The CBS domain protein MJ0729 of Methanocaldococcus jannaschii binds DNA

AbstractThe cystathionine beta-synthase (CBS) domains function as regulatory motifs in several proteins. Elucidating how CBS domains exactly work is relevant because several genetic human diseases have been associated with mutations in those motifs. Here, we show, for the first time, that a CBS domain binds calf-thymus DNA and E-boxes recognized by transcription factors. We have carried out the DNA-binding characterization of the CBS domain protein MJ0729 from Methanocaldococcus jannaschii by biochemical and spectroscopic techniques. Binding induces conformational changes in the protein, and involves the sole tryptophan residue. The apparent dissociation constant for the E-boxes is ∼10μM. These results suggest that CBS domains might interact with DNA

The SARS-CoV-2 Spike Glycoprotein Directly Binds Exogeneous Sialic Acids: A NMR View

[EN] The interaction of the SARS CoV2 spike glycoprotein with two sialic acid-containing trisaccharides (alpha 2,3 and alpha 2,6 sialyl N-acetyllactosamine) has been demonstrated by NMR. The NMR-based distinction between the signals of those sialic acids in the glycans covalently attached to the spike protein and those belonging to the exogenous alpha 2,3 and alpha 2,6 sialyl N-acetyllactosamine ligands has been achieved by synthesizing uniformly C-13-labelled trisaccharides at the sialic acid and galactose moieties. STD-H-1,C-13-HSQC NMR experiments elegantly demonstrate the direct interaction of the sialic acid residues of both trisaccharides with additional participation of the galactose moieties, especially for the alpha 2,3-linked analogue. Additional experiments with the spike protein in the presence of a specific antibody for the N-terminal domain and with the isolated receptor binding and N-terminal domains of the spike protein unambiguously show that the sialic acid binding site is located at the N-terminal domain.This research was funded by the European Research Council (ERC-2017-AdG, project number 788143-RECGLYCA NMR to J.J.B.) and Agencia Estatal de Investigacion (Spain), projects RTI2018-094751-B-C21 to J.J.B. & A.A. and PID2019-107770RA-I00 to J.E.O., and by the Human Frontier Science Program (HFSP; grant LT000747/2018-C to L.U.) and CIBER, an initiative of Instituto de Salud Carlos III (ISCIII), Madrid, Spai

Structural insights into the intracellular region of the human magnesium transport mediator CNNM4

The four member family of “Cyclin and Cystathionine β-synthase (CBS) domain divalent metal cation transport mediators”, CNNMs, are the least-studied mammalian magnesium transport mediators. CNNM4 is abundant in the brain and the intestinal tract, and its abnormal activity causes Jalili Syndrome. Recent findings show that suppression of CNNM4 in mice promotes malignant progression of intestinal polyps and is linked to infertility. The association of CNNM4 with phosphatases of the regenerating liver, PRLs, abrogates its Mg2+-efflux capacity, thus resulting in an increased intracellular Mg2+ concentration that favors tumor growth. Here we present the crystal structures of the two independent intracellular domains of human CNNM4, i.e., the Bateman module and the cyclic nucleotide binding-like domain (cNMP). We also derive a model structure for the full intracellular region in the absence and presence of MgATP and the oncogenic interacting partner, PRL-1. We find that only the Bateman module interacts with ATP and Mg2+, at non-overlapping sites facilitating their positive cooperativity. Furthermore, both domains dimerize autonomously, where the cNMP domain dimer forms a rigid cleft to restrict the Mg2+ induced sliding of the inserting CBS1 motives of the Bateman module, from a twisted to a flat disk shaped dimer.Gobierno Vasco PI2010-17, IE05-147, IE07-202Diputación Foral de Vizcaya 7/13/08/2006/11, 7/13/08/2005/14Ministerio de Ciencia e Innovación BFU2010-17857, CSD2008-00005Ministerio de Economía y Competitividad BFU2013-47531-R, BES-2014-068464, BFU2016-77408-R, BES-2017-08043

Structures of the Inhibitory Receptor Siglec-8 in Complex with a High-Affinity Sialoside Analogue and a Therapeutic Antibody

