Selective C-13-Labels on Repeating Glycan Oligomers to Reveal Protein Binding Epitopes through NMR: Polylactosamine Binding to Galectins

A combined chemo-enzymatic synthesis/NMR-based methodology is presented to identify, in unambiguous manner, the distinctive binding epitope within repeating sugar oligomers when binding to protein receptors. The concept is based on the incorporation of C-13-labels at specific monosaccharide units, selected within a repeating glycan oligomeric structure. No new chemical tags are added, and thus the chemical entity remains the same, while the presence of the C-13-labeled monosaccharide breaks the NMR chemical shift degeneracy that occurs in the non-labeled compound and allows the unique identification of the different components of the oligomer. The approach is demonstrated by a proof-of-concept study dealing with the interaction of a polylactosamine hexasaccharide with five different galectins that display distinct preferences for these entities.This research was funded by European Research Council for financial support (ERC-2017-AdG, project number 788143-RECGLYCANMR). We also thank Agencia Estatal de Investigacion (Spain) for project RTI2018-094751-B-C21 and the Severo Ochoa Excellence Accreditation (SEV-2016-0644

Selective C-13-Labels on Repeating Glycan Oligomers to Reveal Protein Binding Epitopes through NMR: Polylactosamine Binding to Galectins

A combined chemo-enzymatic synthesis/NMR-based methodology is presented to identify, in unambiguous manner, the distinctive binding epitope within repeating sugar oligomers when binding to protein receptors. The concept is based on the incorporation of C-13-labels at specific monosaccharide units, selected within a repeating glycan oligomeric structure. No new chemical tags are added, and thus the chemical entity remains the same, while the presence of the C-13-labeled monosaccharide breaks the NMR chemical shift degeneracy that occurs in the non-labeled compound and allows the unique identification of the different components of the oligomer. The approach is demonstrated by a proof-of-concept study dealing with the interaction of a polylactosamine hexasaccharide with five different galectins that display distinct preferences for these entities.This research was funded by European Research Council for financial support (ERC-2017-AdG, project number 788143-RECGLYCANMR). We also thank Agencia Estatal de Investigacion (Spain) for project RTI2018-094751-B-C21 and the Severo Ochoa Excellence Accreditation (SEV-2016-0644

A generalized approach for NMR studies of lipid-protein interactions based on sparse fluorination of acyl chains.

Sparse lipid fluorination enhances the lipids' 1H signal dispersion, enables clean molecular distinction by 19F NMR, and evinces micelle insertion of proteins via fluorine-induced signal shifts. We present a minimal fluorination scheme, and illustrate the concept on di-(4-fluoro)-heptanoylphosphatidylcholine micelles and solubilised seven-helix transmembrane pSRII protein

The interaction of fluorinated glycomimetics with DC-SIGN: multiple binding modes disentangled by the combination of NMR methods and MD simulations

Fluorinated glycomimetics are frequently employed to study and eventually modulate protein–glycan interactions. However, complex glycans and their glycomimetics may display multiple binding epitopes that enormously complicate the access to a complete picture of the protein–ligand complexes. We herein present a new methodology based on the synergic combination of experimental 19F-based saturation transfer difference (STD) NMR data with computational protocols, applied to analyze the interaction between DC-SIGN, a key lectin involved in inflammation and infection events with the trifluorinated glycomimetic of the trimannoside core, ubiquitous in human glycoproteins. A novel 2D-STD-TOCSYreF NMR experiment was employed to obtain the experimental STD NMR intensities, while the Complete Relaxation Matrix Analysis (CORCEMA-ST) was used to predict that expected for an ensemble of geometries extracted from extensive MD simulations. Then, an in-house built computer program was devised to find the ensemble of structures that provide the best fit between the theoretical and the observed STD data. Remarkably, the experimental STD profiles obtained for the ligand/DC-SIGN complex could not be satisfactorily explained by a single binding mode, but rather with a combination of different modes coexisting in solution. Therefore, the method provides a precise view of those ligand–receptor complexes present in solution.We thank Agencia Estatal de Investigación (Spain) for grants RTI2018-094751-B-C21 and B-C22, CTQ2015-68756-R, and for FPI and FPU fellowships to J.D.M. and P.V., respectively, and for the Severo Ochoa Excellence Accreditation (SEV-2016-0644). J.J.-B. also thanks to the European Research Council (RECGLYCANMR, Advanced Grant no. 788143). S.O. thanks the SFI Award 13/IA/1959Peer reviewe

