystallographic structures of PTG carbohydrate binding module 21 (CBM21) and PTG/PP1 complex, complemented with SAXS analysis of the complex, that will provide a valid information for the rational design of selective compounds targeting interaction of PTG with its partners.

Antibiotic resistance is usually shared between bacteria by using conjugation [1], where a bacterium transfers part of its genome to another bacterium by involving a complex machinery called a secretion system. Conjugative Type IV secretion systems (Conj-T4SSs) are one of the most prevalent ways to share DNA between prokaryotes. Integrative and conjugative elements (or ICEs) are one of the principal types of mobile genetic elements that enable horizontal transmission of genetic information. ICEs encode their own Conj-T4SSs to perform conjugation and have the particularity to be integrated into the chromosome of the host cell. While descriptions of Conj-T4SSs in Gram-negative bacteria are available from CryoEM experiments, no data exist for Gram-positive bacteria yet. In Streptococcus thermophilus, ICESt3 encodes 14 putative proteins involved in the conjugation process. Among them, the Gram-negative VirB8-equivalent protein, called OrfG, is expected to be essential in the plasma membrane part of T4SS since it is supposed to act as an interaction hub for other T4SS proteins. OrfG contains three domains, one transmembrane domain and two soluble ones. Here we present the X-Ray structures of the soluble domains of OrfG. We performed an in-depth analysis of all VirB8-like domains [2] by using existing and new multi-protein structural alignment visualization tools. One of these tools, called MPSA_Viewer, was developed in our lab and significantly improved the visual readability of multi-protein structural alignments, especially when sequence identities are very low. We also analyzed the quaternary structure of OrfG, which consists of a trimeric assembly of interwoven monomers. The trimeric organization seems specific to VirB8-like proteins of Gram-positive bacteria since two other occurrences were found in the structural database. Such intricated assemblies can be biologically relevant, as observed for instance in the prefusion conformation of the 2019-nCoV spike [3]. We also noticed that the variable spacing at the center of these Gram-positive trimers might be compatible with substrate translocation [4] if other conditions are met.

[1] Frost, L. S., Leplae, R., Summers, A.O. & Toussaint, A. (2005) Nat. Rev. Microbiol. 3, 722.

[2] Cappèle, J., Mohamad Ali, A., Leblond-Bourget, N., Mathiot, S., Dhalleine, T., Payot, S., Savko, M. Didierjean, C., Favier, F. & Douzi, B. (2021). Front. Mol. Biosci., 8, 642606.

[3] Wrapp D, Wang, N. Corbett, K.S., Goldsmith, J.A., Hsieh, C.L., Abiona, O., Graham, B.S. & McLellan, J.S. (2020). Science, 367, 1260.

[4] Miletic, S., Fahrenkamp, D., Goessweiner-Mohr, N., Wald, J., Pantel, M., Vesper, O., Kotov, V. & Marlovits, T.C. (2021) Nat. Commun. 12, 1546.

The native SAD phasing method uses the anomalous scattering signals from the S atoms contained in most proteins, the P atoms in nucleic acids, or other light atoms derived from the solution used for crystallization. These signals are very weak and careful data collection is required, which makes this method very difficult. One way to enhance the anomalous signal is to use long-wavelength X-rays; however, these wavelengths are more strongly absorbed by the materials in the pathway. Therefore, a crystal-mounting platform for native SAD data collection that removes solution around the crystals has been developed. This platform includes a novel solution-free mounting tool and an automatic robot, which extracts the surrounding solution, flash-cools the crystal, and inserts the loop into a UniPuck cassette for use in the synchrotron. Eight protein structures (including two new structures) have been successfully solved by the native SAD method from crystals prepared using this platform.

