Influence of the passenger domain of a model autotransporter on the properties of its translocator domain.
Autotransporters are a superfamily of proteins secreted by Gram-negative bacteria including many virulence factors. They are modular proteins composed of an N-terminal signal peptide, a surface-exposed 'passenger' domain carrying the activity of the protein, and a C-terminal 'translocator' domain composed of an alpha-helical linker region and a transmembrane beta-barrel. The translocator domain plays an essential role for the secretion of the passenger domain across the outer membrane; however, the mechanism of autotransport remains poorly understood. The whooping cough agent Bordetella pertussis produces an autotransporter serine-protease, SphB1, which is involved in the maturation of an adhesin at the bacterial surface. SphB1 also mediates the proteolytic maturation of its own precursor. We used SphB1 as a model autotransporter and performed the first comparisons of the biochemical and biophysical properties of an isolated translocator domain with those of the same domain preceded by the C-terminal moiety of its natural passenger. By using cross-linking and dynamic light scattering, we provide evidence that the passenger domain promotes the auto-association of SphB1, although these interactions appear rather labile. Electrophysiological studies revealed that the passenger domain of the autotransporter appears to maintain the translocator channel in a low-conductance conformation, most likely by stabilizing the alpha-helix inside the pore. That the passenger may significantly influence AT physicochemical properties is likely to be relevant for the in vivo maturation and stability of AT proteins.
Dé
Emmanuelle
E
UMR 6522 CNRS, PBM, Plate-forme Protéomique IFRMP23, Université de Rouen, Mont-Saint-Aignan cedex, France. emmanuelle.de@univ-rouen.fr
Saint
Nathalie
N
Glinel
Karine
K
Meli
Albano C
AC
Lévy
Daniel
D
Jacob-Dubuisson
Françoise
F
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001886:Bordetella pertussis / Q000235:genetics / Q000378:metabolism; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D008954:Models, Biological; D008958:Models, Molecular; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C437318:SphB1 protein, Bordetella pertussis