Mechanism of Passenger Cleavage in Autotransporter EspP Explored with QM/MM Molecular Dynamics Simulation

Abstract

EspP is an extracellular serine protease and one of the major virulence factors expressed by E. coli. As in all autotransporters, it contains a C-terminal β-barrel domain embedded in the outer membrane and an N-terminal extracellular passenger domain. EspP belongs to the family of serine protease autotransporters of Enterobacteriaceae (SPATE), whose passengers can cleave various mammalian host proteins. The sequence of SPATE proteins is highly conserved, particularly around the passenger cleavage site. It has been shown that cleavage occurs through asparagine cyclization and that several amino acids in the vicinity of the reacting asparagine are essential for the reaction. Surprisingly, presence of the protein-cleaving serine protease domain or other serines and histidines, commonly employed in enzymatic protein cleavage reactions was not required for cleavage to occur. Therefore, mechanisms involving several aspartate residues and a water molecule have been proposed, although the plausibility of these mechanisms have not been previously explored by computational methods. Here, we employ state of the art hybrid quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations and free energy methods to investigate several paths for asparagine cyclization and the consequent cleavage of the passenger domain in EspP. It is shown that the reaction involves several consecutive proton transfers and that presence of a water molecule and multiple amino acids at the catalytic site greatly reduces the reaction barrier. Our results can be generalized for other SPATE proteins and facilitate development of SPATE inhibitors in order to combat bacterial infections.