Eph (erythropoietin producing hepatocellular) receptor constitutes the largest family of receptor tyrosine kinase. Based on sequence homology and binding partners, Eph receptor and Ephrin ligand are classified into EphA/EphrinA and EphB/EphrinB complexes [1]. Eph-ephrin as a family is ubiquitously expressed in almost all tissue [2]. Both are membrane-bound structures and regulates key physiological events such as cell-cell interaction, cell migration, partitioning and cell adhesion [3]. Eph receptors constitute an extracellular ligand binding domain, a cysteine-rich sushi domain and fibronectin repeat domains. Followed by a transmembrane domain lies the intracellular region of the receptor - juxtamembrane domain, kinase domain (KD) and SAM or PDZ binding domain [4]. Mutations reported in Kinase domain (KD) can affect the overall functionality of the receptor and downstream signalling pathways. Among the different Eph receptors, EphA7 has been recently regarded as a cancer driver gene (cancer gene census, COSMIC database). Similar to other Eph receptors, EphA7 also hold a dual functionality were it can act both as an oncogene and as a tumor suppressor [5, 6]. This dual functionality relate to its varied expression in different cancers. Many clinically important mutations have been reported in EphA7 (cbioportal, cosmic database), among which KD specifically holds hot spot mutations. In the present study, EphA7 mutations, Gly656Arg, Gly656Glu and Asp751His, were selected on the basis of in-silico analysis presented in the cbioportal. Gly656Glu and Gly656Arg are the hotspot mutations and present in the loop connecting two conserved beta sheets at the N – lobe of kinase. The third mutant Asp751His is present on the helix of C – lobe near to the catalytic loop. G656R, G656E, D751H have been crystallized and the structure is solved at a resolution of 3.1Å, 2.6Å, 3.05Å with the Rfactor/Rfree – 0.244/.280, 0.181/0.21, 0.199/0.247 respectively. Significant alterations in kinase domain has been observed due to the mutations that can affect binding affinity of ATP as well as catalytic efficiency of the Kinase Domain. Changes at the secondary structure levels were also observed in the hinge region for Gly656Arg and Asp751His mutants. This can adversely affect the transition of Kinase Domain from open to closed or closed to open confirmations.

[1] U. Nomenclature and T. Ligands, “Unified nomenclature for Eph family receptors and their ligands, the ephrins. Eph Nomenclature Committee.,” Cell, vol. 90, no. 3, pp. 403–404, 1997.

[2] H. Taylor, J. Campbell, and C. D. Nobes, “Ephs and ephrins,” Current Biology. 2017.

[3] E. Stein et al., “Eph receptors discriminate specific ligand oligomers to determine alternative signaling complexes, attachment, and assembly responses,” Genes Dev., vol. 12, no. 5, pp. 667–678, 1998.

[4] J. P. Himanen and D. B. Nikolov, “Eph signaling: A structural view,” Trends in Neurosciences. 2003.

[5] V. Modi and R. L. Dunbrack, “Defining a new nomenclature for the structures of active and inactive kinases,” Proc. Natl. Acad. Sci. U. S. A., vol. 116, no. 14, pp. 6818–6827, 2019.

[6] M. Anderton, E. van der Meulen, M. J. Blumenthal, and G. Schäfer, “The role of the eph receptor family in tumorigenesis,” Cancers (Basel)., vol. 13, no. 2, pp. 1–15, 2021.

[7] N. N. Phan et al., “Overexpressed gene signature of EPH receptor A/B family in cancer patients-comprehensive analyses from the public high-throughput database.,” Int. J. Clin. Exp. Pathol., vol. 13, no. 5, pp. 1220–1242, 2020.

Keywords: RTKs; crystal structure; secondary structural changes; mutations; cancer; change in intramolecular interaction

Acknowledgement – Funding for this study was supported by Annual Scientific Fund from ACTREC-TMC. S.C. is thankful to Council for scientific and industrial research (CSIR) for fellowship. The authors thank the XRD facility at ACTREC for providing necessary support to this study.

