Structural and binding characterization of the LacdiNAc-specific adhesin (LabA; HopD) exodomain from Helicobacter pylori

• Published in: Current research in structural biology Vol. 3; pp. 19 - 29
• Main Authors: Paraskevopoulou, Vasiliki, Schimpl, Marianne, Overman, Ross C., Stolnik, Snow, Chen, Yajie, Nguyen, Linh, Winkler, G. Sebastiaan, Gellert, Paul, Klassen, John S., Falcone, Franco H.
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01-01-2021; Elsevier
• Subjects: Adhesin; Crystal structure; Helicobacter pylori; HopD; LabA; Ligand; Outer membrane porin; TEV cleavage Site; LabA; Outer membrane porin; Helicobacter pylori; TEV cleavage Site; Ligand; HopD; Adhesin; Crystal structure
• ISSN: 2665-928X; 2665-928X
• DOI: 10.1016/j.crstbi.2020.12.004

Helicobacter pylori (H. pylori) uses several outer membrane proteins for adhering to its host's gastric mucosa, an important step in establishing and preserving colonization. Several adhesins (SabA, BabA, HopQ) have been characterized in terms of their three-dimensional structure. A recent addition to the growing list of outer membrane porins is LabA (LacdiNAc-binding adhesin), which is thought to bind specifically to GalNAcβ1-4GlcNAc, occurring in the gastric mucosa. LabA47-496 protein expressed as His-tagged protein in the periplasm of E. coli and purified via subtractive IMAC after TEV cleavage and subsequent size exclusion chromatography, resulted in bipyramidal crystals with good diffraction properties. Here, we describe the 2.06 ​Å resolution structure of the exodomain of LabA from H. pylori strain J99 (PDB ID: 6GMM). Strikingly, despite the relatively low levels of sequence identity with the other three structurally characterized adhesins (20–49%), LabA shares an L-shaped fold with SabA and BabA. The ‘head’ region contains a 4 ​+ ​3 α-helix bundle, with a small insertion domain consisting of a short antiparallel beta sheet and an unstructured region, not resolved in the crystal structure. Sequence alignment of LabA from different strains shows a high level of conservation in the N- and C-termini, and identifies two main types based on the length of the insertion domain (‘crown’ region), the ‘J99-type’ (insertion ~31 ​amino acids), and the H. pylori ‘26695 type’ (insertion ~46 ​amino acids). Analysis of ligand binding using Native Electrospray Ionization Mass Spectrometry (ESI-MS) together with solid phase-bound, ELISA-type assays could not confirm the originally described binding of GalNAcβ1-4GlcNAc-containing oligosaccharides, in line with other recent reports, which also failed to confirm LacdiNAc binding.
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•2.06 Å resolution X-ray structure of LabA shares L-type fold with SabA and BabA, despite low sequence homology.•Avoidance of multiple N-terminal truncations of LabA expressed in E. coli periplasm after redesign of expression construct.•ESI-MS suggests very low binding affinity for lacDiNac or related carbohydrates, calling into question current ligand.•Sequence analysis across different strains indicates 2 types of LabA (‘J99- vs 26695-type’) with high overall conservation.