Structure and function relationship of the autotransport and proteolytic activity of EspP from Shiga toxin-producing Escherichia coli.
BACKGROUND
The serine protease autotransporter EspP is a proposed virulence factor of Shiga toxin-producing Escherichia coli (STEC). We recently distinguished four EspP subtypes (EspPalpha, EspPbeta, EspPgamma, and EspPdelta), which display large differences in transport and proteolytic activities and differ widely concerning their distribution within the STEC population. The mechanisms underlying these functional variations in EspP subtypes are, however, unknown.


RESULTS
The structural basis of proteolytic and autotransport activity was investigated using transposon-based linker scanning mutagenesis, site-directed mutagenesis and structure-function analysis derived from homology modelling of the EspP passenger domain. Transposon mutagenesis of the passenger domain inactivated autotransport when pentapeptide linker insertions occurred in regions essential for overall correct folding or in a loop protruding from the beta-helical core. Loss of proteolytic function was limited to mutations in Domain 1 in the N-terminal third of the EspP passenger. Site-directed mutagenesis demonstrated that His(127), Asp(156) and Ser(263) in Domain 1 form the catalytic triad of EspP.


CONCLUSIONS
Our data indicate that in EspP i) the correct formation of the tertiary structure of the passenger domain is essential for efficient autotransport, and ii) an elastase-like serine protease domain in the N-terminal Domain 1 is responsible for the proteolytic phenotype. Lack of stabilizing interactions of Domain 1 with the core structure of the passenger domain ablates proteolytic activity in subtypes EspPbeta and EspPdelta.
Brockmeyer
Jens
J
Institute for Hygiene and the National Consulting Laboratory on Hemolytic Uremic Syndrome, University of Münster, Münster, Germany. Jens.Brockmeyer@ukmuenster.de
Spelten
Sabrina
S
Kuczius
Thorsten
T
Bielaszewska
Martina
M
Karch
Helge
H
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D021381:Protein Transport; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D022621:Shiga Toxin / Q000096:biosynthesis*; D013329:Structure-Activity Relationship
D029968:Escherichia coli Proteins; D022621:Shiga Toxin; C107189:EspP protein, E coli; D012697:Serine Endopeptidases