NKp30 is an activating receptor on the surface of human natural killer (NK) cells. Its crystal structure has been published previously by Joyce et al. [1], PDB code 3NOI. B7-H6 is an activating immunoligand expressed by some tumor cells. Its structure in complex with NKp30 has been described by Li et al. [2], PDB code 3PV6.

In this study, we present a new crystal structure of NKp30:B7-H6 at resolution 3.1 Å using homogenously glycosylated proteins produced in HEK293S GnTI^- cell lines. The structure has been deposited in the Protein Data Bank under code 6YJP and published [3].

For the structural study, NKp30 was used with complete glycosylation, while B7-H6 was deglycosylated after the first GlcNAc for better crystallization. The new structure showed the same NKp30:B7-H6 interaction interface as observed by Li et al. (3PV6). Similarly, as in the structure of Joyce et al. (3NOI), NKp30 form dimers. However, the dimers of glycosylated NKp30 are different (the glycan presence hinders the formation of the dimers observed in PDB 3NOI), and according to Pisa server validation, the new dimers are more likely biologically relevant.

Furthermore, the asymmetric unit of the new crystal structure contains a dimer of NKp30 placed among two B7-H6 molecules (contacts of chains A-C and B-D_symm). The illustration taken from our paper [3] shows a hypothesis of NKp30 dimer bound between two B7-H6 ligands during contact of the NK cell and the tumor cell.

[1] Joyce, M.G., Tran, P., Zhuravleva, M.A., Jaw, J., Colonna, M., Sun, P.D. (2011) Proc. Natl. Acad. Sci. USA 108, 6223–6228.

[2] Li, Y., Wang, Q., Mariuzza, R.A. (2011). J. Exp. Med. 208, 703–714.

[3] Skořepa, O., Pazicky, S., Kalousková, B., Bláha, J., Abreu, C., Ječmen, T., Rosůlek, M., Fish, A., Sedivy, A., Harlos, K., Dohnálek, J., Skálová, T., Vaněk, O. (2020). Cancers 12, 1998.

This research was funded by Czech Science Foundation (18-10687S), MEYS of the Czech Republic (LTC17065, CZ.02.1.01/0.0/0.0/16_013/0001776), BIOCEV (ERDF CZ.1.05/1.1.00/02.0109), and Charles University (GAUK 927916, SVV 260427/2020). CIISB research infrastructure project LM2015043, funded by MEYS CR, is gratefully acknowledged for the financial support of experiments at the CMS. The authors also acknowledge the support and the use of Instruct-ERIC resources (PID: 1314) and iNEXT (PID: 2322) infrastructures. The Wellcome Centre for Human Genetics is supported by Wellcome Trust grant 203141/Z/16/Z. O.S. and O.V. received short-term scientific mission support from COST Action CA15126.

Infection with the pathobiont Streptococcus pneumoniae (pneumococcus) can cause life-threatening invasive pneumococcal diseases (IPD), including pneumonia, sepsis, and meningitis [1-3]. With the emergence of new pneumococcal strains, there is an urgent need for vaccines that elicit broader population coverage against conserved pneumococcal antigens, irrespective of capsular serotype. Pneumolysin (Ply) is a key pneumococcal virulence factor belonging to a family of cholesterol-dependent cytolysins (CDCs) that disrupts host cell defence mechanisms and immune cell function. This cytotoxin is expressed by virtually all pneumococcal strains and pneumococcal carriage and infection induce natural immunity to Ply. A detoxified form of this protein has therefore been tested as a potential serotype-independent vaccine candidate to protect against IPD [4-6].

In this study, we identified a highly immunogenic human CD4⁺ T cell epitope in pneumolysin, widely presented by a common HLA allotype. The nature of the Ply-specific T cell receptor (TCR) repertoire was evaluated in healthy HLA-typed individuals. HLA-Ply-specific tetramer⁺ CD4⁺ TCRs were cloned, expressed, and purified. The ternary structures of three TCRs, including examples of near-public (B1) and private sequences (B5 and 5F), were solved in complex with HLA-Ply.

All of these TCRs formed stabilizing contacts with solvent-exposed residues in the central region of the peptide via their hypervariable CDR3 loops. The immunodominance of this epitope can therefore be explained by the preferential selection of TCRs capable of this ubiquitous mode of recognition.

