Abstract

Bacterial autotransporters are comprised of an N-terminal 'passenger domain' and a C-terminal barrel (' 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 barrel and revealed that two conserved residues, an aspartate within the domain (Asp¹¹²⁰) and an asparagine (Asn¹⁰²³) 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 Asp¹¹²⁰ and Asn¹⁰²³ 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.