The interaction of the SARS CoV2 spike glycoprotein with two sialic acid‐containing trisaccharides (α2,3 and α2,6 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 α2,3 and α2,6 sialyl N‐acetyllactosamine ligands has been achieved by synthesizing uniformly 13C‐labelled trisaccharides at the sialic acid and galactose moieties. STD‐1H,13C‐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 α2,3‐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.
NMR experiments using 13C‐labelled sialyl‐containing trisaccharides unequivocally demonstrate that the N‐terminal domain of the Spike (S) SARS CoV‐2 glycoprotein directly binds sialic acid residues, highlighting the possibility of additional cellular receptor loci for the viral S protein at our cells.
The 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.
Unprecedent structural details of the glycans of the RBD of SARS‐CoV‐2 spike glycoprotein have been revealed by NMR spectroscopy. Unexpected and non‐previously reported glycoepitopes have been detected. The interaction of the RBD glycoprotein with diverse human lectins has been scrutinised by exploiting the NMR signature of the 13C‐glycans. Our analysis permitted to identify the corresponding glycan epitopes responsible for the interaction with each lectin.
CD22 maintains a baseline level of B-cell inhibition to keep humoral immunity in check. As a B-cell-restricted antigen, CD22 is targeted in therapies against dysregulated B cells that cause ...autoimmune diseases and blood cancers. Here we report the crystal structure of human CD22 at 2.1 Å resolution, which reveals that specificity for α2-6 sialic acid ligands is dictated by a pre-formed β-hairpin as a unique mode of recognition across sialic acid-binding immunoglobulin-type lectins. The CD22 ectodomain adopts an extended conformation that facilitates concomitant CD22 nanocluster formation on B cells and binding to trans ligands to avert autoimmunity in mammals. We structurally delineate the CD22 site targeted by the therapeutic antibody epratuzumab at 3.1 Å resolution and determine a critical role for CD22 N-linked glycosylation in antibody engagement. Our studies provide molecular insights into mechanisms governing B-cell inhibition and valuable clues for the design of immune modulators in B-cell dysfunction.The B-cell-specific co-receptor CD22 is a therapeutic target for depleting dysregulated B cells. Here the authors structurally characterize the ectodomain of CD22 and present its crystal structure with the bound therapeutic antibody epratuzumab, which gives insights into the mechanism of inhibition of B-cell activation.
Induction of broadly neutralizing antibodies (bnAbs) is a major HIV vaccine goal. Germline-targeting immunogens aim to initiate bnAb induction by activating bnAb germline precursor B cells. Critical ...unmet challenges are to determine whether bnAb precursor naïve B cells bind germline-targeting immunogens and occur at sufficient frequency in humans for reliable vaccine responses. Using deep mutational scanning and multitarget optimization, we developed a germline-targeting immunogen (eOD-GT8) for diverse VRC01-class bnAbs. We then used the immunogen to isolate VRC01-class precursor naïve B cells from HIV-uninfected donors. Frequencies of true VRC01-class precursors, their structures, and their eOD-GT8 affinities support this immunogen as a candidate human vaccine prime. These methods could be applied to germline targeting for other classes of HIV bnAbs and for Abs to other pathogens.
•Summary of the overall structure and conformational space of CBS-proteins.•The current knowledge on the structural basis of ligand recognition by CBS-domains is provided.•Analysis of the structural ...effects exerted by bound ligands at CBS-domains.
Cystathionine β-synthase (CBS) domains or CBS motifs are conserved structural domains that are present in thousands of non functionally-related proteins from all kingdoms of life. Their importance is underlined by the range of hereditary diseases associated with mutations in their amino acid sequence. CBS motifs associate in pairs referred to as Bateman modules. In contrast with initial assumptions, it is now well documented that CBS motifs and/or Bateman modules may suffer conformational changes upon binding of adenosine derivatives, metal ions or nucleic acids. The degree and direction of these structural changes depend on the type of ligand, the intrinsic features of the binding sites and the association manner of the Bateman modules. This review aims to provide a summary of the current knowledge on the structural basis of ligand recognition and on the structural effects caused by these ligands in CBS domain containing proteins.
Significance Cystathionine β-synthase (CBS), the pivotal enzyme of the transsulfuration pathway, regulates flux through the pathway to yield compounds, such as cysteine, glutathione, taurine, and H ...₂S, that control cellular redox status and signaling. Our crystal structure of an engineered human CBS construct bound to S -adenosylmethionine (AdoMet) reveals the unique binding site of the allosteric activator and the architecture of the human CBS enzyme in its activated conformation. Together with the basal conformation that we reported earlier, these structures unravel the molecular mechanism of human CBS activation by AdoMet. Current knowledge will allow for modeling of numerous pathogenic mutations causing inherited homocystinuria and for design of compounds modulating CBS activity.
