Figure 7. Molecular Determinants of BabA bg Preference.

(A) Structure of the BabA S831 DL1 grafted hybrid (BabA^AD;DL1-S831; tan, positions 198–199 in magenta) in complex with Le^b6 (magenta), and wild-type 17875 BabA^AD bound to BLe^b7 (light gray, with DL1 region in cyan and positions 198–199 in blue). In the specialist hybrid the replacement of Lys199 with Pro199 results in the inward rotation of residue 198. Replacement of Ser198 of 17875 by Leu198 causes a steric occlusion of the Gal or GalNAc determinants in BLe^b or ALe^b.

(B and C) SPR sensorgrams of binding of BabA^AD;DL1-S831 to a Le^b-coated chip in the presence of competing soluble glycans: Le^b5 (B) or ALe^b5 (C), added in a 2-fold dilution series from 5 mM to 1 μM, colored gray to black.

(D) RIA of Le^b versus ALe^b binding of a generalist (S831G[D]) and specialist clone (S831S), and the control strains S831 and 17875/Leb. Amino acid sequence ofthe DL1 region (shown starting at Cys197) of the babA allele in the two loci of the corresponding strains. Binding and sequencing data are representative for two independent S831G(D) and S831S clones isolated.

(E) H. pylori 17875/Leb demonstrates the generalist phenotype and ability to bind both Alexa 488-labeled Le^b (i) and Alexa 555-labeled ALe^b (ii). Probing of theoriginal sweep population of the specialist H. pylori strain S831 by Alexa 555-labeled ALe^b conjugate demonstrated by fluorescence microscopy the rarepresence of S831 bacterial cells of the generalist phenotype

(F) Competition RIA where Le^b binding to a S831G(D) (blue) and S831S (magenta) clone is performed in competition with nonradiolabeled ALe^b. Increasing concentrations of ALe^b titrate out 34.6% of the Le^b binding signal, demonstrating the concomitant expression of a generalist and a specialist babA variant. Data points show mean ± SD, n = 3. See also Figure S7.