The world is meeting the challenge of the energy crisis, which prompted searching for renewable energy resource. Brown algae are ideal feedstocks for producing biofuels, the promising renewable resource. The sea hare Aplysia kurodai is an excellent model for investigating the biofuel production process. It consumes brown algae as a staple food to release a large amount of glucose from laminarin-depolymerization by the ?-glucosidase in its digestive fluid (akuBGL). However, brown algae produced abundant secondary metabolite as a defense against the herbivores, such as phlorotannin. Phlorotannin inhibits akuBGL activity, which causes a problem in biofuel production from brown algae. Interestingly, Eisenia hydrolysis enhancing protein (EHEP) existed in the digestive fluid of Aplysia kurodai and, was found to protect akuBGL from phlorotannin-inhibition by binding with phlorotannin and precipitating¹. How EHEP bind to phlorotannin, why it can protect akuBGL from inhibition both are unknown.

In this study, we obtained EHEP and akuBGL from digestive fluid of A. kurodai. We determined the structures of EHEP in apo form and complex with an analogue of the phlorotannin, tannic acid by native-SAD method². The structures reveal that EHEP consisted of three chitin-binding domains linked by two long loops (Fig.1) and tannic acid bound at the center of EHEP. Furthermore, we determined the structure of akuBGL and showed it comprised of two GH1 (glycoside hydrolysis family 1) domains linked by the loop (Fig.2). Additionally, the docking analysis of tannic acid with akuBGL was performed, revealing the tannic acid occupies the active pocket of akuBGL. Collectively, we proposed the mechanism of EHEP that protects akuBGL from inhibition.

Human C-reactive protein (CRP) is an innate immune macromolecule of hepatic origin produced in response to inflammatory cytokines. CRP serum concentration is exploited as a clinical biomarker in humans as levels rise rapidly in response to inflammation, infection, or tissue damage [1]. CRP has opsonising abilities and roles in the inflammatory response and activation of complement.

Native human CRP consists of five identical non-covalently bound subunits. The five protomers of CRP are arranged symmetrically around a central pore, consisting of 206 amino acids folded into two antiparallel β-sheets with a flattened jellyroll topology [2]. Each protomer has a calcium dependent ligand binding site and an effector binding site on the opposite sides of the molecule. The ‘recognition’ face binds phosphocholine (PC) in a calcium-dependent manner in a ligand binding site located within a hydrophobic pocket. PC is a principal ligand for CRP; widely expressed on the surface of damaged cell membranes and distributed in lipopolysaccharides of bacteria and other microorganisms [1]. PC binding is mediated by a phosphate-calcium interaction. The opposite ‘effector’ face of CRP accommodates multiple binding sites, for C1q and immunoglobulin Fcγ receptors. The putative C1q binding site is located at the end of a cleft bordered by the pentraxin helix [3].

Although CRP is remarkably stable under physiological conditions, it has been shown that CRP can dissociate into individual subunits to form monomeric CRP (mCRP). Evidence is increasing that monomeric CRP may have a pro-inflammatory role. We have successfully dissociated CRP, in the presence of urea, into mCRP in-vitro and identified a monomeric CRP with the same reactivity as that seen in patient samples [4]. In addition to the presence of urea, the removal of calcium ions to destabilise the protein is required. Monomeric CRP has been produced via urea-induced dissociation, optimised at 3M urea over a ten-week period [4]. This CRP form retains its reversible PC-binding ability. Another form of monomeric CRP has been observed in vitro, produced during excessive denaturing conditions, requiring 8M Urea [5] which does not retain the ability to bind PC. Optimisation of production of these in vitro mCRP forms and crystallisation trials are currently underway.

The stringent response is a fundamental mechanism for bacterial survival and adaptation to a wide range of stress conditions, mediated by the synthesis and hydrolysis of signal molecules collectively referred to as alarmones [1, 2]. Bifunctional enzymes belonging to the RelA‑SpoT homologue (RSH) superfamily are responsible for the synthesis of alarmones guanosine 5'-triphosphate 3'-diphosphate (pppGpp), guanosine 5'-diphosphate 3'-diphosphate (ppGpp), and guanosine 5′-monophosphate 3′-diphosphate (pGpp), and contain both synthetase and hydrolase domains, however, with the hydrolase domain being inactive in many cases. Some organisms additionally contain monofunctional small alarmone synthetases (SAS) and small alarmone hydrolases (SAH), regulation of which has not yet been fully described [3].

