Subdomain 2 of the Autotransporter Pet Is the Ligand Site for Recognizing the Pet Receptor on the Epithelial Cell Surface.
Most autotransporter passenger domains, regardless of their diversity in function, fold or are predicted to fold as right-handed β-helices carrying various loops that are presumed to confer functionality. Our goal here was to identify the subdomain (loop) or amino acid sequence of the Pet passenger domain involved in the receptor binding site on the host cell for Pet endocytosis. Here, we show that d1 and d2 subdomains, as well as the amino acid sequence linking the subdomain d2 and the adjacent β-helix (PDWET), are not required for Pet secretion through the autotransporter system and that none of our deletion mutants altered the predicted long right-handed β-helical structure. Interestingly, Pet lacking the d2 domain (PetΔd2) was unable to bind on the epithelial cell surface, in contrast to Pet lacking d1 (PetΔd1) subdomain or PDWET sequences. Moreover, the purified d1 subdomain, the biggest subdomain (29.8 kDa) containing the serine protease domain, was also unable to bind the cell surface. Thus, d2 sequence (54 residues without the PDWET sequence) was required for Pet binding to eukaryotic cells. In addition, this d2 sequence was also needed for Pet internalization but not for inducing cell damage. In contrast, PetΔd1, which was able to bind and internalize inside the cell, was unable to cause cell damage. Furthermore, unlike Pet, PetΔd2 was unable to bind cytokeratin 8, a Pet receptor. These data indicate that the surface d2 subdomain is essential for the ligand-receptor (Pet-Ck8) interaction for Pet uptake and to start the epithelial cell damage by this toxin.
Department of Cell Biology, Centro de Investigación y de Estudios Avanzados del IPN, Mexico City, Mexico.
Lucia
L
Chavez-Dueñas
Department of Cell Biology, Centro de Investigación y de Estudios Avanzados del IPN, Mexico City, Mexico.
Antonio
A
Serapio-Palacios
Department of Cell Biology, Centro de Investigación y de Estudios Avanzados del IPN, Mexico City, Mexico.
Raul
R
Nava-Acosta
Department of Cell Biology, Centro de Investigación y de Estudios Avanzados del IPN, Mexico City, Mexico fnavarro@cell.cinvestav.mx.
Fernando
F
Navarro-Garcia
D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004768:Enterotoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D053533:Keratin-8 / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D000069320:Type V Secretion Systems / Q000235:genetics / Q000378:metabolism*
D004768:Enterotoxins; D053533:Keratin-8; D008024:Ligands; D000069320:Type V Secretion Systems