The transcription factor p53 controls numerous cellular processes including apoptosis, senescence, DNA repair, and tumor suppression [1]. The function of p53 is closely intertwined with Forkhead box O (FOXO) transcription factors and FOXO protein regulates cellular functions such as cellular homeostasis, oxidative stress resistance, metabolism, and longevity. FOXO protein family has four members: FOXO1, FOXO3, FOXO4, and FOXO6 wherein their activity is tightly regulated by post-translation modification (phosphorylation, acetylation, methylation, and ubiquitination) [2]. The previous study has demonstrated that the physical interaction between FOXO4 and p53 represses apoptosis of senescent cells by upregulating the transcription of p21 gene and maintain the viability of senescent cells [3]. However, the structural aspects of FOXO4:p53 complex formation remain unclear. Therefore, we designed several truncated constructs of FOXO4 and p53 to elucidate the structural details of this complex using analytical ultracentrifugation, NMR, chemical cross-linking, and molecular docking. Our data suggest that the transactivation domain (TAD) of p53 and DNA binding domain of FOXO4 provide overall stability of the complex along with the transient interaction from other regions of FOXO4 and p53. Furthermore, FOXO4:p53 complex formation does not affect the DNA binding affinity of FOXO4, thereby this interaction presumably allows co-localization of both FOXO4 and p53 transcription factors in the promoter region. Our finding further promotes future research for drug development aiming for the selective elimination of senescent cells.

[1] Boutelle, A.M., Attardi, L.D. (2021) Rev. Trends Cell Biology. 31, 298-310.

[2] Obsil T., Obsilova V. (2011) Rev. Biochimica et Biophysical Acta (BBA)-Molecular cell research. 1813, 1946-1953.

[3] Baar M.P., Brandt R.M.C., Putavet D.A., et al. (2017) Cell. 169, 132-147.

This study was supported by Czech Science Foundation Grant No. 21-02080S and the Grant agency of the Charles University (project number: 1002119).

The interactions between metal ions and nucleic acids have attracted considerable interest for their involvement in structure formation and folding of nucleic acids, and their possible roles in catalytic activity of nucleic acids. The structural and thermodynamic properties of the binding with the perfectly matched duplex DNA have been reported for many metal ions, but few studies have been reported for the interaction of metal ions with the mismatched base pair duplex DNA. We found that the addition of Hg²⁺ significantly increased the thermal stability of the duplex DNA with the T:T mismatched base pair [1, 2], and the combination of Hg²⁺ and the duplex DNA with the T:T mismatched base pair was highly specific for metal ion-DNA interactions. Isothermal titration calorimetry (ITC) demonstrated that Hg²⁺ specifically bound to the T:T mismatched base pair at 1:1 molar ratio with a binding constant of 10⁶ M^-1 [2]. We also found that the addition of Ag⁺ significantly increased the thermal stability of the duplex DNA with the C:C mismatched base pair [3, 4], and the combination of Ag⁺ and the duplex DNA with the C:C mismatched base pair was highly specific for metal ion-DNA interaction. ITC demonstrated that Ag⁺ specifically bound to the C:C mismatched base pair at 1:1 molar ratio with a binding constant of 10⁶ M^-1 [4]. Recently, we analyzed the possibility of specific binding between metal ion and 5-fluorouracil (5-FdU)-modified mismatched base pair in duplex DNA. Thermal stability analyses revealed that Hg²⁺ and Ag⁺ could specifically bind to the T:5-FdU and the C:5-FdU mismatched base pair in duplex DNA, respectively. Here, we determined the crystal structures of two duplex DNAs including mercury-mediated T-Hg-(5F-dU) base pair and silver-mediated C-Ag-(5F-dU) base pair.

Palindromic DNA fragments designed to contain T or C:5-FdU mismatched base pair in the center were used for crystallization. Both fragments were cocrystallized with metal ions by the sitting drop vapor diffusion method, and x-ray diffraction data were collected at the Beamline BL-5A of the Photon Factory. Experimental phases of duplex DNAs containing mercury- and silver-mediated base pairs were obtained by single wavelength anomalous dispersion (SAD) phasing with mercury atoms and the molecular replacement method, respectively. The structure of duplex DNA containing mercury-mediated T-Hg-(5F-dU) base pair was determined at 2.85 Å resolution and revealed that two duplex DNA molecules present in an asymmetric unit. Hg²⁺ specifically bound to the T:5-FdU mismatched base pair at 1:1 ratio with taking linear coordination to N3 atoms of T and 5-FdU residues to form T-Hg-(5F-dU) base pair. The structure of duplex DNA containing silver-mediated C-Ag-(5F-dU) base pair was determined at 2.20 Å resolution and revealed that a single-stranded DNA present in an asymmetric unit; two single-stranded DNAs forming a duplex DNA in the crystal is related by crystallographic 2-fold axis. One Ag⁺ is specifically inserted between C:5-FdU mismatches to form a C-Ag-(5F-dU) base pair by taking linear coordination to N3 atoms of C and 5-FdU residues as same as Hg²⁺. In addition, a hydrogen bond is formed between O4 atom of 5-FdU and N4 atoms of C residue, implying a contribution to stability by forming a stronger bond network.

In this study, we demonstrated the structural basis for the novel interaction of metal ions with the mismatched base pair including 5-FdU. Structural information for specific binding modes between chemically modified mismatched base pairs and metal ions can be expected to be applied to various fields such as environment, medicine and nanotechnology.