Here, we present a 1.2 Å structure of the Leptospira levetii small alarmone hydrolase (SAH) and a 1.8 Å structure of Corynebacterium glutamicum small alarmone hydrolase, together with an analysis of similarities and differences with the known bifunctional Rel enzyme structures. We show that the SAH structures contain common features typical for hydrolases, such as a metal ion binding site and the highly conserved histidine–aspartate (HD) motif. Analysis of the structures using PISA surprisingly revealed they both form dimers, in contrast to earlier reports. Moreover, there is a distinct difference in the dimerization interface, despite a high degree of structure conservation between the monomers. Dimer formation was confirmed experimentally using size exclusion chromatography multi-angle light scattering (SEC-MALS) and small angle X-ray scattering (SAXS). The structures of the two small alarmone hydrolase representatives allowed for a structural analysis and comparison with known bifunctional hydrolase structures (RelSeq from Streptococcus equisimilis and RelTh from Thermus thermophilus) [4, 5]. We demonstrate that while the key residues involved in substrate coordination are clearly conserved, there are some notable differences in several secondary structure elements. Intriguingly, we find differences in the position of helices involved in the regulation of activity in bifunctional hydrolases, that adopt an unanticipated position in the SAH structures. Taken together, our data shed new light on how small alarmone hydrolases are regulated as well as how they differ from the larger bifunctional enzymes. In the future, this will help us further understand the role of monofunctional SAH enzymes and in turn bring forward a better understanding of the stringent response in bacteria.

Bacterial chromosomes contain large numbers of toxin-antitoxin (TA) systems, consisting of a gene encoding a toxic protein and a gene encoding an antitoxin which can be an RNA or a protein [1, 2]. In the largest known group of type II TA systems, vapBC, the toxin VapC is an endoribonuclease belonging to the PIN (PilT N-terminal) domain family, which is very conserved structurally and found in all domains of life. VapC is a known RNase and targets for VapCs include various tRNAs and rRNAs. The VapB antitoxin inhibits the toxin with its C terminus domain and contains a DNA binding domain in the N terminus [1].

TA systems have been implicated in bacterial tolerance to antibiotics, which can eventually lead to antibiotic resistance and it is considered a major health challenge by the WHO [3]. Previously, it was experimentally observed when a bacterial strain developed antibiotic tolerance in the presence of ampicillin during intermittent exposure, the development of tolerance increase the risk of the strain also developing antibiotic resistance [4]. In strains of Escherichia coli KLY where tolerance was observed, it was shown that mutations in a gene encoding VapB were present [4].

However, it is not known how or if TA systems are involved in creating tolerance and potentially affect antibiotic resistance. Here, we determine the structure of VapBC from E. coli KLY to 2.8 Å using x-ray crystallography. The structure is overall very similar to the previously determined structure of a VapBC complex from Shigella flexneri 2a, where the VapBC is encoded on the pMYSH6000 plasmid. This VapBC was shown to form a hetero-octameric complex containing four VapB and four VapC proteins [5]. The VapBC from E. coli KLY differs from the VapBC in S. flexneri 2a in six positions. The two VapBs have two amino acids difference and the VapCs have four amino acid differences among them.

In the near future, structures representing several VapBC complexes with naturally occurring tolerance mutations will be determined. We envisage that the information gained will allow us to hypothesize the possible functional implications of the mutations and the possible effect this could have on the bacterium and whether this could explain an antibiotic tolerant phenotype.