Double Monoubiquitination Modifies the Molecular Recognition Properties of p15PAF Promoting Binding to the Reader Module of Dnmt1

The proliferating cell nuclear antigen (PCNA)-associated factor p15PAF is a nuclear protein that acts as a regulator of DNA repair during DNA replication. The p15PAF gene is overexpressed in several types of human cancer, and its function is regulated by monoubiquitination of two lysines (K15 and K24) at the protein N-terminal region. We have previously shown that p15PAF is an intrinsically disordered protein which partially folds upon binding to PCNA and independently contacts DNA through its Nterminal tail. Here we present an NMR conformational characterization of p15PAF monoubiquitinated at both K15 and K24 via a disulfide bridge mimicking the isopeptide bond. We show that doubly monoubiquitinated p15PAF is monomeric, intrinsically disordered, and binds to PCNA as nonubiquitinated p15PAF does but interacts with DNA with reduced affinity. Our SAXS-derived conformational ensemble of doubly monoubiquitinated p15PAF shows that the ubiquitin moieties, separated by eight disordered residues, form transient dimers because of the high local effective ubiquitin concentration. This observation and the sequence similarity with histone H3 N-terminal tail suggest that doubly monoubiquitinated p15PAF is a binding target of DNA methyl transferase Dnmt1, as confirmed by calorimetry. Therefore, doubly monoubiquitinated p15PAF directly interacts with PCNA and recruits Dnmt1 for maintenance of DNA methylation during replication.Spanish Ministerio de Economía y Competitividad and the Fondo Europeo de Desarrollo Regional (MINECO/FEDER) [CTQ2017-83810-R to F.J.B.]; Labex EpiGenMed, an “Investissements d”avenir’ program [ANR-10-LABX-12-01 to PB]. MOSTMicro [LISBOA-01-0145-FEDER-007660 to T.N.C. and H.M.]. A.G.M. acknowledges Spanish MINECO for predoctoral contract BE-2015-075847, and the CIC bioGUNE acknowledges MINECO for the Severo Ochoa accreditation Sev-2016-0644. The CBS-Montpellier is a member of France-BioImaging (FBI) and the French Infrastructure for Integrated Structural Biology (FRISBI), two national infrastructures supported by the French National Research Agency (ANR-10-INSB-04-01 and ANR-10-INSB- 05, respectively)

Structure of p15PAF–PCNA complex and implications for clamp sliding during DNA replication and repair

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Side chain to main chain hydrogen bonds stabilize a polyglutamine helix in a transcription factor

Polyglutamine (polyQ) tracts are low-complexity regions and their expansion is linked to certain neurodegenerative diseases. Here the authors combine experimental and computational approaches to find that the length of the androgen receptor polyQ tract correlates with its helicity and show that the polyQ helical structure is stabilized by hydrogen bonds between the Gln side chains and main chain carbonyl groups

What is the sugar code?

54 p.-11 fig.A code is defined by the nature of the symbols, which are used to generate information-storing combinations (e.g. oligo- and polymers). Like nucleic acids and proteins, oligo- and polysac-charides are ubiquitous, and they are a biochemical platform for establishing molecular mes-sages. Of note, the letters of the sugar code system (third alphabet of life) excel in coding ca-pacity by making an unsurpassed versatility for isomer (code word) formation possible by var-iability in anomery and linkage position of the glycosidic bond, ring size and branching. The enzymatic machinery for glycan biosynthesis (writers) realizes this enormous potential for building a large vocabulary. It includes possibilities for dynamic editing/erasing as known from nucleic acids and proteins. Matching the glycome diversity, a large panel of sugar receptors (lectins) has developed based on more than a dozen folds. Lectins ‘read’ the glycan-encoded information. Hydrogen/coordination bonding and ionic pairing together with stacking and C-H/- interactions as well as modes of spatial glycan presentation underlie the selectivity and specificity of glycan-lectin recognition. Modular design of lectins together with glycan display and the nature of the cognate glycoconjugate account for the large number of post-binding events. They give an entry to the glycan vocabulary its functional, often context-dependent meaning(s), hereby building the dictionary of the sugar codeFunding by the NIH grant CA242351 (to M.C.), the SFI Investigator Programme Awards 16/IA/4419 (to P.V.M.) and 13/IA/1959 & 16/RC/3889 (to S.O.) as well as by the grant BFU 2016-77835-R of the Spanish Ministry of Economy, Industry and Competitiveness (to A.R.).Peer reviewe

