Multivalency in lectins-a crystallographic,modelling and light-scattering study involving peanut lectin and a bivalent ligand

Multivalency is believed to be important in the activity of lectins, although definitive structural studies on it have been few and far between. We have now studied the complexation of tetravalent peanut lectin with a synthetic compound containing two terminal lactose moieties, using a combination of crystallography, dynamic light scattering and modelling. Light scattering indicates the formation of an apparent dimeric species and also larger aggregates of the tetrameric lectin in the presence of the bivalent ligand. Crystals of presumably crosslinked lectin molecules could be obtained. They diffract poorly, but X-ray data from them are good enough to define the positions of the lectin molecules. Extensive modelling on possible crosslinking modes of protein molecules by the ligand indicated that systematic crosslinking could lead to crystalline arrays. The studies also provided a rationale for crosslinking in the observed crystal structure. The results obtained provide further insights into the general problem of multivalency in lectins. They indicate that crosslinking involving multivalent lectins and multivalent carbohydrates could lead to an ensemble of a finite number of distinct periodic arrays rather than a unique array

Nucleic Acids Res

The non-coding RNA 7SK is the scaffold for a small nuclear ribonucleoprotein (7SKsnRNP) which regulates the function of the positive transcription elongation factor P-TEFb in the control of RNA polymerase II elongation in metazoans. The La-related protein LARP7 is a component of the 7SKsnRNP required for stability and function of the RNA. To address the function of LARP7 we determined the crystal structure of its La module, which binds a stretch of uridines at the 3'-end of 7SK. The structure shows that the penultimate uridine is tethered by the two domains, the La-motif and the RNA-recognition motif (RRM1), and reveals that the RRM1 is significantly smaller and more exposed than in the La protein. Sequence analysis suggests that this impacts interaction with 7SK. Binding assays, footprinting and small-angle scattering experiments show that a second RRM domain located at the C-terminus binds the apical loop of the 3' hairpin of 7SK, while the N-terminal domains bind at its foot. Our results suggest that LARP7 uses both its N- and C-terminal domains to stabilize 7SK in a closed structure, which forms by joining conserved sequences at the 5'-end with the foot of the 3' hairpin and has thus functional implications

Ribosomal RNA 2′O-methylation as a novel layer of inter-tumour heterogeneity in breast cancer

International audienceRecent epitranscriptomics studies unravelled that ribosomal RNA (rRNA) 2′O-methylation is an additional layer of gene expression regulation highlighting the ribosome as a novel actor of translation control. However, this major finding lies on evidences coming mainly, if not exclusively, from cellular models. Using the innovative next-generation RiboMeth-seq technology, we established the first rRNA 2′O-methylation landscape in 195 primary human breast tumours. We uncovered the existence of compulsory/stable sites, which show limited inter-patient variability in their 2′O-methylation level, which map on functionally important sites of the human ribosome structure and which are surrounded by variable sites found from the second nucleotide layers. Our data demonstrate that some positions within the rRNA molecules can tolerate absence of 2′O-methylation in tumoral and healthy tissues. We also reveal that rRNA 2′O-methylation exhibits intra- and inter-patient variability in breast tumours. Its level is indeed differentially associated with breast cancer subtype and tumour grade. Altogether, our rRNA 2′O-methylation profiling of a large-scale human sample collection provides the first compelling evidence that ribosome variability occurs in humans and suggests that rRNA 2′O-methylation might represent a relevant element of tumour biology useful in clinic. This novel variability at molecular level offers an additional layer to capture the cancer heterogeneity and associates with specific features of tumour biology thus offering a novel targetable molecular signature in cancer

Identification of Mg 2+ ions next to nucleotides in cryo-EM maps using electrostatic potential maps