Human sialic acid binding immunoglobulin-like lectin-8 (Siglec-8) is an inhibitory receptor that triggers eosinophil apoptosis and can inhibit mast cell degranulation when engaged by specific monoclonal antibodies (mAbs) or sialylated ligands. Thus, Siglec-8 has emerged as a critical negative regulator of inflammatory responses in diverse diseases, such as allergic airway inflammation. Herein, we have deciphered the molecular recognition features of the interaction of Siglec-8 with the mAb lirentelimab (2C4, under clinical development) and with a sialoside mimetic with the potential to suppress mast cell degranulation. The three-dimensional structure of Siglec-8 and the fragment antigen binding (Fab) portion of the anti-Siglec-8 mAb 2C4, solved by X-ray crystallography, reveal that 2C4 binds close to the carbohydrate recognition domain (V-type Ig domain) on Siglec-8. We have also deduced the binding mode of a high-affinity analogue of its sialic acid ligand (9-N-napthylsufonimide-Neu5Ac, NSANeuAc) using a combination of NMR spectroscopy and X-ray crystallography. Our results show that the sialoside ring of NSANeuAc binds to the canonical sialyl binding pocket of the Siglec receptor family and that the high affinity arises from the accommodation of the NSA aromatic group in a nearby hydrophobic patch formed by the N-terminal tail and the unique G–G′ loop. The results reveal the basis for the observed high affinity of this ligand and provide clues for the rational design of the next generation of Siglec-8 inhibitors. Additionally, the specific interactions between Siglec-8 and the N-linked glycans present on the high-affinity receptor FcεRIα have also been explored by NMR.This work was supported by operating grant PID2019-107770RA-I00 (J.E.-O.) from the Agencia Estatal Investigación of Spain and by the European Research Council (ERC-2017-AdG, 788143-RECGLYCANMR to J.J.-B.). We also thank the Marie-Skłodowska-Curie actions (ITN Glytunes grant agreement no. 956758 to J.E.-O and ITN BactiVax under grant agreement no. 860325 to U.A.). Additional funding was provided by CIBER, an initiative of Instituto de Salud Carlos III (ISCIII), Madrid, Spain. We also thank the Ikerbasque Basque Foundation of Science and the Spanish Ministry of Economy, Industry and Competitiveness (for the postdoctoral contract Juan de la Cierva Incorporación to J.E-O). X-ray diffraction experiments described in this paper were performed using the XALOC synchrotron beamline at ALBA (Spain) and PXIII in Swiss Light Source (Switzerland)

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

Structural insights into Siglec-15 reveal glycosylation dependency for its interaction with T cells through integrin CD11b

