CIISB Research Highlights Archive

Double-strand breaks (DSBs) are the most severe type of DNA damage. Long et al. show that hSSB1 is modified and forms a trimeric SOSS1 complex that comes to DSBs in an R-loop-dependent manner. At DSBs, SOSS1 and RNA polymerase II form liquid-like repair compartments. Depletion of the SOSS1 impairs DNA repair.

Monika Gullerova Research Group

Significance

Double-strand breaks (DSBs) are the most severe type of DNA damage. Previously, we demonstrated that RNA polymerase II (RNAPII) phosphorylated at the tyrosine 1 (Y1P) residue of its C-terminal domain (CTD) generates RNAs at DSBs. However, the regulation of transcription at DSBs remains enigmatic. Here, we show that the damage-activated tyrosine kinase c-Abl phosphorylates hSSB1, enabling its interaction with Y1P RNAPII at DSBs. Furthermore, the trimeric SOSS1 complex, consisting of hSSB1, INTS3, and c9orf80, binds to Y1P RNAPII in response to DNA damage in an R-loop-dependent manner. Specifically, hSSB1, as a part of the trimeric SOSS1 complex, exhibits a strong affinity for R-loops, even in the presence of replication protein A (RPA). Our in vitro and in vivo data reveal that the SOSS1 complex and RNAPII form dynamic liquid- like repair compartments at DSBs. Depletion of the SOSS1 complex impairs DNA repair, underscoring its biological role in the R-loop-dependent DNA damage response.

Long, QL; Sebesta, M; Sedova, K; Haluza, V; Alagia, A; Liu, ZC; Stefl, R; Gullerova, M: The phosphorylated trimeric SOSS1 complex and RNA polymerase II trigger liquid-liquid phase separation at double-strand breaks.

Cell Reports 42, 113439, https://doi.org/10.1016/j.celrep.2023.113489

c) Three-dimensional (3D) reconstruction showing an electron density map with γ-conglutin model in top and side views (PDB: 4pph) fitted as a ring-like hexamer assemble.

Jaroslaw Czubinsky Research Group

Significance

Despite extensive research carried out on lupin seed γ-conglutin neither its mechanism of action as a hypoglycaemic nutraceutical compound nor its physiological role for the plant has been unveil. This article revealed a pH-dependent reversible association/dissociation equilibrium involving monomer, dimer and hexamer of Lupinus angustifolius γ-conglutin. The interaction between different oligomeric forms of this protein is reversible, and spectroscopic studies showed that the intact structure of γ-conglutin was preserved under the tested environmental conditions tested (pH 4.5–7.5). The obtained results prove that the hexameric form was preferred under basic conditions and was stabilised by a number of bonds formed upon association of individual protomers. The simultaneous occurrence of several oligomeric forms at a given pH value was shown, and their share was strongly driven by protein concentration. The main changes in oligomerisation of γ-conglutin take place in a pH range of 4.5–6.0, correlating with the pKa_R values of the amino acid residues of His (6.0), Glu (4.1), and Asp (3.9). Moreover, a structural model of the protein in hexamer assembly was obtained based on small-angle X-ray scattering (SAXS) and negative staining cryo-electron microscopy (cryo-EM) analyses. The presented study provides essential knowledge about the colloidal dynamics and stability of γ-conglutin in solution, improving our understanding of fundamental environmental factors that could affect the health-promoting activity of this lupin seed protein.

Czubinksi, J., Kubíčková, M., et al. pH-Dependent oligomerisation of γ-conglutin: A key element to understand its molecular mechanism of action.

Food Hydrocol. 147, Part A, 109386 (2024) https://doi.org/10.1016/j.foodhyd.2023.109386

a, b Mapping the binary interaction between RSM and RPA32C by NMR titrations. ¹⁵N labeled RSM titrated with zero to fourfold molar addition of RPA32C (a) and the reverse (b).

Kostas Tripsianes and Lumír Krejčí Research Groups

Significance

Biomolecular polyelectrolyte complexes can be formed between oppositely charged intrinsically disordered regions (IDRs) of proteins or between IDRs and nucleic acids. Highly charged IDRs are abundant in the nucleus, yet few have been functionally characterized. Here, we show that a positively charged IDR within the human ATP-dependent DNA helicase Q4 (RECQ4) forms coacervates with G-quadruplexes (G4s). We describe a three-step model of charge-driven coacervation by integrating equilibrium and kinetic binding data in a global numerical model. The oppositely charged IDR and G4 molecules form a complex in the solution that follows a rapid nucleation-growth mechanism leading to a dynamic equilibrium between dilute and condensed phases. We also discover a physical interaction with Replication Protein A (RPA) and demonstrate that the IDR can switch between the two extremes of the structural continuum of complexes. The structural, kinetic, and thermodynamic profile of its interactions revealed a dynamic disordered complex with nucleic acids and a static ordered complex with RPA protein. The two mutually exclusive binding modes suggest a regulatory role for the IDR in RECQ4 function by enabling molecular handoffs. Our study extends the functional repertoire of IDRs and demonstrates a role of polyelectrolyte complexes involved in G4 binding.

Papageorgiou, A.C., Pospisilova, M., Cibulka, J. et al. Recognition and coacervation of G-quadruplexes by a multifunctional disordered region in RECQ4 helicase.

Nat Commun 14, 6751 (2023). https://doi.org/10.1038/s41467-023-42503-z

Crystal structures of DhaA223 and DhaA231. (a) Cartoon representations of DhaA223 (8OE2, red) and DhaA231 (PDB ID: 8OE6, dark blue) crystal structures aligned to the DhaA115 (PDB ID: 6SP5, gray). Residues of the catalytic pentad are shown as sticks. Stabilizing mutations are shown as spheres (pink spheres indicate mutations suggested by both FireProt and PROSS). (b) The structural context of selected stabilizing mutations. Newly formed stabilizing interactions involving other residues or water molecules (red spheres) are depicted by yellow dashed lines.

