title: Comparative analysis of the biochemical and functional properties of C-terminal domains of autotransporters.
abstract: Autotransporters (ATs) are the largest group of proteins secreted by Gram-negative bacteria and include many virulence factors from human pathogens. ATs are synthesized as large precursors with a C-terminal domain that is inserted in the outer membrane (OM) and is essential for the translocation of an N-terminal passenger domain to the extracellular milieu. Several mechanisms have been proposed for AT secretion. Self-translocation models suggest transport across a hydrophilic channel formed by an internal pore of the β-barrel or by the oligomerization of C-terminal domains. Alternatively, an assisted-translocation model suggests that transport employs a conserved machinery of the bacterial OM such as the Bam complex. In this work we have investigated AT secretion by carrying out a comparative study to analyze the conserved biochemical and functional features of different C-terminal domains selected from ATs of gammaproteobacteria, betaproteobacteria, alphaproteobacteria, and epsilonproteobacteria. Our results indicate that C-terminal domains having an N-terminal α-helix and a β-barrel constitute functional transport units for the translocation of peptides and immunoglobulin domains with disulfide bonds. In vivo and in vitro analyses show that multimerization is not a conserved feature in AT C-terminal domains. Furthermore, we demonstrate that the deletion of the conserved α-helix severely impairs β-barrel folding and OM insertion and thereby blocks passenger domain secretion. These observations suggest that the AT β-barrel without its α-helix cannot form a stable hydrophilic channel in the OM for protein translocation. The implications of our data for an understanding of AT secretion are discussed.
authors.affiliation: Department of Microbial Biotechnology, Centro Nacional de Biotecnología, CSIC, Campus de la Universidad Autónoma de Madrid (UAM), Cantoblanco, Madrid 28049, Spain.
authors.forename: Elvira
authors.initials: E
authors.lastname: Marín
authors.forename: Gustavo
authors.initials: G
authors.lastname: Bodelón
authors.forename: Luis Ángel
authors.initials: LÁ
authors.lastname: Fernández
mesh_terms: D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010957:Plasmids; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000502:physiology*; D021381:Protein Transport; D011994:Recombinant Proteins
chemical_list: D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins
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