1H, 13C, 15N backbone assignment of the human heat-labile enterotoxin B-pentamer and chemical shift mapping of neolactotetraose binding

The major virulence factor of enterotoxigenic Escherichia coli is the heat-labile enterotoxin (LT), an AB5 toxin closely related to the cholera toxin. LT consists of six subunits, the catalytically active A-subunit and five B-subunits arranged as a pentameric ring (LTB), which enable the toxin to bind to the epithelial cells in the intestinal lumen. LTB has two recognized binding sites; the primary binding site is responsible for anchoring the toxin to its main receptor, the GM1-ganglioside, while the secondary binding site recognizes blood group antigens. Herein, we report the 1H, 13C, 15N main chain assignment of LTB from human isolates (hLTB; 103 a.a. per subunit, with a total molecular mass of 58.5 kDa). The secondary structure was predicted based on 13C′, 13Cα, 13Cβ, 1HN and 15N chemical shifts and compared to a published crystal structure of LTB. Neolactotetraose (NEO) was titrated to hLTB and chemical shift perturbations were measured. The chemical shift perturbations were mapped onto the crystal structure, confirming that NEO binds to the primary binding site of hLTB and competes with GM1-binding. Our new data further lend support to the hypothesis that binding at the primary binding site is transmitted to the secondary binding site of the toxin, where it may influence the binding to blood group antigens. This research was first published in Biomolecular NMR Assignments. © Springer Verlag

Quantum chemical modeling of the reaction path of chorismate mutase based on the experimental substrate/product complex

Chorismate mutase is a well‐known model enzyme, catalyzing the Claisen rearrangement of chorismate to prephenate. Recent high‐resolution crystal structures along the reaction coordinate of this enzyme enabled computational analyses at unprecedented detail. Using quantum chemical simulations, we investigated how the catalytic reaction mechanism is affected by electrostatic and hydrogen‐bond interactions. Our calculations showed that the transition state (TS) was mainly stabilized electrostatically, with Arg90 playing the leading role. The effect was augmented by selective hydrogen‐bond formation to the TS in the wild‐type enzyme, facilitated by a small‐scale local induced fit. We further identified a previously underappreciated water molecule, which separates the negative charges during the reaction. The analysis includes the wild‐type enzyme and a non‐natural enzyme variant, where the catalytic arginine was replaced with an isosteric citrulline residue

Dataset for: Quantum chemical modeling of the reaction path of chorismate mutase based on the experimental substrate/product complex

Chorismate mutase is a well-known model enzyme, catalyzing the Claisen rearrangement of chorismate to prephenate. Recent high-resolution crystal structures along the reaction coordinate of this enzyme enable computational analyses at unprecedented detail. Using quantum chemical simulations, we have investigated how the catalytic reaction mechanism is affected by electrostatic and hydrogen bond interactions. Our calculations showed that the transition state was mainly stabilized electrostatically, with Arg90 playing the leading role. The effect was augmented by selective hydrogen bond formation to the transition state in the wild-type enzyme, facilitated by a small-scale local induced fit. We further identified a previously underappreciated water molecule, which separates the negative charges during the reaction. The analysis includes the wild-type enzyme and a non-natural enzyme variant, where the catalytic arginine was replaced with an isosteric citrulline residue

High-Resolution Crystal Structures Elucidate the Molecular Basis of Cholera Blood Group Dependence

Cholera is the prime example of blood-group-dependent diseases, with individuals of blood group O experiencing the most severe symptoms. The cholera toxin is the main suspect to cause this relationship. We report the high-resolution crystal structures (1.1–1.6 Å) of the native cholera toxin B-pentamer for both classical and El Tor biotypes, in complexes with relevant blood group determinants and a fragment of its primary receptor, the GM1 ganglioside. The blood group A determinant binds in the opposite orientation compared to previously published structures of the cholera toxin, whereas the blood group H determinant, characteristic of blood group O, binds in both orientations. H-determinants bind with higher affinity than A-determinants, as shown by surface plasmon resonance. Together, these findings suggest why blood group O is a risk factor for severe cholera

Inter-Enzyme Allosteric Regulation of Chorismate Mutase in Corynebacterium glutamicum: Structural Basis of Feedback Activation by Trp

Corynebacterium glutamicum is widely used for the industrial production of amino acids, nucleotides, and vitamins. The shikimate pathway enzymes DAHP synthase (CgDS, Cg2391) and chorismate mutase (CgCM, Cgl0853) play a key role in the biosynthesis of aromatic compounds. Here we show that CgCM requires the formation of a complex with CgDS to achieve full activity, and that both CgCM and CgDS are feedback regulated by aromatic amino acids binding to CgDS. Kinetic analysis showed that Phe and Tyr inhibit CgCM activity by inter-enzyme allostery, whereas binding of Trp to CgDS strongly activates CgCM. Mechanistic insights were gained from crystal structures of the CgCM homodimer, tetrameric CgDS, and the heterooctameric CgCM–CgDS complex, refined to 1.1, 2.5, and 2.2 Å resolution, respectively. Structural details from the allosteric binding sites reveal that DAHP synthase is recruited as the dominant regulatory platform to control the shikimate pathway, similar to the corresponding enzyme complex from Mycobacterium tuberculosis

Data collection and refinement statistics.

