Graphical abstract

The interaction of human galectin‐1 with a variety of oligosaccharides, from di‐(N‐acetyllactosamine) to tetra‐saccharides (blood B type‐II antigen) has been scrutinized by using a combined approach of different NMR experiments, molecular dynamics (MD) simulations, and isothermal titration calorimetry. Ligand‐ and receptor‐based NMR experiments assisted by computational methods allowed proposing three‐dimensional structures for the different complexes, which explained the lack of enthalpy gain when increasing the chemical complexity of the glycan. Interestingly, and independently of the glycan ligand, the entropy term does not oppose the binding event, a rather unusual feature for protein‐sugar interactions. CLEANEX‐PM and relaxation dispersion experiments revealed that sugar binding affected residues far from the binding site and described significant changes in the dynamics of the protein. In particular, motions in the microsecond‐millisecond timescale in residues at the protein dimer interface were identified in the presence of high affinity ligands. The dynamic process was further explored by extensive MD simulations, which provided additional support for the existence of allostery in glycan recognition by human galectin‐1.

Pharmaceutical industry is progressively replacing the chemical synthesis of cholesterol‐lowering agents by enzymatic processes. The directed evolution of acyltransferase LovD was a breakthrough in the synthesis of simvastatin, although little is known about how the in vitro evolution path raised up an engineered variant (LovD9) with excellent biocatalytic properties (high catalytic efficiency and stability under reaction conditions). In this study, we unveil how different mutation clusters scattered across LovD9 primary sequence specifically contribute to enhance both enzyme kinetics and stability. To this aim, simvastatin synthetic and hydrolytic activities, kinetic parameters and thermostability of several engineered variants were assessed. Through a rational combination of those clusters of mutations, we generated the variant LovD−BuCh2 whose catalytic efficiency is around 90 % of that obtained with LovD9 but with 15 less mutations. Supported by molecular dynamics simulations, this work demonstrates the cumulative effect of mutations at both the active site and the substrate entrance channel to enhance binding of the acyl donor and speed up the acyl transfer step from the acyl‐enzyme complex to monacolin J acid, while simultaneously minimizing detrimental side‐reaction pathways, substrate inhibition and increasing thermostability.

The chemistry of diazo compounds has generated a huge breadth of applications in the field of organic synthesis. Their versatility combined with their tunable reactivity, stability, and chemoselectivity makes diazo compounds desirable reagents for chemical biologists. Here, we describe a method for the precise installation of diazo handles on proteins and antibodies in a mild and specific approach. Subsequent 1,3-cycloaddition reactions with strained alkynes enable both bioimaging through an in-cell “click” reaction and probing of the cysteine proteome in cell lysates. The selectivity and efficiency of these processes makes these suitable reagents for chemical biology studies.

Laccases are in increasing demand as innovative solutions in the biorefinery fields. Here, we combine mutagenesis with structural, kinetic, and in silico analyses to characterize the molecular features that cause the evolution of a hyperthermostable metallo-oxidase from the multicopper oxidase family into a laccase (k cat 273 s–1 for a bulky aromatic substrate). We show that six mutations scattered across the enzyme collectively modulate dynamics to improve the binding and catalysis of a bulky aromatic substrate. The replacement of residues during the early stages of evolution is a stepping stone for altering the shape and size of substrate-binding sites. Binding sites are then fine-tuned through high-order epistasis interactions by inserting distal mutations during later stages of evolution. Allosterically coupled, long-range dynamic networks favor catalytically competent conformational states that are more suitable for recognizing and stabilizing the aromatic substrate. This work provides mechanistic insight into enzymatic and evolutionary molecular mechanisms and spots the importance of iterative experimental and computational analyses to understand local-to-global changes.

The interaction of human galectin 8 and its two separate N-terminal and C-terminal carbohydrate recognition domains (CRD) to their natural ligands has been analysed using a synergistic combination of experimental...

The tandem-repeat Galectin-4 (Gal-4) contains two different domains covalently linked through a short flexible peptide. Both domains have been shown to bind preferentially to A and B histo blood group antigens with different affinities, although the binding details are not yet available. The biological relevance of these associations is unknown, although it could be related to its attributed role in pathogen recognition. The presentation of A and B histo blood group antigens in terms of peripheral core structures differs among tissues and from that of the antigen-mimicking structures produced by pathogens. Herein, the binding of the N-terminal domain of Gal-4 toward a group of differently presented A and B oligosaccharide antigens in solution has been studied through a combination of NMR, isothermal titration calorimetry (ITC), and molecular modeling. The data presented in this paper allow the identification of the specific effects that subtle chemical modifications within this antigenic family have in the binding to the N-terminal domain of Gal-4 in terms of affinity and intermolecular interactions, providing a structural-based rationale for the observed trend in the binding preferences.

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 Xray 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 Nterminal 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 Nlinked glycans present on the high-affinity receptor FcεRIα have also been explored by NMR.