Zbyněk Prokop and David Bednář Research Groups

Significance

Thermostability is an essential requirement for the use of enzymes in the bioindustry. Here, we compare different protein stabilization strategies using a challenging target, a stable haloalkane dehalogenase DhaA115. We observe better performance of automated stabilization platforms FireProt and PROSS in designing multiple-point mutations over the introduction of disulfide bonds and strengthening the intra- and the inter-domain contacts by in silico saturation mutagenesis. We reveal that the performance of automated stabilization platforms was still compromised due to the introduction of some destabilizing mutations. Notably, we show that their prediction accuracy can be improved by applying manual curation or machine learning for the removal of potentially destabilizing mutations, yielding highly stable haloalkane dehalogenases with enhanced catalytic properties. A comparison of crystallographic structures revealed that current stabilization rounds were not accompanied by large backbone re-arrangements previously observed during the engineering stability of DhaA115. Stabilization was achieved by improving local contacts including protein–water interactions. Our study provides guidance for further improvement of automated structure-based computational tools for protein stabilization.

Kunka, A., Marques, S. M., Havlásek, M., Vašina, M,, Velátová, N., Cengelová, L., Kovář, J., Damborský, J., Marek, M., Bednář*. D., and Prokop*, Z. Advancing Enzyme’s Stability and Catalytic Efficiency through Synergy of Force-Field Calculations, Evolutionary Analysis, and Machine Learning

ACS Catal. 2023, 13, 19, 12506–12518, https://doi.org/10.1021/acscatal.3c02575

Micro-CT images of P0 pups with control and low iron diet, containing 178.58 mg iron/kg or 5.16 mg iron/kg, respectively. The kidney (red), interscapular brown adipose tissue (IBAT) (yellow), liver (green), and adrenal glands (orange) are segmented using 3D Visualization software and superimposed onto the pups.

Julian Petersen and Igor Adamyeko Research Group

Significance

In this study, we use comparative genomics to uncover a gene with uncharacterized function (1700011H14Rik/C14orf105/CCDC198), which we hereby name FAME (Factor Associated with Metabolism and Energy). We observe that FAME shows an unusually high evolutionary divergence in birds and mammals. Through the comparison of single nucleotide polymorphisms, we identify gene flow of FAME from Neandertals into modern humans. We conduct knockout experiments on animals and observe altered body weight and decreased energy expenditure in Fame knockout animals, corresponding to genome-wide association studies linking FAME with higher body mass index in humans. Gene expression and subcellular localization analyses reveal that FAME is a membrane-bound protein enriched in the kidneys. Although the gene knockout results in structurally normal kidneys, we detect higher albumin in urine and lowered ferritin in the blood. Through experimental validation, we confirm interactions between FAME and ferritin and show co-localization in vesicular and plasma membranes.

Petersen, J., Englmaier, L., Artemov, A.V. et al. A previously uncharacterized Factor Associated with Metabolism and Energy (FAME/C14orf105/CCDC198/1700011H14Rik) is related to evolutionary adaptation, energy balance, and kidney physiology.

Nat Commun. 14, 3092 (2023). https://doi.org/10.1038/s41467-023-38663-7

a, The view of the C₂S₂ supercomplex from the lumenal side with indicated subunits of light-harvesting antenna, Lhcb5, Lhcb8 and the S-LHCII trimer, bound to the dimeric core complex. b, The side view of the C₂S₂ supercomplex along the membrane plane. c, Assigned subunits of the core complex.

Roman Kouřil Research Group

Significance

The heart of oxygenic photosynthesis is the water-splitting photosystem II (PSII), which forms supercomplexes with a variable amount of peripheral trimeric light-harvesting complexes (LHCII). Our knowledge of the structure of green plant PSII supercomplex is based on findings obtained from several representatives of green algae and flowering plants; however, data from a non-flowering plant are currently missing. Here we report a cryo-electron microscopy structure of PSII supercomplex from spruce, a representative of non-flowering land plants, at 2.8 Å resolution. Compared with flowering plants, PSII supercomplex in spruce contains an additional Ycf12 subunit, Lhcb4 protein is replaced by Lhcb8, and trimeric LHCII is present as a homotrimer of Lhcb1. Unexpectedly, we have found α-tocopherol (α-Toc)/α-tocopherolquinone (α-TQ) at the boundary between the LHCII trimer and the inner antenna CP43. The molecule of α-Toc/α-TQ is located close to chlorophyll a614 of one of the Lhcb1 proteins and its chromanol/quinone head is exposed to the thylakoid lumen. The position of α-Toc in PSII supercomplex makes it an ideal candidate for the sensor of excessive light, as α-Toc can be oxidized to α-TQ by high-light-induced singlet oxygen at low lumenal pH. The molecule of α-TQ appears to shift slightly into the PSII supercomplex, which could trigger important structure–functional modifications in PSII supercomplex. Inspection of the previously reported cryo-electron microscopy maps of PSII supercomplexes indicates that α-Toc/α-TQ can be present at the same site also in PSII supercomplexes from flowering plants, but its identification in the previous studies has been hindered by insufficient resolution.

Opatíková, M., Semchonok, D.A., Kopečný, D. et al. Cryo-EM structure of a plant photosystem II supercomplex with light-harvesting protein Lhcb8 and α-tocopherol.

Nat. Plants 9, 1359–1369 (2023). https://doi.org/10.1038/s41477-023-01483-0