A Single Amino Acid Substitution Changes the Self-Assembly Status of a Type IV Piliation Secretin

Abstract

eSecretins form large multimeric complexes in the outer membranes of many Gram-negative bacteria, where they function as dedicated gateways that allow proteins to access the extracellular environment. Despite their overall relatedness, different secretins use different specific and general mechanisms for their targeting, assembly, and membrane insertion. We report that all tested secretins from several type II secretion systems and from the filamentous bacteriophage f1 can spontaneously multimerize and insert into liposomes in an in vitro transcription-translation system. Phylogenetic analyses indicate that these secretins form a group distinct from the secretins of the type IV piliation and type III secretion systems, which do not autoassemble in vitro. A mutation causing a proline-to-leucine substitution allowed PilQ secretins from two different type IV piliation systems to assemble in vitro, albeit with very low efficiency, suggesting that autoassembly is an inherent property of all secretins.

Bacterial Secretins Form Constitutively Open Pores Akin to General Porins

“…2B) (22,38). We have already proven PulD 28-42/259-660 to be useful for mutant screening with reduced multimerization (which was exploited again as described below) (24,25). Both PulD fl and PulD 28-42/259-660 formed abundant multimers within 30 min (Fig.…”

“…Having created a versatile assay to estimate the pore sizes of channel-forming proteins, we next examined the permeation characteristics of other secretins that insert spontaneously into liposomes (24). Of all of the secretins examined, only OutD from E. carotovora caused calcein leakage at a rate similar to that of PulD (Fig.…”

“…pCHAP9724 containing a P252L substitution in the filamentous phage secretin pIV was obtained by mutagenesis using pCHAP9147 (24) and primers DIS43 and DIS44. PulD 28-42/259-660 ⌬Gate2 (pCHAP9715) was constructed from pCHAP3716 in two PCR steps.…”

“…The existence and properties of a pore in a resting-state secretin complex are the subjects of this report. Many membrane proteins, including PulD and some other secretins, can be efficiently synthesized and assembled into liposomes in a cell-free system (22)(23)(24). Here, we investigated the permeability properties of PulD, XcpQ, OutD, and pIV inserted into liposomes preloaded with small reporter molecules.…”

“…Here, we investigated the permeability properties of PulD, XcpQ, OutD, and pIV inserted into liposomes preloaded with small reporter molecules. A miniaturized version of the assay was used to screen a previously characterized PulD mutant library to identify residues influencing pore size (24,25). Tn10)] was maintained in LB medium at 30°C.…”