1. Bendtsen, K.L. and D.E. Brodersen, Higher-Order Structure in Bacterial VapBC Toxin-Antitoxin Complexes. Subcell Biochem, 2017. 83: p. 381-412.

2. Harms, A., et al., Toxins, Targets, and Triggers: An Overview of Toxin-Antitoxin Biology. Molecular Cell, 2018. 70(5): p. 768-784.

3. Antimicrobial resistance. Available from: https://www.who.int/health-topics/antimicrobial-resistance.

4. Levin-Reisman, I., et al., Antibiotic tolerance facilitates the evolution of resistance. Science, 2017. 355(6327): p. 826-830.

5. Dienemann, C., et al., Crystal structure of the VapBC toxin-antitoxin complex from Shigella flexneri reveals a hetero-octameric DNA-binding assembly. J Mol Biol, 2011. 414(5): p. 713-22.

The cholera causative Vibrio cholerae can adapt rapidly to changing environments via sensory proteins like inner membran regulator ToxR, transducing signals from its periplasmic sensory domain to its cytoplasmic effector domain. ToxR thus activates, co-activates or represses numerous genes in V. cholerae, among them also virulence associated genes. Previously studies suggested that inner membrane protein ToxS plays a crucial role in the activity of ToxR.

The NMR structure of the periplasmic domain of ToxR (ToxRp) reveals the formation of a four stranded β sheet stacked against a long α-helix (Gubensäk et al. 2020). C236, in the middle of the helix forms a disulphide bond with C293 at the C-terminal end. NMR dynamic studies showed that under reducing conditions ToxRp adapts two conformations: one resembling the oxidized form, and a second one revealing strong dynamics proposing an unstructured form. The long C-terminal stretch, including C293, seems to be unstructured and highly flexible under reducing conditions, thereby suggesting an explanation for the increased proteolytic sensitivity of reduced ToxR (ToxRp-red) that was previously reported in V. cholerae (Lembke et al. 2018; Lembke et al. 2020).

By using a combination of NMR, SEC-MALS, and Fluorescence Anisotropy we could identify the formation of a strong heterodimer of the periplasmic domains of inner membrane proteins ToxR and ToxS (ToxRSp) independent on the redox state of ToxRp. Our results reveal that ToxRp binds ToxSp in a 1:1 fashion with a dissociation constant of 11.6nM. Additionally, by monitoring the proteolytic cleavage of ToxRp with NMR we provide a direct evidence of ToxS protective function.

The versatile functions of ToxR propose separate control mechanisms, in order to regulate the activity of ToxR as direct activator, co-activator or repressor. We propose that ToxR activity is mainly controlled by its stability. The reduction of ToxRp cysteines represent one possibility to decrease ToxR stability. This regulation is controlled by periplasmic oxidoreductases DsbA and DsbC in-vivo (Fengler et al. 2012; Lembke et al. 2018; Lembke et al. 2020). The interaction with ToxS represents another possibility to increase ToxR stability by directly protecting ToxRp from proteases DegPS (Lembke et al. 2018; Pennetzdorfer et al. 2019). Binding of ToxS seems to be independent from ToxRp cysteines. Previous work has shown that V. cholerae alkalinizes its surrounding in the late stationary phase, which decreases the interaction between ToxRS and subsequently leads to a loss of function of ToxR due to proteolysis (Almagro-Moreno et al. 2015a; Midgett et al. 2017) Furthermore, our experiments show that binding of ToxSp does not trigger dimerization of ToxRp via disulphide bonds under the applied conditions. Therefore, our data also support the theory that dimerization of ToxR, in order to induce transcription, is activated by the presence of DNA (Midgett et al. 2020; Lembke et al. 2020).

Financial support by the the Austrian Science Fund FWF project T-1239 to Nina Gubensäk is gratefully acknowledged.

