The interaction was measured in HBS buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 0.005% Tween20) and data was analyzed using the NT Analysis software (NanoTemper Technologies). Formation, crystallization and framework determination of the complexes between FedF15C165 and different inhibitory nanobodies Complexes between FedF15C165 and the different nanobodies were obtained by adding an excess of FedF15C165 to the appropriate nanobody and subsequently weight the Fexaramine mixture on a pre-packed Ni-NTA column (GE Healthcare). chain and side chain atoms are depicted in stick representation with oxygen and nitrogen atoms colored respectively in reddish and blue.(TIF) pone.0114691.s002.tif (338K) GUID:?0E7D55DC-861B-419C-80E5-8B2E11FFBE35 S3 Figure: NbFedF9 directly competes with the sugar binding site around the FedF surface. Shown is a comparison of the binding site of blood group A type 1 hexasaccharide (A6-1) (left) and NbFedF9 (right) around the FedF surface. Residues His88 and Arg117 can be seen to interact both with NbFedF9 and A6-1, and these residues are named. FedF is usually depicted in cartoon representation and colored gray, whereas NbFedF9 and A6-1 are depicted in cartoon and stick representations, respectively, and colored yellow. Interacting residues are shown in stick model with oxygen and nitrogen atoms colored reddish and blue, respectively. Hydrogen bonds are highlighted as reddish dotted lines.(TIF) pone.0114691.s003.tif (211K) GUID:?F2DB9C38-557D-4B85-831F-E8392974BCE9 S4 Figure: Details on the interaction between FedF15C165 and NbFedF6 or NbFedF7 that induce a conformational change in the D-E loop . NbFedF6 (A)(B) and NbFedF7 (C)(D) are colored yellow and interact at the interface between the two -linens of the immunoglobulin-like fold of FedF (grey). Direct hydrogen bonds (dashed lines, colored reddish) are created by residues of both nanobodies and FedF15C165 or indirect hydrogen bonds by a connecting intermediary water molecule (dashed lines, orange). Amino acid residues involved in the conversation are named and indicated by either a black (nanobodies) or dark blue (FedF) label. Water molecules are depicted as spheres and colored green. Interacting main chain and side chain atoms are depicted in stick representation with oxygen and nitrogen atoms colored respectively in reddish and blue.(TIF) pone.0114691.s004.tif (422K) Fexaramine GUID:?213CDD20-A1F1-4873-BE6C-DAABE1DF6684 S5 Figure: Electron density maps of the interface of Fexaramine the different FedF15C165-nanobody complexes. Electron density map at 1.6 sigma of the interaction interface of the FedF15C165-NbFedF6, FedF15C165-NbFedF7 and FedF15C165-NbFedF9 complexes.(TIF) pone.0114691.s005.tif (351K) GUID:?E3BD8702-8387-4DE7-959A-EA7708D81E1A Data Availability StatementThe authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. All PDB files are available from your PDB database (accession figures 4W6W, 4W6X and 4W6Y). Abstract Post-weaning diarrhea and edema disease caused by F18 fimbriated are important diseases in newly weaned piglets and lead to severe production losses in farming industry. Protective treatments against these infections have thus far limited efficacy. In this study we generated nanobodies directed against the lectin domain name of the F18 fimbrial adhesin FedF and showed in an adherence assay that four unique nanobodies inhibit the attachment of F18 fimbriated bacteria to piglet enterocytes. Crystallization of the FedF lectin domain name with the most potent inhibitory nanobodies revealed their mechanism of action. These either competed with the binding of the blood group antigen receptor around the FedF surface or induced a conformational switch in which the CDR3 region of the nanobody displaces the D-E loop adjacent to the binding site. This D-E loop was previously shown to be required for the conversation between F18 fimbriated bacteria and blood group antigen receptors in a membrane context. This work demonstrates the feasibility of inhibiting the attachment of fimbriated pathogens by employing nanobodies directed against the adhesin domain name. Introduction In farming industry enterotoxigenic (ETEC) and Fexaramine Shiga toxin generating (STEC) are important pathogens [1], [2] causing severe mortality and severe production losses [3]. Common to both classes of pathogenic is the presence of two crucial virulence factors: (1) adherence factors (often fimbriae) in order to mediate the attachment to specific receptors, usually glycans, followed by colonization of the intestinal tract and (2) the production of one or multiple Rabbit Polyclonal to BRCA2 (phospho-Ser3291) Fexaramine toxins that induce disease symptoms [2]. In piglets ETEC and STEC strains expressing F18 fimbriae are associated with respectively post-weaning diarrhoea and edema disease [4], [5]. After the.