Metabolic labelling of cholesteryl glucosides in Helicobacter pylori reveals how the uptake of human lipids enhances bacterial virulence.

Helicobacter pylori infects approximately half of the human population and is the main cause of various gastric diseases. This pathogen is auxotrophic for cholesterol, which it converts upon uptake to various cholesteryl α-glucoside derivatives, including cholesteryl 6'-acyl and 6'-phosphatidyl α-glucosides (CAGs and CPGs). Owing to a lack of sensitive analytical methods, it is not known if CAGs and CPGs play distinct physiological roles or how the acyl chain component affects function. Herein we established a metabolite-labelling method for characterising these derivatives qualitatively and quantitatively with a femtomolar detection limit. The development generated an MS/MS database of CGds, allowing for profiling of all the cholesterol-derived metabolites. The subsequent analysis led to the unprecedented information that these bacteria acquire phospholipids from the membrane of epithelial cells for CAG biosynthesis. The resulting increase in longer or/and unsaturated CAG acyl chains helps to promote lipid raft formation and thus delivery of the virulence factor CagA into the host cell, supporting the idea that the host/pathogen interplay enhances bacterial virulence. These findings demonstrate an important connection between the chain length of CAGs and the bacterial pathogenicity

[[alternative]]The Preparation of Nitriles

[[abstract]]不開放

Dissecting the Structure–Activity Relationship of Galectin–Ligand Interactions

Galectins are β-galactoside-binding proteins. As carbohydrate-binding proteins, they participate in intracellular trafficking, cell adhesion, and cell–cell signaling. Accumulating evidence indicates that they play a pivotal role in numerous physiological and pathological activities, such as the regulation on cancer progression, inflammation, immune response, and bacterial and viral infections. Galectins have drawn much attention as targets for therapeutic interventions. Several molecules have been developed as galectin inhibitors. In particular, TD139, a thiodigalactoside derivative, is currently examined in clinical trials for the treatment of idiopathic pulmonary fibrosis. Herein, we provide an in-depth review on the development of galectin inhibitors, aiming at the dissection of the structure–activity relationship to demonstrate how inhibitors interact with galectin(s). We especially integrate the structural information established by X-ray crystallography with several biophysical methods to offer, not only in-depth understanding at the molecular level, but also insights to tackle the existing challenges

Structural Basis Underlying the Binding Preference of Human Galectins-1, -3 and -7 for Galβ1-3/4GlcNAc

<div><p>Galectins represent β-galactoside-binding proteins and are known to bind Galβ1-3/4GlcNAc disaccharides (abbreviated as LN1 and LN2, respectively). Despite high sequence and structural homology shared by the carbohydrate recognition domain (CRD) of all galectin members, how each galectin displays different sugar-binding specificity still remains ambiguous. Herein we provided the first structural evidence of human galectins-1, 3-CRD and 7 in complex with LN1. Galectins-1 and 3 were shown to have higher affinity for LN2 than for LN1, while galectin-7 displayed the reversed specificity. In comparison with the previous LN2-complexed structures, the results indicated that the average glycosidic torsion angle of galectin-bound LN1 (ψ^LN1 ≈ 135°) was significantly differed from that of galectin-bound LN2 (ψ^LN2 ≈ -108°), i.e. the GlcNAc moiety adopted a different orientation to maintain essential interactions. Furthermore, we also identified an Arg-Asp/Glu-Glu-Arg salt-bridge network and the corresponding loop (to position the second Asp/Glu residue) critical for the LN1/2-binding preference.</p></div

Pairwise comparison of the β-galactoside-recognition site in the LN1-hGal1/3-CRD/7 and LN2-hGal1/3-CRD/7 complexes.

<p>(A-C) Structures of hGal1 (PDB ID: 1W6P), hGal3-CRD (1KJL) and hGal7 (5GAL) in complex with LN2 (all shown in gray) were superimposed, respectively, with the LN1 (in yellow)-containing structures of hGal1 (pink), hGal3-CRD (cyan) and hGal7 (green). (D) Diagrams delineate the different interaction geometries of LN1 (left) and LN2 (right) with respect to hGal1/3-CRD/7. Definition and values of the glycosidic torsion angles (ϕ and ψ) for LN1 and LN2 molecules are also listed. (E-G) Close-up view of the unique salt bridge networks in hGal1 (E), hGal3-CRD (F) and hGal7 (G), with a superposition of their LN2 and LN1-complex structures. Polar interactions among LN2-complexed structures are shown in gray dash lines while the interactions among amino acid residues in LN1-loaded structures are indicated by colored dash lines according to hGal1 (pink), 3-CRD (blue) and 7 (green), respectively, and all interactions related to galectin/LN1 complexes formation are colored in yellow.</p

LNs-binding preference for hGal1, 3 and 7.