Cystathionine β-synthase (CBS) controls the flux of sulfur from methionine to cysteine, a precursor of glutathione, taurine, and H ₂S. CBS condenses serine and homocysteine to cystathionine with the ...help of three cofactors, heme, pyridoxal-5′-phosphate, and S-adenosyl- l -methionine. Inherited deficiency of CBS activity causes homocystinuria, the most frequent disorder of sulfur metabolism. We present the structure of the human enzyme, discuss the unique arrangement of the CBS domains in the C-terminal region, and propose how they interact with the catalytic core of the complementary subunit to regulate access to the catalytic site. This arrangement clearly contrasts with other proteins containing the CBS domain including the recent Drosophila melanogaster CBS structure. The absence of large conformational changes and the crystal structure of the partially activated pathogenic D444N mutant suggest that the rotation of CBS motifs and relaxation of loops delineating the entrance to the catalytic site represent the most likely molecular mechanism of CBS activation by S-adenosyl- l -methionine. Moreover, our data suggest how tetramers, the native quaternary structure of the mammalian CBS enzymes, are formed. Because of its central role in transsulfuration, redox status, and H ₂S biogenesis, CBS represents a very attractive therapeutic target. The availability of the structure will help us understand the pathogenicity of the numerous missense mutations causing inherited homocystinuria and will allow the rational design of compounds modulating CBS activity.
Monoclonal antibodies constitute one of the largest groups of drugs to treat cancers and immune disorders, and are guiding the design of vaccines against infectious diseases. Fragments ...antigen-binding (Fabs) have been preferred over monoclonal antibodies for the structural characterization of antibody–antigen complexes due to their relatively low flexibility. Nonetheless, Fabs often remain challenging to crystallize because of the surface characteristics of complementary determining regions and the residual flexibility in the hinge region between the variable and constant domains. Here, we used a variable heavy-chain (VHH) domain specific for the human kappa light chain to assist in the structure determination of three therapeutic Fabs that were recalcitrant to crystallization on their own. We show that this ligand alters the surface properties of the antibody–ligand complex and lowers its aggregation temperature to favor crystallization. The VHH crystallization chaperone also restricts the flexible hinge of Fabs to a narrow range of angles, and so independently of the variable region. Our findings contribute a valuable approach to antibody structure determination and provide biophysical insight into the principles that govern the crystallization of macromolecules.
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•A variable heavy-chain domain (VHH) specifically binds the human kappa light chain.•VHH binding decreases the aggregation temperature of Fabs.•Fab crystallizability is enhanced by crystal-packing contacts mediated by the VHH.•A restricted range of elbow angles is observed for Fabs in complex with VHH.•Three therapeutic antibody structures are revealed from Fab-VHH complexes.
Human sialic-acid-binding immunoglobulin-like lectin-9 (Siglec-9) is a glycoimmune checkpoint receptor expressed on several immune cells. Binding of Siglec-9 to sialic acid containing glycans ...(sialoglycans) is well documented to modulate its functions as an inhibitory receptor. Here, we first assigned the amino acid backbone of the Siglec-9 V-set domain (Siglec-9
), using well-established triple resonance three-dimensional nuclear magnetic resonance (NMR) methods. Then, we combined solution NMR and molecular dynamic simulation methods to decipher the molecular details of the interaction of Siglec-9 with the natural ligands α2,3 and α2,6 sialyl lactosamines (SLN), sialyl Lewis X (sLeX), and 6-O sulfated sLeX and with two synthetically modified sialoglycans that bind with high affinity. As expected, Neu5Ac is accommodated between the F and G β-strands at the canonical sialic acid binding site. Addition of a heteroaromatic scaffold 9
-5-(2-methylthiazol-4-yl)thiophene sulfonamide (MTTS) at the C9 position of Neu5Ac generates new interactions with the hydrophobic residues located at the G-G' loop and the N-terminal region of Siglec-9. Similarly, the addition of the aromatic substituent (5-
(1-benzhydryl-1
1,2,3-triazol-4-yl)methyl (BTC)) at the C5 position of Neu5Ac stabilizes the conformation of the long and flexible B'-C loop present in Siglec-9. These results expose the underlying mechanism responsible for the enhanced affinity and specificity for Siglec-9 for these two modified sialoglycans and sheds light on the rational design of the next generation of modified sialoglycans targeting Siglec-9.
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.
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