International audienceCryo electron microscopy (cryo-EM) can produce maps of macromolecules that have resolutions that are sufficiently high that structural details such as chemical modifications, water molecules and bound metal ions can be discerned. However, those accustomed to interpreting the electron-density maps of macromolecules produced by X-ray crystallography need to be careful when assigning features such as these in cryo-EM maps because cations, for example, interact far more strongly with electrons than they do with X-rays. Using simulated electrostatic potential (ESP) maps as a tool led us to re-examine a recent cryo-EM map of the human ribosome, and we realized that some of the ESP peaks originally identified as novel groups covalently bonded to the N7, O6 or O4 atoms of several guanines, adenines or uridines, respectively, in this structure are likely to instead represent Mg 2+ ions coordinated to these atoms, which provide only partial charge compensation compared with Mg 2+ ions located next to phosphate groups. In addition, direct evidence is provided for a variation in the level of 2′- O ribose methylation of nucleotides in the human ribosome. ESP maps can thus help in identifying ions next to nucleotide bases, i.e. at positions that can be difficult to address in cryo-EM maps due to charge effects, which are specifically encountered in cryo-EM. This work is particularly relevant to nucleoprotein complexes and shows that it is important to consider charge effects when interpreting cryo-EM maps, thus opening possibilities for localizing charges in structures that may be relevant for enzymatic mechanisms and drug interactions

Peanut agglutinin, a lectin with an unusual quaternary structure and interesting ligand binding properties

Lectins are multivalent proteins which play their biological role through the ability to specifically bind different carbohydrate structures. This ability has also led to their myriad applications. They occur in all forms of life. Among plant lectins, those from leguminous plants constitute the most thoroughly studied family. Most of the well-characterized legume lectins can be classified as mannose (Man)/glucose (Glc) specific or galactose (Gal)/N-acetylgalactosamine (GalNAc) specific. Tetrameric, non-glycosylated peanut agglutinin (PNA) is the most thoroughly investigated member of the Gal/GalNAc specific family of legume lectins. Its structure indicated that open quaternary association also needed to be considered when dealing with multimeric proteins. The structure also helped to establish legume lectins as a family of proteins in which small alterations in essentially the same tertiary structure lead to large changes in quaternary association. It provides an explanation for the exclusive specificity of PNA for galactose at the primary site. The structures of the relevant complexes demonstrated that the specificity of the lectin for T-antigen at the disaccharide level is caused by a water-bridge, thus establishing water-mediated interactions as a strategy for generating ligand specificity. In disaccharide complexes, the second residue interacts directly with the protein only when the glycosidic linkage is β1-3 or β1-4. Studies on the interactions of PNA with a bivalent ligand provide valuable insights into the multivalency of lectins. The availability of detailed structural information on the lectin permitted rational re-design of its combining site. A comparison of the available structural data on PNA led to the elucidation of the limited plasticity of the lectin molecule and its relation to function. An exploration of its hydration shell brought out the importance of conserved water molecules in the structure and function of PNA. Interestingly, while the Man/Glc specific concanavalin A, the most thoroughly studied lectin to date, represents the conventional wisdom on legume lectins, PNA, perhaps the next most extensively studied legume lectin, largely highlights departures from it

Visualization of chemical modifications in the human 80S ribosome structure

Comment in RNA modifications: Ribosomes get decorated. [Nat Chem Biol. 2017]International audienceChemical modifications of human ribosomal RNA (rRNA) are introduced during biogenesis and have been implicated in the dysregulation of protein synthesis, as is found in cancer and other diseases. However, their role in this phenomenon is unknown. Here we visualize more than 130 individual rRNA modifications in the three-dimensional structure of the human ribosome, explaining their structural and functional roles. In addition to a small number of universally conserved sites, we identify many eukaryote- or human-specific modifications and unique sites that form an extended shell in comparison to bacterial ribosomes, and which stabilize the RNA. Several of the modifications are associated with the binding sites of three ribosome-targeting antibiotics, or are associated with degenerate states in cancer, such as keto alkylations on nucleotide bases reminiscent of specialized ribosomes. This high-resolution structure of the human 80S ribosome paves the way towards understanding the role of epigenetic rRNA modifications in human diseases and suggests new possibilities for designing selective inhibitors and therapeutic drugs