<p>¹To quantitatively evaluate LN1- or LN2-binding preference of hGal1, 3 and 7, LN1/LN2 ratios (<i>K</i>_<i>d</i>^<i>LN1</i><i>/K</i>_<i>d</i>^<i>LN2</i>) were calculated based on the <i>K</i>_<i>d</i> values presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125946#pone.0125946.t001" target="_blank">Table 1</a>.</p><p>²The values were obtained based on the <i>K</i>_<i>d</i>^<i>BI</i> values determined by Biolayer Interferometry.</p><p>³The values were obtained based on the <i>K</i>_<i>d</i>^<i>FP</i> values determined by FP-based competition assays.</p><p>⁴It was reported by Hirabayashi, J. <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125946#pone.0125946.ref012" target="_blank">12</a>].</p><p>⁵The CRD domain was used instead of full-length hGal3.</p><p>LNs-binding preference for hGal1, 3 and 7.</p

Water-mediated interactions of hGal1 and hGal3 with the LN2 molecules.

<p>(A and B) Stereoview of LN2 molecule bound in the carbohydrate-recognition site of hGal1 (PDB ID: 1W6P) and hGal3-CRD (1KJL), respectively. LN2 ligand is depicted as gray stick model. The water (blue sphere) is coordinated by the H-bonds (green dashes) from N2 atom of LN2 and amino acid residues of galectins. <i>2F</i>_<i>o</i><i>-F</i>_<i>c</i> omit electron density (gray mesh) of the water molecules are highlighted and contoured at 1σ. Unique salt bridge network of hGal1 and hGal3-CRD are indicated as yellow dashes. (C and D) Structural superposition of LN1 and LN2 complex structures from hGal1 (C) and hGal3-CRD (D). The C5-hydroxyl group of LN1 (yellow sticks) in hGal1 and hGal3-CRD complexes makes close contact with the coordinated water in LN2-hGal1 andLN2-hGal3-CRD complex with a distance of 2.2 and 2.1 Å, respectively. (E) Structure of hGal7 (shown in color green) in complex with LN2 (orange) is superimposed with LN2-hGal3-CRD complex structure (all in gray). Most LN2-contacting residues in hGal7 (such as Arg53^hGal7, Trp69^hGal7, Glu72^hGal7 and Arg74^hGal7) are well superimposed with those of hGal3, except the Glu58^hGal7 residue. Location of Glu58^hGal7 is distinctive from Glu165^hGal3/ Asp54^hGal1 and distance between Glu58^hGal7 and N2 atom of LN2 is too far for them to coordinate a water molecule in between.</p

Dissociation constants (<i>K</i>_<i>d</i> in μM) of human galectins-1, 3, 7 and mutants for Galβ1-3/4GlcNAc disaccharides¹,².

<p>¹The values are determined by Biolayer Interferometry at 300 K.</p><p>²The values are determined by fluorescence anisotropy at 277 K.</p><p>³The binding is too weak to be determined by Biolayer Interferometry.</p><p>Dissociation constants (<i>K</i>_<i>d</i> in μM) of human galectins-1, 3, 7 and mutants for Galβ1-3/4GlcNAc disaccharides<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125946#t001fn001" target="_blank">¹</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125946#t001fn002" target="_blank">²</a>.</p

Structural basis for the hydrolytic activity of the transpeptidase-like protein DpaA to detach Braun’s lipoprotein from peptidoglycan

ABSTRACT The peptidoglycan layer is a defining characteristic of bacterial cells, providing them with structural support and osmotic protection. In Escherichia coli, this layer is linked to the outer membrane via the abundant membrane-anchored protein Lpp, known as Braun’s lipoprotein, with LD-transpeptidases LdtA, LdtB, and LdtC catalyzing the attachment. However, one distinctive member of the YkuD-type transpeptidase family, LdtF (recently renamed DpaA), carries out the opposite reaction of detaching Lpp from the peptidoglycan layer. In this study, we report the crystal structure of DpaA, which reveals the enzyme’s ability to cleave, rather than form, the Lpp-peptidoglycan linkage. Assays with purified peptidoglycan-Lpp as the substrate and chemically synthesized compounds suggest that DpaA’s shallow L-shaped active site can only accommodate and cleave the peptidoglycan-Lpp cross-link with a constrained conformation. This study provides insights into how homologous Ldt enzymes can perform opposing chemical reactions. IMPORTANCE Cross-linking reaction of Braun's lipoprotein (Lpp) to peptidoglycan (PG) is catalyzed by some members of the YkuD family of transpeptidases. However, the exact opposite reaction of cleaving the Lpp-PG cross-link is performed by DpaA, which is also a YkuD-like protein. In this work, we determined the crystal structure of DpaA to provide the molecular rationale for the ability of the transpeptidase-like protein to cleave, rather than form, the Lpp-PG linkage. Our findings also revealed the structural features that distinguish the different functional types of the YkuD family enzymes from one another

Structural comparisons among hGal1, 3-CRD and 7.

<p>(A) S4-S6 β-strands of hGal1 (pink), 3-CRD (blue) and 7 (green) are superimposed. The unique salt bridge network of hGal1 (R48-D54-E71-R73), 3-CRD (R162-E165-E184-R186) and 7 (R53-E58-E72-R74) are shown in stick models. (B) Structure-based sequence alignment of S4-S6 β-strands of hGal1, 3-CRD and 7. Secondary structures were designated according to the resolved x-ray structures. The highly conserved LNs-interacting residues among hGal1, 3-CRD and 7 are indicated by asterisks. Residues involved in unique salt bridge network of hGal1, 3-CRD and 7 are colored in either red (Glu/Asp) or blue (Arg).</p