[1] Adams, W., Bhowmick, R., Bou Ghanem, E. N., Wade, K., Shchepetov, M., Weiser, J. N., McCormick, B. A., Tweten, R. K. & Leong, J. M. (2020). J. Immunol. 204, 101.

[2] Backhaus, E., Berg, S., Andersson, R., Ockborn, G., Malmström, P., Dahl, M., Nasic, S. & Trollfors, B. (2016). BMC Infect. Dis. 16, 367.

[3] Kaur, R., Surendran, N., Ochs, M. & Pichichero, M. (2014). Infect. Immun. 82, 5069.

[4] Salha, D., Szeto, J., Myers, L., Claus, C., Sheung, A., Tang, M., Ljutic, B., Hanwell, D., Ogilvie, K., Ming, M., Messham, B., van den Dobbelsteen, G., Hopfer, R., Ochs, M. M. & Gallichan, S. (2012). Infect. Immun. 80, 2212.

[5] van de Garde, M. D. B., van Westen, E., Poelen, M. C. M., Rots, N. Y. & van Els, A. C. M. (2019). Infect. Immun. 87, e00098.

[6] Rossjohn, J., Gilbert, R. J. C., Crane, D., Morgan, P. J., Mitchell, T. J., Rowe, A. J., Andrew, P. W., Paton, J. C., Tweten, R. K. & Parker, M. W. (1998). J. Mol. Biol. 284, 1223.

We wish to acknowledge Josien Lanfermeijer from the RIVM/IIV for technical support, Frans Reubsaet from RIVM/IDS for providing clinical bacterial isolates, Sanofi-Pasteur for providing the peptide, and the NIH Tetramer Core Facility. We thank the staff at the Monash Macromolecular Crystallisation Facility and the Australian Synchrotron (beamlines MX1 and MX2) for assistance with crystallisation and data collection, respectively.

An increasing number of surface-exposed ligands and receptors acting on immune cells are being considered as a starting point in drug development applications. As they are dedicated to manipulate a wide range of immune responses, accurately predicting their molecular interactions will be necessary for the development of safe and effective therapeutics to enhance immune responses and vaccination. Here, we focused on characterization of human CD160 and HVEM immune receptors whose mutual engagement leads to bidirectional signaling (e.g., T cell inhibition, natural killer cell activation, or mucosal immunity). In particular, our study report on the molecule preparation, characterization and initial crystallographic analysis of CD160-HVEM complex and both HVEM and CD160 in ligand-free form. Despite the importance of the CD160-HVEM immune signaling and its therapeutic relevance, the structural and mechanistic basis underlying CD160-HVEM engagement has some controversial evidence. Some newer studies reported CD160 molecule in monomeric form [1-3], while older reports provided evidence on multimeric form acting on immune cells [4, 5]. In our study, the native non-linked CD160-HVEM complex was co-expressed in the baculovirus-insect host; purified to homogeneity by anion-exchange chromatography to provide missing evidence of trimeric form in solution. The CD160-HVEM crystallized in orthorhombic space group with unit cell parameters that could accommodate one trimeric complex (3:3) in asymmetric unit. Crystals of CD160-HVEM complex, CD160 trimer and HVEM monomer (reported in two space groups) diffracted to a minimum Bragg spacing of 2.8, 3.1 and 1.9/2.1 Å resolution, respectively.

[1] Liu, W.; Garrett, S. C.; Fedorov, E. V.; Ramagopal, U. A.; Garforth, S. J.; Bonanno, J. B.; Almo, S. C. Structure (2019), 27 (8), 1286-1295 e4.

[2] Kojima, R.; Kajikawa, M.; Shiroishi, M.; Kuroki, K.; Maenaka, K. J Mol Biol (2011), 413 (4), 762-72.

[3] Stiles, K. M.; Whitbeck, J. C.; Lou, H.; Cohen, G. H.; Eisenberg, R. J.; Krummenacher, C. J Virol (2010), 84 (22), 11646-60.