Novel FAD-dependent oxidoreductase from a lignocellulose-degrading fungus Chaetomium thermophilum (CtFDO) belongs to glucose-methanol-choline (GMC) superfamily of oxidoreductases which act on the hydroxyl groups of non-activated alcohols, carbohydrates and sterols. GMC superfamily enzymes share a two-domain character, the FAD-binding motif GxGxxG, and usually His-His or His-Asn active-site pair [1]. The crystal structure of CtFDO reveals a unique His-Ser active-site pair a an active-site pocket, which is, compared to known structures of GMC oxidoreductases, unusually large, wide-open, and extended beyond the pyrimidine moiety of FAD. These features of the active-site pocket indicate a different type of substrate than common for GMC oxidoreductases.

A large activity screening with about 1000 compounds including substrates of GMC oxidoreductases showed CtFDO to be inactive toward these compounds. To identify chemical groups of putative substrates and predict the substrates specificity of CtFDO, we utilized the technique of crystallographic fragment screening, which resulted in series of six complexes binding small inorganic and aromatic moieties inside the active-site pocket of CtFDO (Fig. 1). The size of the pocket together with preference for binding of aromatic moieties indicate polyaromatic nature of the putative substrate with molecular weight likely greater than 500 Da.

Figure 1. Crystal structure of CtFDO with color-coded substrate-binding (yellow) and the FAD-binding (blue) domains. The FAD cofactor is shown as sticks with black C atoms and the active-site pocket with salmon surface. (b) Three-dimensional superposition of the active sites of the CtFDO complexes binding four fragments from Frag Xtal Screen (Jena Bioscience) and two other compounds. The active site pocket is displayed as salmon mesh, selected surrounding residues and FAD as sticks with gray C atoms, and the ligands with red, cyan, blank, yellow, green, purple C atoms. Tthe molecular graphics were created using PyMOL (Schrödinger).

[1] Sützl, L. Foley, G., Gillam, E. M. J., Bodén, M., Haltrich, D. (2019). Biotechnol Biofuels 12: 118.

[2] Švecová, L. Østergaard, L. H., Skálová, T., Schnorr, K., Koval’, T., Kolenko, P., Stránský, J., Sedlák, D., Dušková, J., Trundová, M., Hašek, J., Dohnálek, J. (2021). Acta Cryst. D77, 755-775.

The work was supported by the institutional support of IBT CAS, v.v.i. (RVO: 86652036), ERDF (CZ.02.1.01/0.0/0.0/15_003/0000447, CZ.02.1.01/0.0/0.0/16_013/0001776 and CZ.1.05/1.1.00/02.0109), MEYS CR (LM2018127 and CZ.02.1.01/0.0/0.0/16_019/0000778) and by the Grant Agency of the Czech Technical University in Prague (SGS19/189/ OHK4/3T/14).

This poster describes structure-assisted design of inhibitors of human carbonic anhydrase IX (CA IX) based on three-dimensional carborane and cobalt bis(dicarbollide) clusters. CA IX enzyme is known to play crucial role in cancer cell proliferation and formation of metastases. The new class of potent and selective CA IX inhibitors combines structural motif of bulky inorganic cluster with an alkylsulfamido or alkylsulfonamido anchor group for Zn²⁺ atom in the enzyme active site. Detailed structure-activity relationship (SAR) study of a large series containing 50 compounds is corroborated by almost 50 high resolution structures of compounds bound to CA IX active site and the active site of CA II. Structural features of the cluster-containing inhibitors that important for efficient and selective inhibition of CA IX activity were uncovered and used in structure-assisted design. Preclinical evaluation of selected compounds revealed low toxicity, favourable pharmacokinetics and ability to reduce tumour growth. Cluster-containing inhibitors of CA IX can thus be considered as promising candidates for drug development and/ or for combination therapy in boron neutron capture therapy.