<p>Data collection and refinement statistics.</p

Structures and nomenclatures of the oligosaccharides in this article.

<p>Lewis-y blood group determinants H-tetra-BGA, B-penta-BGA, A-penta-BGA and the related human milk oligosaccharide A-penta-HMO. All of these have type-2 core structures. Note that H-tetra-HMO and A-penta-HMO were referred to as H-tetra and A-penta in our previous publications [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005567#ppat.1005567.ref027" target="_blank">27</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005567#ppat.1005567.ref029" target="_blank">29</a>]. Carbohydrate symbols follow the nomenclature of the Consortium for Functional Glycomics (Nomenclature Committee, Consortium for Functional Glycomics (functionalglycomics.org/static/consortium/Nomenclature.shtml); d-galactose (Gal)–yellow circle, <i>N</i>-acetylgalactosamine (GalNAc)–yellow square, d-glucose (Glc)–blue circle, <i>N</i>-acetylglucosamine (GlcNAc)–blue square, l-fucose (Fuc)–red triangle.</p

Blood group antigen binding to the cholera toxin.

<p>(<b>A</b>) X-ray structure of cCTB in complex with A-penta-BGA (PDB ID: 5ELD); side and top views. The B-pentamer is colored by subunit, and the ligands are shown in stick representation. Blood group determinants are bound to the lateral side of the toxin (modeled into four of the five secondary binding sites), and galactose to the primary binding site (in three of the sites), facing the cell membrane. (<b>B-G</b>) Close-up views of the blood group antigen binding sites. Oligosaccharide residues are labeled in italics, amino acid residues in bold, and residues from neighboring subunits are indicated with a hash (#). (<b>B-C</b>) Interactions of the blood group A determinant with cCTB and ET CTB. Biotype-specific residues are highlighted in orange and green sticks. Water molecules are shown as red spheres, and hydrogen bonds are depicted as red dashed lines. (<b>D-E</b>) Electron density. <i>σ</i>_A-weighted <i>F</i>_o − <i>F</i>_c maps (green mesh; contoured at 3.0<i>σ</i>) are shown for the blood group A determinant and a human milk oligosaccharide. The maps were generated before insertion of the ligands. Circles indicate special features: the GlcNAc’s <i>N</i>-acetyl group, and the less-defined electron density for GalNAc in A-penta-BGA. (<b>F-G</b>) Interaction of the blood group H determinant with cCTB and ET CTB (two orientations). Yellow residues mark the reducing-end GlcNAc, α and β anomers are labeled. Water molecules are depicted as red spheres, hydrogen bonds represented by red dashed lines. In B, C, F and G, only those H-bonds are shown that have favorable angles, maximum bond lengths of 3.5 Å and that are conserved in all binding sites.</p

Inter-Enzyme Allosteric Regulation of Chorismate Mutase in <i>Corynebacterium glutamicum</i>: Structural Basis of Feedback Activation by Trp

<i>Corynebacterium glutamicum</i> is widely used for the industrial production of amino acids, nucleotides, and vitamins. The shikimate pathway enzymes DAHP synthase (CgDS, Cg2391) and chorismate mutase (CgCM, Cgl0853) play a key role in the biosynthesis of aromatic compounds. Here we show that CgCM requires the formation of a complex with CgDS to achieve full activity, and that both CgCM and CgDS are feedback regulated by aromatic amino acids binding to CgDS. Kinetic analysis showed that Phe and Tyr inhibit CgCM activity by inter-enzyme allostery, whereas binding of Trp to CgDS strongly activates CgCM. Mechanistic insights were gained from crystal structures of the CgCM homodimer, tetrameric CgDS, and the heterooctameric CgCM–CgDS complex, refined to 1.1, 2.5, and 2.2 Å resolution, respectively. Structural details from the allosteric binding sites reveal that DAHP synthase is recruited as the dominant regulatory platform to control the shikimate pathway, similar to the corresponding enzyme complex from <i>Mycobacterium tuberculosis</i>