Cleavage of a bacterial autotransporter by an evolutionarily convergent autocatalytic mechanism.
Bacterial autotransporters are comprised of an N-terminal 'passenger domain' and a C-terminal beta barrel ('beta domain') that facilitates transport of the passenger domain across the outer membrane. Following translocation, the passenger domains of some autotransporters are cleaved by an unknown mechanism. Here we show that the passenger domain of the Escherichia coli O157:H7 autotransporter EspP is released in a novel autoproteolytic reaction. After purification, the uncleaved EspP precursor underwent proteolytic processing in vitro. An analysis of protein topology together with mutational studies strongly suggested that the reaction occurs inside the beta barrel and revealed that two conserved residues, an aspartate within the beta domain (Asp(1120)) and an asparagine (Asn(1023)) at the P1 position of the cleavage junction, are essential for passenger domain cleavage. Interestingly, these residues were also essential for the proteolytic processing of two distantly related autotransporters. The data strongly suggest that Asp(1120) and Asn(1023) form an unusual catalytic dyad that mediates self-cleavage through the cyclization of the asparagine. Remarkably, a very similar mechanism has been proposed for the maturation of eukaryotic viral capsids.
Dautin
Nathalie
N
Genetics and Biochemistry Branch, National Institutes of Health, Bethesda, MD 20892, USA.
Barnard
Travis J
TJ
Anderson
D Eric
DE
Bernstein
Harris D
HD
D000595:Amino Acid Sequence; D001216:Asparagine / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism; D002384:Catalysis; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D003902:Detergents; D004926:Escherichia coli / Q000378:metabolism*; D019453:Escherichia coli O157 / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019143:Evolution, Molecular; D008954:Models, Biological; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D012996:Solutions; D013388:Succinimides / Q000378:metabolism
D003902:Detergents; D011498:Protein Precursors; D012996:Solutions; D013388:Succinimides; D001224:Aspartic Acid; D001216:Asparagine