Nearly one-third of the proteins require metal ions to accomplish their functions, making them obligatory for the growth and survival of microorganisms in varying environmental niches [1, 2]. In prokaryotes, besides their involvement in various cellular and physiological processes, metal ions stimulate the uptake of citrate molecules. Citrate is a source of carbon and energy and is reported to be transported by secondary transporters. In Gram-positive bacteria, citrate molecules are transported in complex with divalent metal ions, whereas in Gram-negative bacteria, they are translocated by Na⁺/citrate symporters (CitS) [3, 4]. Interestingly, the presence of a secondary transporter allowing the translocation of divalent metal ion-complexed citrate in Gram-negative bacteria has not been reported till date. In this study, we report the presence of a novel divalent metal ion-complexed citrate uptake system that belongs to the primary active ABC transporter superfamily. For the uptake, the metal ion-complexed citrate molecules are sequestered by substrate-binding proteins (SBPs) and transferred to transmembrane domains (TMDs) for their transport [1, 2]. Since SBPs are involved in maintaining the selectivity and specificity of the substrate(s) and directionality of the transport, they have been reported to be pivotal. This study reports the crystal structures of an Mg²⁺-citrate-binding protein (MctA) from a Gram-negative thermophilic bacteria Thermus thermophilus HB8 in both apo and holo forms at a resolution range of 1.63 to 2.50 Å. Despite binding various divalent metal ions, MctA follows the coordination geometry to bind its physiological metal ion, Mg²⁺. The results also suggest a novel subclassification of cluster D SBPs, known to bind and transport divalent metal ion-complexed citrate molecules. Comparative assessment of the open and closed conformations of the wild-type and mutant proteins of MctA suggests a gating mechanism of ligand entry following an “asymmetric domain movement” of the N-terminal domain (NTD) for substrate binding.

[1] Mandal, S. K., Nayak, S. G. & Kanaujia, S. P. (2021). Int. J. Biol. Macromol.185, 324.

[2] Mandal, S. K., Adhikari, R., Sharma, A., Chandravanshi, M., Gogoi, P. & Kanaujia, S. P (2019). Metallomics. 11, 597.

[3] Kim, J. W., Kim, S., Kim, S., Lee, H., Lee, J. O. & Jin, M. S. (2017). Sci. Rep. 7, 1.

[4] Wohlert, D., Grotzinger, M. J., Kuhlbrandt, W. & Yildiz, O. (2015). Elife. 4, e09375.

Acknowledgements: This work was supported in part by a grant from Department of Biotechnology (DBT), Government of India (Sanction Order No.: BT/PR16065/NER/95/61/2015). SKM acknowledges the Ministry of Human Resource and Development (MHRD), Government of India, for his research scholarship.

In Gram-negative bacteria, the maintenance of lipid asymmetry (Mla) system is involved in the transport of phospholipids (PLs) between the inner membrane (IM) and outer membrane (OM), thereby maintaining OM asymmetry [1]. The Mla system is a multi-component intermembrane machinery which is composed of three main constituents- an OM MlaA-OmpC/F complex, a free-floating periplasmic protein MlaC and an IM ATP-binding cassette (ABC) transporter complex MlaFEDB [2]. MlaC, which serves as the solute-binding protein (SBP), has been reported to have atypical structural features [3]. However, an in-depth investigation highlighting the peculiarities and the mechanism of ligand binding is still lacking. This study reports, for the first time, the crystal structure of MlaC from Escherichia coli at a resolution of 2.5 Å in a quasi-open state and in complex with a PL. The analysis reveals that MlaC comprises two major domains viz, NTF2-like (D1) and AAA helical-bundle (D2). Each domain can be divided into two subdomains (D1R1, D1R2; D2R1, D2R2) that are arranged in a discontinuous fashion. Further, MlaC would follow a reverse mechanism of binding pocket opening and the subdomains exhibit specific movements that aid in ligand binding and orientation. Based on extensive structural analysis, a novel mechanism of ligand binding is proposed that has not been observed for any known SBP till date. Additionally, the study also highlights the unique ancestries of MlaC and other atypical SBPs that are involved in OM biogenesis. The work firmly establishes MlaC to be a one-of-a-kind transporter protein that plays critical role in maintaining OM asymmetry.

