Bacterial Secretins Form Constitutively Open Pores Akin to General Porins

Abstract

e Proteins called secretins form large multimeric complexes that are essential for macromolecular transit across the outer membrane of Gram-negative bacteria. Evidence suggests that the channels formed by some secretin complexes are not tightly closed, but their permeability properties have not been well characterized. Here, we used cell-free synthesis coupled with spontaneous insertion into liposomes to investigate the permeability of the secretin PulD. Leakage assays using preloaded liposomes indicated that PulD allows the efflux of small fluorescent molecules with a permeation cutoff similar to that of general porins. Other secretins were also found to form similar pores. To define the polypeptide region involved in determining the pore size, we analyzed a collection of PulD variants and studied the roles of gates 1 and 2, which were previously reported to affect the pore size of filamentous phage f1 secretin pIV, in assembly and pore formation. Liposome leakage and a novel in vivo assay showed that replacement of the conserved proline residue at position 443 in PulD by leucine increased the apparent size of the pore. The in vitro approach described here could be used to study the pore properties of membrane proteins whose production in vivo is toxic.

Independent Domain Assembly in a Trapped Folding Intermediate of Multimeric Outer Membrane Secretins

“…The ability of the PulD 28-42/259-660 variants to form a pore was measured by the leakage of self-quenched calcein from preloaded liposomes upon PulD insertion (Disconzi et al, 2014). The fluorescence increase observed following the synthesis of PulD 28-42/259-660 WT, T470V, and T470S indicated efficient calcein leakage from these liposomes due to the formation of a pore (Disconzi et al, 2014), consistent with their ability to promote PulA secretion. Calcein fluorescence remained low upon synthesis of PulD , indicating the absence of a pore.…”

Biogenesis of Pseudomonas aeruginosa type IV pili and regulation of their function

“…Secretins form a 50–80 Å diameter channel that allows the polymerized fibre to pass through the outer membrane (Korotkov et al ., ). In the absence of a pilus fibre, the secretin pore is closed by loops that are part of the secretin structure, or in some systems, by a plug protein that physically blocks the channel (Korotkov et al ., ; Disconzi et al ., ;). These components inhibit the release of periplasmic proteins while the secretin is in its resting state, unoccupied by pili (Disconzi et al ., ).…”

“…In the absence of a pilus fibre, the secretin pore is closed by loops that are part of the secretin structure, or in some systems, by a plug protein that physically blocks the channel (Korotkov et al ., ; Disconzi et al ., ;). These components inhibit the release of periplasmic proteins while the secretin is in its resting state, unoccupied by pili (Disconzi et al ., ). Secretins consist of two regions; a variable N‐terminal periplasmic region responsible for protein–protein and/or protein–peptidoglycan interactions and substrate recognition, and a highly conserved C‐terminal domain that forms the physical pore in the membrane (Bouley et al ., ; Balasingham et al ., ; Tammam et al ., ).…”

The trans‐envelope architecture and function of the type 2 secretion system: new insights raising new questions

“…The secretin pore is composed of a GspD multimer that forms a gated OM channel constitutively open for small (∼600 Da) molecules (Koster et al ., ; Bitter et al ., ; Disconzi et al ., ). The mature GspD monomer has six domains: the periplasmic N0‐, N1‐, N2‐ and N3‐domains, followed by the beta‐barrel C‐domain, OM‐embedded at its distal end, and the C‐terminal S‐domain, also in the periplasm (Fig.…”