Autotransporters are a large family of secreted proteins found in Gram-negative bacteria that are typically involved in virulence. They consist of two domains, an N-terminal passenger domain and a C-terminal β-domain. The β-domain is embedded in the outer membrane while the passenger domain is secreted to the extracellular space and contains the effector function 1
. Passenger domains range in size but are usually large and can be over 3000 residues in length 2
. Some of their virulence functions include promoting actin based motility 3; 4
, biofilm formation 5
, and cell vacuolation 6
. There are two known subtypes of autotransporters, the monomeric autotransporters (also referred to as classical/conventional autotransporters) and the trimeric autotransporters.
For the monomeric autotransporters, the β-domain is comprised of a 12-stranded β-barrel that sits in the outer membrane and is formed by the C-terminal 250 – 300 residues 7; 8; 9; 10; 11; 12
. A single polypeptide segment that passes through the barrel pore connects the surface exposed passenger to the first β-strand of the barrel. For the trimeric autotransporters, the β-domain is formed by ~70 residues located at the C-terminus. This domain contains a 4-stranded β-sheet that oligomerizes to form a trimeric 12-stranded β-barrel that is structurally very similar to the monomeric autotransporter barrels 8
. For the trimeric autotransporters, the surface exposed passenger domain is also trimeric with each monomer connected to the β-domain by a polypeptide segment that passes through the barrel pore.
Passenger domain translocation is not well understood for either the monomeric or trimeric autotransporters. For the monomeric autotransporters, translocation models showing the passenger domain using the central pore of the β-domain or a pore created by the Bam complex have been proposed 9
(Supplementary Figure S1
). The Bam complex (also known as the Omp85 or YaeT complex) assembles β-barrel outer membrane proteins into the outer membrane 13
. In both models, a hairpin intermediate is present during translocation for the monomeric autotransporters because the C-terminus of the passenger is surface exposed while its N-terminus is still in the periplasm 14; 15
. Folding above the cell surface, at the tip of the hairpin, would simultaneously prevent the passenger from slipping back through the translocation pore and provide energy to pull the N-terminal portion of the passenger from the periplasm to the cell surface.
After translocation, some monomeric autotransporters cleave their passenger domains allowing them to be released from the cell surface 16
. For members of the SPATE (Serine Protease Autotransporters of Enterobacteriaceae
) family of autotransporters, cleavage occurs by asparagine cyclization inside the β-domain pore 17
and can take less than a minute 16
. In peptides, asparagine cyclization is slow (t1
= days) and an alternate cyclization pathway that results in deamidation of the asparagine rather than cleavage is favored 18
. For this study we wanted to further examine the cleavage mechanism of the SPATE autotransporter EspP produced by E. coli
O157:H7. Specifically, we wanted to visualize the active site prior to cleavage in order to determine how its conformation and the residues that surround it could facilitate asparagine cyclization. To do this we solved the pre-cleavage structures of three non-cleavable mutants of EspP. While our study was underway, the pre-cleavage structure of another SPATE, Hbp, was reported 10
. The structures of Hbp and EspP are very similar and, since they are both SPATEs, they share the same cleavage mechanism. Comparison of the Hbp and three EspP pre-cleavage structures allowed us to see a conformational change that is likely an artifact that occurs when aspartate is substituted for the active site asparagine. This substitution was used by Tajima et al.
to trap Hbp in its pre-cleavage state and it affects the proposed catalytic water molecule in their cleavage mechanism. We used molecular dynamics simulations to reveal potential alternate catalytic waters in EspP and Hbp that could increase the nucleophilicity of the active site asparagine to initiate cyclization. We report a modified version of the cleavage mechanism where a catalytic water molecule is positioned by one to three conserved acidic residues near the cleavage site. Additionally, further analysis of the EspP and Hbp pre-cleavage structures revealed how the active site asparagine side chain is sterically constrained to rotamers favorable for cyclization and its carboxamide group is correctly oriented over its main chain carbonyl carbon by electrostatics. Finally, we also identified a potential proton transfer event that would reduce the energy required for cleavage.