Funding Information: This work was supported by the European Research Council (ERC-2017-AdG, 788143-RECGLYCANMR to J.J.-B; ERC-2018-StG 804236-NEXTGEN-IO to A.P.) and the Marie-Skłodowska-Curie actions (ITN Glytunes grant agreement No 956758 to K.S.; ITN BactiVax under grant agreement no. 860325 to U.A. and ITN DIRNANO grant agreement No 956544 to F.C.). X-ray diffraction experiments described in this paper were performed using beamlines XALOC synchrotron at ALBA (Spain) and PXIII in Swiss Light Source (Switzerland). F.M., C.S. and H.C. acknowledge Fundação para a Ciência e a Tecnologia (FCT-Portugal) for funding projects: PTDC/BIA-MIB/31028/2017 and UCIBIO project (UIDP/04378/2020 and UIDB/04378/2020) and Associate Laboratory Institute for Health and Bioeconomy—i4HB project (LA/P/0140/2020), to the CEEC contracts 2020.00233.CEECIND and 2020.03261.CEECIND for F.M. and H.C., respectively, and to PhD grant 2022.11723.BD of C.S. The NMR spectrometers are part of the National NMR Network (PTNMR) and are partially supported by Infrastructure Project No 22161 (co-financed by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC). F.M. and J.J.-B. acknowledge to the European funding for the GLYCOTwinning project (No. 101079417) and -COST Action GLYCONANOPROBES. A.P.’s research is funded by “La Caixa” Foundation (HR21-00925), AECC (LABAE211744PALA), Fundación FERO, Ikerbasque, and BIOEF EITB MARATOIA BIO19/CP/002. We thank Agencia Estatal de Investigación of Spain for grants PID2019-107956RA-I00 (A.P.), PID2019-107770RA-I00 (J.E.-O.), RTI2018-099592-B-C21 (F.C.), ID2020-114178GB (R.B. and J.D.S.), RYC2018-024183-I (A.P.), and the Severo Ochoa Center of Excellence Accreditation CEX2021-001136-S, all funded by MCIN/AEI/10.13039/501100011033 and by El FSE invierte en tu futuro, as well as CIBERES, and initiative of Instituto de Salud Carlos III (ISCIII, Spain) A.A.-V. receives funding from “La Caixa” Foundation (ID 100010434, LCF/BQ/DR20/11790022). A. B. (AECC Bizkaia Scientific Foundation, PRDVZ19003BOSC). F.C. acknowledges the Mizutani Foundation for Glycoscience (Grant 220115). Funding Information: This work was supported by the European Research Council (ERC-2017-AdG, 788143-RECGLYCANMR to J.J.-B; ERC-2018-StG 804236-NEXTGEN-IO to A.P.) and the Marie-Skłodowska-Curie actions (ITN Glytunes grant agreement No 956758 to K.S.; ITN BactiVax under grant agreement no. 860325 to U.A. and ITN DIRNANO grant agreement No 956544 to F.C.). X-ray diffraction experiments described in this paper were performed using beamlines XALOC synchrotron at ALBA (Spain) and PXIII in Swiss Light Source (Switzerland). F.M., C.S. and H.C. acknowledge Fundação para a Ciência e a Tecnologia (FCT-Portugal) for funding projects: PTDC/BIA-MIB/31028/2017 and UCIBIO project (UIDP/04378/2020 and UIDB/04378/2020) and Associate Laboratory Institute for Health and Bioeconomy—i4HB project (LA/P/0140/2020), to the CEEC contracts 2020.00233.CEECIND and 2020.03261.CEECIND for F.M. and H.C., respectively, and to PhD grant 2022.11723.BD of C.S. The NMR spectrometers are part of the National NMR Network (PTNMR) and are partially supported by Infrastructure Project No 22161 (co-financed by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC). F.M. and J.J.-B. acknowledge to the European funding for the GLYCOTwinning project (No. 101079417) and -COST Action GLYCONANOPROBES. A.P.’s research is funded by “La Caixa” Foundation (HR21-00925), AECC (LABAE211744PALA), Fundación FERO, Ikerbasque, and BIOEF EITB MARATOIA BIO19/CP/002. We thank Agencia Estatal de Investigación of Spain for grants PID2019-107956RA-I00 (A.P.), PID2019-107770RA-I00 (J.E.-O.), RTI2018-099592-B-C21 (F.C.), ID2020-114178GB (R.B. and J.D.S.), RYC2018-024183-I (A.P.), and the Severo Ochoa Center of Excellence Accreditation CEX2021-001136-S, all funded by MCIN/AEI/10.13039/501100011033 and by El FSE invierte en tu futuro, as well as CIBERES, and initiative of Instituto de Salud Carlos III (ISCIII, Spain) A.A.-V. receives funding from “La Caixa” Foundation (ID 100010434, LCF/BQ/DR20/11790022). A. B. (AECC Bizkaia Scientific Foundation, PRDVZ19003BOSC). F.C. acknowledges the Mizutani Foundation for Glycoscience (Grant 220115). Publisher Copyright: © 2023, The Author(s).Sialic acid-binding Ig-like lectin 15 (Siglec-15) is an immune modulator and emerging cancer immunotherapy target. However, limited understanding of its structure and mechanism of action restrains the development of drug candidates that unleash its full therapeutic potential. In this study, we elucidate the crystal structure of Siglec-15 and its binding epitope via co-crystallization with an anti-Siglec-15 blocking antibody. Using saturation transfer-difference nuclear magnetic resonance (STD-NMR) spectroscopy and molecular dynamics simulations, we reveal Siglec-15 binding mode to α(2,3)- and α(2,6)-linked sialic acids and the cancer-associated sialyl-Tn (STn) glycoform. We demonstrate that binding of Siglec-15 to T cells, which lack STn expression, depends on the presence of α(2,3)- and α(2,6)-linked sialoglycans. Furthermore, we identify the leukocyte integrin CD11b as a Siglec-15 binding partner on human T cells. Collectively, our findings provide an integrated understanding of the structural features of Siglec-15 and emphasize glycosylation as a crucial factor in controlling T cell responses.publishersversionpublishe

Current Status on Therapeutic Molecules Targeting Siglec Receptors

The sialic acid-binding immunoglobulin-type of lectins (Siglecs) are receptors that recognize sialic acid-containing glycans. In the majority of the cases, Siglecs are expressed on immune cells and play a critical role in regulating immune cell signaling. Over the years, it has been shown that the sialic acid-Siglec axis participates in immunological homeostasis, and that any imbalance can trigger different pathologies, such as autoimmune diseases or cancer. For all this, different therapeutics have been developed that bind to Siglecs, either based on antibodies or being smaller molecules. In this review, we briefly introduce the Siglec family and we compile a description of glycan-based molecules and antibody-based therapies (including CAR-T and bispecific antibodies) that have been designed to therapeutically targeting Siglecs

Purification, crystallization and preliminary crystallographic analysis of protein MJ1225 from Methanocaldococcus jannaschii, a putative archaeal homologue of γ-AMPK

A preliminary crystallographic analysis at 2.3 Å resolution of protein MJ1225 from M. jannaschii, a putative archaeal homolog of γ-AMPK, is described