Visualizing the Role of 2’-OH rRNA Methylations in the Human Ribosome Structure

Chemical modifications of RNA have recently gained new attention in biological sciences. They occur notably on messenger RNA (mRNA) and ribosomal RNA (rRNA) and are important for various cellular functions, but their molecular mechanism of action is yet to be understood in detail. Ribosomes are large ribonucleoprotein assemblies, which synthesize proteins in all organisms. Human ribosomes, for example, carry more than 200 modified nucleotides, which are introduced during biogenesis. Chemically modified nucleotides may appear to be only scarcely different from canonical nucleotides, but modifications such as methylations can in fact modulate their chemical and topological properties in the RNA and alter or modulate the overall translation efficiency of the ribosomes resulting in dysfunction of the translation machinery. Recent functional analysis and high-resolution ribosome structures have revealed a large repertoire of modification sites comprising different modification types. In this review, we focus on 2′-O-methylations (2′-O-Me) and discuss the structural insights gained through our recent cryo electron microscopy (cryo-EM) high-resolution structural analysis of the human ribosome, such as their locations and their influence on the secondary and tertiary structures of human rRNAs. The detailed analysis presented here reveals that ribose conformations of the rRNA backbone differ when the 2′-OH hydroxyl position is methylated, with 3′-endo conformations being the default and the 2′-endo conformations being characteristic in that the associated base is flipped-out. We compare currently known 2′-O-Me sites in human rRNAs evaluated using RiboMethSeq and cryo-EM structural analysis and discuss their involvement in several human diseases

Atomic model building and refinement into high-resolution cryo-EM maps

International audienc

Structural studies on peanut lectin complexed with disaccharides involving different linkages: further insights into the structure and interactions of the lectin

Crystal structures of peanut lectin complexed with Gal$_\beta$ 1-3Gal, methyl-T-antigen, Gal$_\beta$1-6Gal\alpha NAc, Gal\alpha1-3Gal and Gal\alpha1-6Glc and that of a crystal grown in the presence of Gal\alpha1-3Gal$_\beta$1-4Gal have been determined using data collected at 100 K. The use of water bridges as a strategy for generating carbohydrate specificity was previously deduced from the complexes of the lectin with lactose (Gal$_\beta$1-4Glc) and T-antigen (Gal$_\beta$1-3GalNAc). This has been confirmed by the analysis of the complexes with Gal$_\beta$1-3Gal and methyl-Tantigen (Gal$_\beta$1-3GalNAc-\alpha-OMe). A detailed analysis of lectin–sugar interactions in the complexes shows that they are more extensive when the $_\beta$-anomer is involved in the linkage. As expected, the second sugar residue is ill-defined when the linkage is $1\rightarrow 6$. There are more than two dozen water molecules which occur in the hydration shells of all structures determined at resolutions better than 2.5 \AA. Most of them are involved in stabilizing the structure, particularly loops. Water molecules involved in lectin–sugar interactions are also substantially conserved. The lectin molecule is fairly rigid and does not appear to be affected by changes in temperature

Multivalency in lectins A crystallographic, modelling and light-scattering study involving peanut lectin and a bivalent ligand

Multivalency is believed to be important in the activity of lectins, although definitive structural studies on it have been few and far between. We have now studied the complexation of tetravalent peanut lectin with a synthetic compound containing two terminal lactose moieties, using a combination of crystallography, dynamic light scattering and modelling. Light scattering indicates the formation of an apparent dimeric species and also larger aggregates of the tetrameric lectin in the presence of the bivalent ligand. Crystals of presumably crosslinked lectin molecules could be obtained. They diffract poorly, but X-ray data from them are good enough to define the positions of the lectin molecules. Extensive modelling on possible crosslinking modes of protein molecules by the ligand indicated that systematic crosslinking could lead to crystalline arrays. The studies also provided a rationale for crosslinking in the observed crystal structure. The results obtained provide further insights into the general problem of multivalency in lectins. They indicate that crosslinking involving multivalent lectins and multivalent carbohydrates could lead to an ensemble of a finite number of distinct periodic arrays rather than a unique array