[1] Ono, A. & Togashi, H. (2004). Angew. Chem. Int. Ed. 43, 4300-4302.

[2] Torigoe, H., Ono, A. & Kozasa, T. (2010). Chem. Eur. J. 16, 13218-13225.

[3] Ono, A., Cao, S., Togashi, H., Tashiro, M., Fujimoto, T., Machinami, T., Oda, S., Miyake, Y., Okamoto, I. & Tanaka, Y. (2008) Chem. Commun. 4825-4827.

[4] Torigoe, H., Okamoto, I., Dairaku, T., Tanaka, Y., Ono, A. & Kozasa, T. (2012) Biochimie 94, 2431-2440.

Tick-borne encephalitis virus (TBEV) is a major human pathogen, transmitted by ticks from family Ixodidae. TBEV is an enveloped virus with a ~ 11 kb positive-sense single-strand RNA genome, encoding a single 375 kDa polyprotein. During infection, the polyprotein is cleaved into three structural and seven non-structural (NS) proteins. While the structural proteins are involved in assembly of new virions, the non-structural proteins are responsible for virus replication.

Non-structural protein 5 (NS5) is a large bi-functional conserved protein comprising two domains connected by a highly flexible linker, which is important for the activity as well as determines the overall shape of the protein. N-terminal methyltransferase (MTase) domain is the capping enzyme. The C-terminal RNA-dependent RNA polymerase (RdRp) is crucial for virus replication.

This project aims at structure determination and functional studies of TBEV NS5 protein. Various gene constructs were designed and cloned: NS5 full length, RdRp domain and MTase domain. Expression and purification of individual products have been optimized and pure proteins were used for initial crystallization screening, cryo-EM analysis and functional assays.

So far, we have obtained cryo-EM data for RdRp domain, using Titan Krios equipped with Falcon 4 camera and Relion processing pipeline yielded a reconstruction of 6 Å resolution. Tiny protein crystals of RdRp grew in several crystallization conditions. Furthermore, fluorescence-based binding assays revealed substrate affinity and specificity.

Surfactant Protein D (SP-D) is a member of the collectin family of proteins and acts as part of the innate immune system, the first line of defence, in the lung. The collectins recognise and bind specific structural features conserved amongst pathogens, particularly bacterial and fungal cell surface lipopolysaccharides (LPS) and viral glycans. SP-D can immobilise and form aggregates of pathogens that are more recognisable to neutrophils, along with triggering the rest of the immune system. High-resolution ligand-bound crystal structures of a biologically and therapeutically active recombinant homotrimeric fragment of native human SP-D (rfhSP-D) complexed with simple disaccharides have shown ligand binding to take place through the coordination of a Ca²⁺ ion and binding site residues to a mannose-type O3’ and O4’ pair of hydroxyls of the bound sugar[1].

Further work has taken place with fragments isolated from the lipopolysaccharide (LPS) of Haemophilus influenzae Eagan and Salmonella enterica Minnesota R5 through mild acid hydrolysis to cleave the bulky, hydrophobic lipid A[2,3]. These showed preferential binding by rfhSP-D of the LPS inner core through coordination of the calcium to the Hep O6’ and O7’ sidechain hydroxyls. These ligand-bound structures also highlight that hSP-D has the flexibility and versatility to recognise alternative LPS epitopes when the preferred core heptose is unavailable. In one subunit (subunit A) of the Salmonella LPS bound structure, the proximity of a crystal contact prevents the preferred inner core binding mode seen in the other chains, resulting in binding through the terminal glucose.

Crystals were also frozen at varying time-stages of the ligand soak, and analysis of these structures provides insight into the changes in the interaction between the protein and ligand over time as binding with the LPS progresses. The definition of the bound ligand in the electron density is seen to increases as the soaking time progresses. Comparison of the crystal structures over time also shows that in chain B the non-bound terminal glucose of the oligosaccharide, initially aligned planar with Pro319, rotates by 90° around the Glc-HepII glycosidic bond as binding progresses. There are also structural changes as depletion of the tertiary calcium site progresses over the time course of the ligand soak.

[1] Shrive AK, Tharia HA, Strong P, Kishore U, Burns I, Rizkallah PJ, et al. (2003) ‘High-resolution Structural Insights into Ligand binding and Immune Cell Recognition by Human Lung Surfactant Protein D’. J Mol Biol.; 331(2):509–23

[2] Littlejohn JR, da Silva RF, Neale WA, Smallcombe CC, Clark HW, Mackay R-MA, et al. (2018) ‘Structural definition of hSP-D recognition of Salmonella enterica LPS inner core oligosaccharides reveals alternative binding modes for the same LPS’. PLoS One. 13(6)

[3] Clark, H.W., Mackay, R.M., Deadman, M.E., Hood, D.W., Madsen, J., Moxon, R., Townsend, J.P., Reid, K.B.M., Ahmed, A., Shaw, A.J., Greenhough, T.J., Shrive, A.K. (2016). ‘Crystal Structure of a Complex of Surfactant Protein D (SP-D) and Haemophilus influenzae Lipopolysaccharide Reveals Shielding of Core Structures in SP-D-Resistant Strains’. Infection and Immunity, 84 (5), 1585 - 1592.