[4] Anumanthan, A.; Bensussan, A.; Boumsell, L.; Christ, A. D.; Blumberg, R. S.; Voss, S. D.; Patel, A. T.; Robertson, M. J.; Nadler, L. M.; Freeman, G. J. J Immunol (1998), 161 (6), 2780-90.

[5] Maiza, H.; Leca, G.; Mansur, I. G.; Schiavon, V.; Boumsell, L.; Bensussan, A. J Exp Med (1993), 178 (3), 1121-6.

Keywords: CD160/BY55; HVEM/TNFRSF14; immune receptor; immunological synapse; receptor-ligand interaction

This research was funded by the contribution of the Slovak Research and Development Agency under the project APVV-14-0839 and continuous project APVV-19-0376; and the contribution of the Scientific Grant Agency of the Slovak Republic under the grant VEGA-02/0020/18 and VE-GA-02/0060/21. IN was Marie Curie Fellow financed by programme SASPRO co-funded by European Union and the Slovak Academy of Sciences. The part of the research team was supported by Interreg V-A SK-AT cooperation programme by project CAPSID under the contract No. NFP305010V235 co-financed by European Regional Development Fund.

Integral membrane proteins suchg as GPCR’s, ion-channels or transporters are drug targets for more than 60% of all approved drugs. Structure based drug discovery on soluble proteins is managed well within the project timelines and portfolio changes in pharmaceutical industry, but transmembrane proteins are still underexplored because of their challenges to be expressed, purified and made them work for high resolution structure determination and ligand characterization by biophysical methods.

The presentation includes recent advances in the technologies and their application to relevant drug targets.

Construct engineering, application of in meso in situ serial X-ray crystallography (IMISX) is exemplified with the GPCR structure of CCR2 in complex with an antagonist ligand. This study is combined with detailed binding characterization using grating-coupled interferometry (GCI, Creoptix) to facilitate drug design with binding kinetic, affinity. Furthermore, the crosstalk between allosteric and orthosteric ligand binding could be investigated.

The structure of the human TRPV4 ion-channel with bound small molecule agonist shows activation of the channel opening with a significant structural change enabling direct observation of agonist pharmacology by high resolution cryo-EM analysis. Next example is LPTDE, a clinically validated antibiotics drug target. Due to limited size of 120 kDa and the monomeric b-sheet transmembrane architecture, the leadXpro proprietary tool of Pro-Macrobodies was essential for the successful EM structure at 2.9 A resolution.

The outlook at future perspectives includes further advances in cryo-EM and the application of serial X-ray crystallography using femtosecond pulsed Free Electron Lasers (FEL) for determination of room temperature structures and observation of structural dynamic of ligand binding and associated conformational changes. All new developments in structural biology will further enhance the impact to the design of candidate drug compounds.

Selected references:

Botte M. et al. Cryo-EM structural studies of the agonist complexed human TRPV4 ion-channel reveals novel structural rearrangements resulting in an open-conformation (2020), https://doi.org/10.1101/2020.10.13.334797

Nass, K., et al. Advances in long-wavelength native phasing at X-ray free-electron lasers. IUCrJ, 2020

https://doi.org/10.1107/S2052252520011379

Cheng, R.K.Y., Towards an optimal sample delivery method for serial crystallography at XFEL, Crystals, 2020, 10, 215;

Rufer, A, Hennig, M., Biophysical assessment of target protein quality in structure‐based drug discovery. https://onlinelibrary.wiley.com/doi/book/10.1002/9781118681121

Apel, A-C., Crystal structure of CC chemokine receptor 2A in complex with an orthosteric antagonist provides insights for the design of selective antagonists, Structure 27, (2019)