[1] Malinverni, J. C. & Silhavy, T. J. (2009). Proc. Natl. Acad. Sci. U.S.A. 106, 8009. [2] Coudray, N., Isom, G. L., MacRae, M. R., Saiduddin, M. N., Bhabha, G. & Ekiert, D.C. (2020). Elife 9, e62518. [3] Yero, D., Díaz-Lobo, M., Costenaro, L., Conchillo-Solé, O., Mayo, A., Ferrer-Navarro, M., Vilaseca, M., Gibert, I. & Daura, X. (2021). Commun. Biol. 4, 1.

Acknowledgements: This work is supported by Science and Engineering Research Board (SERB), Department of Science and Technology, Government of India (Grant/Award Number: ECR/2018/000013). AD acknowledges the Ministry of Human Resource and Development (MHRD), Government of India.

Fasciolosis caused by the liver fluke Fasciola hepatica is a worldwide spread parasitic disease of ruminant and an emerging human disease. Cystatin superfamily of cysteine protease inhibitors is composed of intracellular type 1 cystatins (stefins), secreted type 2 cystatins, and multidomain type 2 cystatins. Helminth parasites secrete type 2 cystatins to modulate host immune responses for successful parasitism, except for F. hepatica that lacks type 2 cystatin genes.

This work is focused on F. hepatica type 1 cystatin FhCY2. It was localized to gastroderm and tegument and was surprisingly detected in the excretory/secretory products. We demonstrated that recombinant FhCY2 is a broad-selective inhibitor of host cysteine cathepsins as well as cysteine cathepsins of F. hepatica, suggesting its dual role in the regulation of exogenous and endogenous proteolytic systems. Furthermore, we solved the crystal structure of FhCY2 at 1.6 Å. The structural and phylogenetic analyses revealed that FhCY2 has the sequence and fold of type 1 cystatins but also the signal peptide and disulfides typical for type 2 cystatins, combining all hallmarks in an unprecedented way. We propose that FhCY2 is an evolutionary upgrade of type 1 cystatins for secretion that occurred in F. hepatica (and Fasciolidae family in general) in the absence of type 2 cystatins.

Protein self-association is an extremely common phenomenon in biology. However, light-driven protein homo-oligomerization has so far only been described in a few classes of photoreceptors, most notably plant cryptochromes and phytochromes. The characterization of light-induced protein oligomerization is challenging due to the need of synchronizing sample irradiation with data acquisition. Using a combination of carefully chosen methods, we hereby show that EL222, a bacterial transcription factor belonging to the light-oxygen-voltage (LOV) family, can form clusters in a concentration and power dependent manner. In the dark state, the DNA-binding helix-turn-helix module of EL222 is caged by the adjacent LOV domain. Blue-light excitation of the embedded flavin mononucleotide (FMN) cofactor triggers a cascade of protein conformational changes leading to uncaging of the HTH domain, EL222 dimerization, and interaction with its target DNA. Our time-resolved small-angle neutron scattering (SANS) experiments revealed the kinetics of EL222 assembly into high-order oligomers upon illumination and their subsequent disassembly in the dark. The light-induced changes in EL222 size and shape were found to be fully reversible and the photorecovery rate was in line with the well-known FMN photocycle. Further experiments employing fluorescence correlation (FCS) spectroscopy supported the SANS observations and allowed us to gain more insight into the photoinduced oligomerization kinetics. Analyses of the fluorescence traces and FCS curves pointed to the co-existence of multiple diffusing species in EL222 samples illuminated continuously. Moreover, we identified putative protein-protein interaction interfaces and the role of DNA in the aggregation process. Taken together, our hybrid SANS/FCS approach suggests a plausible mechanism of multimer formation in irradiated EL222.