Scott, L. G. | Search bioRxiv

bioRxiv2022

Pre-B cell receptor acts as a selectivity switch for Galectin-1 at the pre-B cell surface

Galectins are glycan binding proteins translating the sugar-encoded information of cellular glycoconjugates into many physiological activities including immunity, cell migration, and signaling. During early B lymphocytes (BL) development at the pre-B cell stage, BL express the pre-B cell receptor (pre-BCR) and are supported by mesenchymal stromal cells secreting Galectin-1 (Gal-1). Gal-1 interacts with glycosylated receptors from stromal and pre-B cell surfaces but also with the pre-BCR through a direct carbohydrate-independent contact. How this interaction might interplay with the glycan-decoding function of Gal-1 is unknown. Here, we investigated Gal-1 binding to cell surface ligands using NMR spectroscopy on native membranes. We showed that pre-BCR regulates Gal-1 binding to specifically target 2,3-sialylated receptors on pre-B cells. Upon pre-BCR interaction, dynamic changes resulted in additional contacts with 2,3-sialylated glycans converting Gal-1 from an exo- to an endo-type lectin. Remarkably, this selectivity switch is able to promote pre-B cell survival. Altogether, we shed light on a new mechanism allowing fine-tuning of Galectin specificity at the cell surfaces.

biochemistry↗

bioRxiv2020

Accumulation of an unprecedented 5'-deoxyadenos-4'-yl radical unmasks the kinetics of the radical-mediated C-C bond formation step in MoaA catalysis

Radical S-adenosyl-L-methionine (SAM) enzymes catalyze various free radical-mediated reactions. In these enzymes, the rate-determining SAM cleavage kinetically masks all the subsequent steps. Due to this kinetic masking, detailed mechanistic characterization of radical transformations catalyzed by these enzymes is very difficult. Here, we report a successful kinetic characterization of the radical C-C bond formation catalyzed by a MoaA radical SAM enzyme. MoaA catalyzes an unprecedented 3',8-cyclization of GTP into 3',8-cyclo-7,8-dihydro-GTP (3',8-cH2GTP) during the molybdenum cofactor (Moco) biosynthesis. Through a series of EPR and biochemical characterization, we found that MoaA accumulates a 5'-deoxyadenos-4'-yl radical (5'-dA-C4'*) under the turnover conditions, and forms (4'S)-5'-deoxyadenosine ((4'S)-5'-dA), which is a C-4' epimer of the naturally occurring (4'R)-5'-dA. Together with kinetic characterizations, these observations revealed the presence of a shunt pathway in which an on-pathway intermediate, GTP C-3' radical, abstracts H-4' atom from 5'-dA to transiently generate 5'-dA-C4'* that is subsequently reduced stereospecifically to yield (4'S)-5'-dA. Detailed kinetic characterization of the shunt and the main pathways provided the comprehensive view of MoaA kinetics, and determined the rate of the on-pathway 3',8-cyclization step as 2.7 {+/-} 0.7 s-1. Together with DFT calculations, this observation suggested that the 3',8-cyclization is accelerated by 6 [~] 9 orders of magnitude by MoaA. Potential contributions of the active-site amino acid residues, and their potential relationships with human Moco deficiency disease are discussed. This is the first determination of the magnitude of catalytic rate acceleration by a radical SAM enzyme, and provides the foundation for understanding how radical SAM enzymes achieve highly specific radical catalysis.

biochemistry↗