Immunoglobulin G (IgG) glycosylation critically modulates antibody effector functions. Streptococcus pyogenes secretes a unique endo-β- N -acetylglucosaminidase, EndoS2, which deglycosylates the conserved N -linked glycan at Asn297 on IgG Fc to eliminate its effector functions and evade the immune system. EndoS2 and specific point mutants have been used to chemoenzymatically synthesize antibodies with customizable glycosylation for gain of functions. EndoS2 is useful in these schemes because it accommodates a broad range of N -glycans, including high-mannose, complex, and hybrid types; however, its mechanism of substrate recognition is poorly understood. We present crystal structures of EndoS2 alone and bound to complex and high-mannose glycans; the broad N -glycan specificity is governed by critical loops that shape the binding site of EndoS2. Furthermore, hydrolytic experiments, domain-swap chimeras, and hydrogen–deuterium exchange mass spectrometry reveal the importance of the carbohydrate-binding module in the mechanism of IgG recognition by EndoS2, providing insights into engineering enzymes to catalyze customizable glycosylation reactions.

Endoglycosidase S (EndoS) is a bacterial endo-β-N-acetylglucosaminidase that specifically catalyzes the hydrolysis of the β-1,4 linkage between the first two N-acetylglucosamine residues of the biantennary complex-type N-linked glycans of IgG Fc regions. It is used for the chemoenzymatic synthesis of homogeneously glycosylated antibodies with improved therapeutic properties, but the molecular basis for its substrate specificity is unknown. Here, we report the crystal structure of the full-length EndoS in complex with its oligosaccharide G2 product. The glycoside hydrolase domain contains two well-defined asymmetric grooves that accommodate the complex-type N-linked glycan antennae near the active site. Several loops shape the glycan binding site, thereby governing the strict substrate specificity of EndoS. Comparing the arrangement of these loops within EndoS and related endoglycosidases, reveals distinct-binding site architectures that correlate with the respective glycan specificities, providing a basis for the bioengineering of endoglycosidases to tailor the chemoenzymatic synthesis of monoclonal antibodies.

MAT2 complex is expressed in nearly all tissues and is essential in providing the necessary SAMe flux for methylation of DNA and various proteins including histones. X-ray crystallography and solution X-ray scattering structures of this complex show an unexpected stoichiometry, offering a unique mechanism of regulation and thus providing a gateway for structure-based drug design in anticancer therapies including liver disease.

The principal methyl donor of the cell, S-adenosylmethionine (SAMe), is produced by the highly conserved family of methionine adenosyltranferases (MATs) via an ATP-driven process. These enzymes play an important role in the preservation of life, and their dysregulation has been tightly linked to liver and colon cancers. We present crystal structures of human MATα2 containing various bound ligands, providing a "structural movie" of the catalytic steps. High-to atomic-resolution structures reveal the structural elements of the enzyme involved in utilization of the substrates methionine and adenosine and in formation of the product SAMe. MAT enzymes are also able to produce S-adenosylethionine (SAE) from substrate ethionine. Ethionine, an S-ethyl analog of the amino acid methionine, is known to induce steatosis and pancreatitis. We show that SAE occupies the active site in a manner similar to SAMe, confirming that ethionine also uses the same catalytic site to form the product SAE.methionine adenosyltransferase | cell growth | liver cancer | X-ray crystallography | methylation

Salp15, a salivary protein of Ixodes ticks, inhibits the activation of naïve CD4 T cells. Treatment with Salp15 results in the inhibition of early signaling events and the production of the autocrine growth factor, interleukin-2. The fate of the CD4 T cells activated in the presence of Salp15 or its long-term effects are, however, unknown. We now show that Salp15 binding to CD4 is persistent and induces a long-lasting immunomodulatory effect. The activity of Salp15 results in sustained diminished cross-antigenic antibody production even after interruption of the treatment with the protein. Transcriptionally, the salivary protein provokes an acute effect that includes known activation markers, such as Il2 or Cd44, and that fades over time. The long-term effects exerted by Salp15 do not involve the induction of either anergy traits nor increased populations of regulatory T cells. Similarly, the treatment with Salp15 does not result in B cell anergy or the generation of myeloid suppressor cells. However, Salp15 induces the increased expression of the ectoenzyme, CD73, in regulatory T cells and increased production of adenosine. Our study provides a profound characterization of the immunomodulatory activity of Salp15 and suggests that its long-term effects are due to the specific regulation of CD73.