Antibody labeling has become a useful tool for determining three-dimensional (3D) structures of protein molecules and their complexes. Antibody fragments such as antigen-binding fragments (Fabs) serve as crystallization chaperones to promote lattice formation in X-ray crystallography. On the other hand, Fabs can be used as fiducial marks to aid the particle picking and alignment in cryo-electron microscopy (EM), which is difficult to apply to small protein molecules. However, establishing antibodies that bind stably to their respective targets is a prerequisite for utilizing the antibody labeling. To expand its applicability, we have developed an alternative strategy by inserting an exogenous epitope into targets and subsequently preparing complexes with antibody fragments. Specifically, we utilized a monoclonal antibody NZ-1, which has been established by immunizing rat with a 14-residue peptide segment (PA14) from human podoplanin.It has been shown that a 12-residue peptide segment (PA12) lacking two N-terminal residues of PA14 binds to NZ-1 with an extremely high affinity.^[1] In addition, a previous crystallographic analysis of the NZ-1 Fab complexed with the PA14 peptide has revealed that the PA12 part adopts a bent loop-like conformation within the antigen-binding pocket of NZ-1. Based on these observations, we first attempted to develop the NZ-1 labeling technique using the PA12 tag. We inserted the PA12 tag into the PDZ tandem (PDZ-N and PDZ-C) located in the periplasmic region of intramembrane protease. In fact, the PA12-inserted PDZ tandem formed a stable complex with the NZ-1 Fab.^[2] However, our structural analysis also showed that the complex formation through the inserted PA12 tag inevitably caused structural changes around the insertion site on the target. Therefore, we next attempted to utilize the PA14 tag, instead of PA12, which contains Glu-Gly residues upstream of PA12. We expected that the two additional residues could serve as a buffer region to tolerate structural changes on the target because they are flexible in the above-mentioned crystal structure of the NZ-1 Fab complexed with the PA14 peptide. As a result, the PA14-inserted PDZ tandem also produced co-crystals with the NZ-1 Fab, where the complex formation had less impact on the folding of the target PDZ domains as compared to the NZ-1 labeling with the PA12 tag.^[3] In addition, molecular-dynamics (MD) simulations have also suggested that PA14-inserted PDZ domains stably bind to the NZ-1 Fab with no significant structural changes. To demonstrate that our improved NZ-1 labeling technique could be applied to EM analysis, we also performed negative stain EM on the NZ-1-labeled full-length intramembrane protease. We actually obtained 3D models of the complex and succeeded in approximating the spatial arrangement of the PDZ domains based on the docking mode of the NZ-1 Fab.

[1] Fujii, Y., Kaneko, M., Neyazaki, M., Nogi, T., Kato, Y. & Takagi, J. (2014). Protein Expr. Purif. 95, 240-247.

[2] Tamura, R., Oi, R., Akashi, S., Kaneko, M., Kato, Y. & Nogi, T. (2019). Protein Sci. 28, 823-836.

[3] Tamura-Sakaguchi, R., Aruga, R., Hirose, M., Ekimoto, T., Miyake, T., Hizukuri, Y., Oi, R., K.Kaneko, M., Kato, Y., Akiyama, Y., Ikeguchi, M., Iwasaki, K. & Nogi, T. (2021). Acta Cryst. D77, 645-662

Yersinia entomophaga is a naturally occurring, Gram negative insect pathogen, first isolated from the diseased larvae of the New Zealand grass grub C. zealandica a decade ago [1]. Its main virulence determinant is YenTc, a heterogenous 2.4 MDa toxin complex that is a prototypical example of the ABC or Tc family of predominantly insecticidal toxins. YenTc is unique amongst members of this family, in that it is the only member of this class of toxins characterised to date that has a broad target host range, and which exhibits potent oral activity towards susceptible hosts without relying on a nematode symbiont for delivery. This has positioned YenTc as a potentially high value target for the development of novel biopesticides based on this class of toxins.

