The pore size of the autotransporter domain is critical for the active translocation of the passenger domain

The autotransporter mode of surface presentation in Gram-negative bacteria requires a hypothetical C-terminal β-barrel which makes up an aqueous channel in the outer membrane. PalA is a Pseudomonas sp. autotransporter lipolytic protein. PalA is a 66 kDa protein that is composed of two parts, the N-terminal region (Ala 1–Ala 296) similar to the GDSL lipases and the C-terminal region (Leu 320–Phe 612) to the autotransporter. In this report, we provide biochemical and structural evidence demonstrating that the pore size of the β-barrel conduit is important in delivering the N-terminal domain to the cell surface. Among all the autotransporter domains two strictly conserved residues (Pro 478 and Gly 576 in PalA) are converted to other various residues using site-directed mutagenesis. This investigation was made into the different pore-size mutants, affecting the folding of N-terminal domain. Wild β-domain contains a cavity of ∼2 nm diameter that is optimal for the active conformation of the N-terminal domains. However, deviation from the proper size of the pore, whether it is larger or smaller, is not suitable for the proper folding of the N-terminal catalytic domain.