ADP-glucose pyrophosphorylase (AGPase) controls bacterial glycogen and plant starch biosynthetic pathways, the most common carbon storage polysaccharides in nature. AGPase activity is allosterically regulated by a series of metabolites in the energetic flux within the cell. Very recently, we reported the first crystal structures of the paradigmatic AGPase from (AGPase) in complex with its preferred physiological negative and positive allosteric regulators, adenosine 5'-monophosphate (AMP) and fructose 1,6-bisphosphate (FBP), respectively. However, understanding the molecular mechanism by which AMP and FBP allosterically modulates AGPase enzymatic activity still remains enigmatic. Here we found that single point mutations of key residues in the AMP-binding site decrease its inhibitory effect but also clearly abolish the overall AMP-mediated stabilization effect in wild-typeAGPase. Single point mutations of key residues for FBP binding did not revert the AMP-mediated stabilization. Strikingly, an AGPase-R130A mutant displayed a dramatic increase in activity when compared with wild-typeAGPase, and this increase correlated with a significant increment of glycogen content The crystal structure ofAGPase-R130A revealed unprecedented conformational changes in structural elements involved in the allosteric signal transmission. Altogether, we propose a model in which the positive and negative energy reporters regulate AGPase catalytic activity via intra- and interprotomer cross-talk, with a "sensory motif" and two loops, RL1 and RL2, flanking the ATP-binding site playing a significant role. The information reported herein provides exciting possibilities for industrial/biotechnological applications.

Glycosyltransferases (GTs) are a key family of enzymes that catalyze the synthesis of glycosidic bonds in all living organisms. The reaction involves the transfer of a glycosyl moiety and can proceed with retention or inversion of the anomeric configuration. To date, the catalytic mechanism of retaining GTs is a topic of great controversy, particularly for those enzymes containing a putative nucleophilic residue in the active site, for which the occurrence of a double-displacement mechanism has been suggested. We report native ternary complexes of the retaining glycosyltransferase α-1,3-galactosyltransferase (α3GalT) from Bos taurus, which contains such a nucleophile in the active site, in a productive mode for catalysis in the presence of its sugar donor UDP-Gal, the acceptor substrate lactose, and the divalent cation cofactor. This new experimental evidence supports the occurrence of a front-side substrate-assisted S i-type reaction for α3GalT, and suggests a conserved common catalytic mechanism among retaining GTs.

Glycogen and starch are the major carbon and energy reserve polysaccharides in nature, providing living organisms with a survival advantage. The evolution of the enzymatic machinery responsible for the biosynthesis and degradation of such polysaccharides, led the development of mechanisms to control the assembly and disassembly rate, to store and recover glucose according to cell energy demands. The tetrameric enzyme ADP-glucose pyrophosphorylase (AGPase) catalyzes and regulates the initial step in the biosynthesis of both α-polyglucans. AGPase displays cooperativity and allosteric regulation by sensing metabolites from the cell energy flux. The understanding of the allosteric signal transduction mechanisms in AGPase arises as a long-standing challenge. In this work, we disclose the cryoEM structures of the paradigmatic homotetrameric AGPase from Escherichia coli ( Ec AGPase), in complex with either positive or negative physiological allosteric regulators, fructose-1,6-bisphosphate (FBP) and AMP respectively, both at 3.0 Å resolution. Strikingly, the structures reveal that FBP binds deeply into the allosteric cleft and overlaps the AMP site. As a consequence, FBP promotes a concerted conformational switch of a regulatory loop, RL2, from a “locked” to a “free” state, modulating ATP binding and activating the enzyme. This notion is strongly supported by our complementary biophysical and bioinformatics evidence, and a careful analysis of vast enzyme kinetics data on single-point mutants of Ec AGPase. The cryoEM structures uncover the residue interaction networks (RIN) between the allosteric and the catalytic components of the enzyme, providing unique details on how the signaling information is transmitted across the tetramer, from which cooperativity emerges. Altogether, the conformational states visualized by cryoEM reveal the regulatory mechanism of Ec AGPase, laying the foundations to understand the allosteric control of bacterial glycogen biosynthesis at the molecular level of detail.