Previous work from our lab has yielded structures of the pore-forming A component determined by cryo-EM [2,3], the toxin-encapsulating BC cage [4] of YenTc determined by X-ray crystallography, as well as the co-expressed chitinase enzymes Chi1 and Chi2 also determined by X-ray crysatllography [4,5], the latter of which we show are structurally incorporated into the complex. Moreover, these chitinases exhibit significant structural mobility and appear to play a role in glycan recognition at the host cell surface. Our most recent work has led to the determination (using cryo-EM) of structures for the complete YenTc holotoxin assembly in both pre-pore and pore states. Comparing these structures to those of related toxins (primarily derived from the nematode symbiont Photorhabdus luminescens), has helped us to elucidate the overall mechanism of toxin packaging, translocation and delivery. We used the knowledge derived from these structures to guide a Hidden Markov Model-based bioinformatic analysis that led to the identification of more than 800 putative toxin complexes in diverse bacterial genome sequences. Phylogenetic analysis of these putative toxins led us to conclude that ABC toxins cluster into 4 subtypes, and illuminate a model for the evolution of these subtypes in response to host adaptation. Finally, as part of this analysis, we identified an orphan subunit located outside the pathogenicity island of YenTc, and solved the structure of the cytotoxic effector encoded within this subunit using X-ray crystallography. This is, to our knowledege, the first cytotoxic effector associated with an ABC toxin to have it's structure determined.

[1] Hurst, M.R.H. et al. (2011) Int J Syst Evol Microbiol, 61(4) 844-849.

[2] Landsberg, M.J. et al. (2011) PNAS, 108(51) 20544-20549

[3] Piper, S.J. et al. (2019) Nature Commun, 10(2019) 1952.

[4] Busby, J.N. et al. (2013) Nature, 501(7468) 547-550.

[5] Busby, J.N. et al. (2011) J Mol Biol, 415(2) 359-371.

Toll-like receptors (TLRs) detect pathogens and endogenous danger, initiating immune responses that lead to the production of pro-inflammatory cytokines. At the same time, TLR-mediated inflammation is associated with a number of pathological states, including infectious, autoimmune, inflammatory, cardiovascular and cancer-related disorders. This dual role of the pathways has attracted widespread interest from pharmaceutical industries. Cytoplasmic signaling by TLRs starts by their TIR (Toll/interleukin-1 receptor) domain interacting with TIR domain-containing adaptor proteins MyD88, MAL, TRIF and TRAM. Combinatorial recruitment of these adaptors via TIR:TIR domain interactions orchestrates downstream signaling pathways, leading to induction of the pro-inflammatory genes. Although many constituents of the TLR pathways have been identified, the available information on their coordinated interactions is limited. Such information is crucial for a mechanistic understanding of TLR signaling, development of therapeutic strategies, and understanding of the molecular basis of the consequences for human disease of adaptor polymorphic variants. We have discovered that TIR domains can form large assemblies. We hypothesized that TIR domain signaling occurs through a mechanism involving higher-order assembly formation. In this study we aim to determine the molecular architecture of higher-order assemblies formed by TIR domains with a focus on TRAM-TRIF assemblies in the TLR4 and TLR3 pathway.

Innate immunity represents a typical and widely distributed form of immunity. Innate immune responses are the first line of defense against pathogens, which can help destroy invaders invertebrate animals, invertebrates, and plants. The innate immune system recognizes microorganisms via pattern-recognition receptors (PRRs). The family of Toll-like receptors (TLRs) is a distinct group of PRRs. They detect the microbial components known as pathogen-associated molecular patterns (PAMPs), activate downstream transcription factors such as nuclear factor-κB (NF-κB), resulting in a pro-inflammatory response [1]. 10 TLRs has been identified in the human TLR family. In humans, TLR2 can form heterodimers with TLR1 and TLR6 when binding different types of ligands. The cytoplasmic Toll/interleukin-1 receptor (TIR) domain can be found in all TLRs and is responsible for transmitting extracellular signals to intracellular cytoplasmic TIR domain-containing adaptor proteins through TIR: TIR domain interactions, thus initiating downstream signaling. Two TIR-domain containing adaptor proteins, Myeloid differentiation primary response 88 (MyD88) and MyD88 adaptor-like (MAL) mediate downstream signaling in TLR2-TLR1/6 signaling pathway. It has been previously demonstrated that higher-order assembly formation occurs in the TLR4 signalling pathway [2]. The mechanism, which is known as signaling by cooperative assembly formation (SCAF), may occur in all TLR signal transduction. To date, the transduction mechanisms of TLR2-TLR1/6 signalling are still unclear. My project is to determine the structural basis of higher-order assemblies formed by TIR domains with a focus on assemblies in the TLR2-TLR1/6 signalling.