Structural Characterization of N-Linked Glycans in the Receptor Binding Domain of the SARS-CoV-2 Spike Protein and their Interactions with Human Lectins

info:eu-repo/grantAgreement/WT/Physiological Sciences/095700 ERC‐2017‐AdG, 788143‐RECGLYCANMR grant 200077 grant RTI2018‐094751‐B‐C21 GC2018‐098996‐B‐I00 RTI2018‐099592‐B‐C22 RTI2018‐101269‐B‐I00 SEV‐2016‐0644 IF/00780/2015 PTDC/BIA‐MIB/31028/2017 UCIBIO UIDB/04378/2020 Infrastructure project 22161 PD/BD/142847/2018The glycan structures of the receptor binding domain of the SARS-CoV2 spike glycoprotein expressed in human HEK293F cells have been studied by using NMR. The different possible interacting epitopes have been deeply analysed and characterized, providing evidence of the presence of glycan structures not found in previous MS-based analyses. The interaction of the RBD 13C-labelled glycans with different human lectins, which are expressed in different organs and tissues that may be affected during the infection process, has also been evaluated by NMR. In particular, 15N-labelled galectins (galectins-3, -7 and -8 N-terminal), Siglecs (Siglec-8, Siglec-10), and C-type lectins (DC-SIGN, MGL) have been employed. Complementary experiments from the glycoprotein perspective or from the lectin's point of view have permitted to disentangle the specific interacting epitopes in each case. Based on these findings, 3D models of the interacting complexes have been proposed.publishersversionpublishe

A glutamine-based single α-helix scaffold to target globular proteins

The binding of intrinsically disordered proteins to globular ones can require the folding of motifs into α-helices. These interactions offer opportunities for therapeutic intervention but their modulation with small molecules is challenging because they bury large surfaces. Linear peptides that display the residues that are key for binding can be targeted to globular proteins when they form stable helices, which in most cases requires their chemical modification. Here we present rules to design peptides that fold into single α-helices by instead concatenating glutamine side chain to main chain hydrogen bonds recently discovered in polyglutamine helices. The resulting peptides are uncharged, contain only natural amino acids, and their sequences can be optimized to interact with specific targets. Our results provide design rules to obtain single α-helices for a wide range of applications in protein engineering and drug design.We thank Luis Serrano for help with the Agadir predictions and helpful discussions, Ben Lehner and Ernest Giralt for helpful discussions and the ICTS NMR facility, managed by the scientific and technological centers of the University of Barcelona (CCiT UB), for their help in NMR. B.M. acknowledges funding from the Asociación Española contra el Cáncer (FCAECC project #POSTD211371MATE). C.G. acknowledges a graduate fellowship from MINECO (PRE2018-084684). M.S.-N. acknowledges funding from MINECO (PID2020-119810RB-I00). M.S.-N. holds a Ramón y Cajal contract (RYC2018-024759-I) from the Spanish Ministry of Science, Innovation, and Universities. X.S. acknowledges funding from AGAUR (2017 SGR 324), MINECO (BIO2015-70092-R and PID2019-110198RB-I00), and the European Research Council (CONCERT, contract number 648201). B.B.K acknowledges funding from the Novo Nordisk Foundation (#NNF18OC0033926). M.O. acknowledges funding from the Instituto Nacional de Bioinformática, The EU BioExcel Centre of Excellence for HPC and the Spanish Ministry of Science (PID2021-122478NB-I00) and the Instituto de Salud Carlos III–Instituto Nacional de Bioinformatica (ISCIII PT 17/0009/0007 co-funded by the Fondo Europeo de Desarrollo Regional). M.O. is an ICREA Academy scholar and J.A. is a Juan de la Cierva fellow. M.C. was supported by institutional funds of the Max Planck Society. This project has been carried out using the resources of CSUC. IRB Barcelona is the recipient of a Severo Ochoa Award of Excellence from MINECO (Government of Spain).Peer reviewe