Secretins revealed: structural insights into the giant gated outer membrane portals of bacteria

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Highlights

  • Secretins are outer membrane pores that passage large macromolecular substrates.

  • Recent cryo-EM structures reveal a unique, double-walled β-barrel architecture.

  • Central secretin domain is conserved across various bacterial secretion systems.

  • Additional motifs common to the secretin family clarify assembly and function.

The acquisition and evolution of customized and often highly complex secretion systems allows Gram-negative bacteria to efficiently passage large macromolecules across both inner and outer membranes and, in some cases, that of the infected host. Essential to the virulence and ultimate survival of the many pathogenic species that encode them, secretion systems export a wide variety of effector proteins and DNA as well as the downstream extracellular filaments of the secretion apparatus themselves. Although these customized secretion systems differ in their cytosolic and inner membrane components, several commonly rely on the secretin family of giant pores to allow these large substrates to traverse the outer membrane. Recently, several near-atomic resolution cryo-EM secretin structures have unveiled the first insights into the unique structural motifs required for outer membrane localization, assembly, hallmark ultrastable nature, spontaneous membrane insertion, and mechanism of action  including the requisite central gating needed to prevent deleterious passage of periplasmic contents to the extracellular space.

Introduction

While the inner and outer membranes of the Gram-negative bacterial envelope provide vital structural support and environmental protection, they pose a substantial barrier for transport into and out of the cytosol. As bacteria rely on extracellular secretion for survival and virulence, they have evolved complex protein secretion systems to transport specific substrates through their multi-layered envelope. Many of these envelope-spanning nanomachines share a conserved outer membrane channel, termed the secretin pore. Research interest in secretin pores was first ignited when d’Enfert et al. found that knockout of the T2SS secretin PulD prevented secretion of pullalanase, a saccharide debranching enzyme [1]. PulD was later revealed to share sequence homology in its C-terminal region (termed the secretin domain) with outer membrane proteins from other bacterial species and secretion systems, coining the secretin family of proteins [2, 3]; members of this family share a remarkable stability to temperature, denaturing agents, and detergents [4, 5, 6]. These massive, necessarily gated outer membrane portals are essential for secretion of folded substrates in the type II secretion system (T2SS) [7], the needle of the type III secretion system (T3SS) and subsequent virulence effectors passaged therein [8], the pilus of the type IV pilus system (T4PS) [9], and filamentous phage [10] (see Figure 1).

It has been long recognized by primary sequence analysis and supporting genetic/low resolution EM data that secretins from diverse secretion systems and bacterial species have a similar apparent domain organization [6, 11]. They are made up of a single polypeptide, wherein a proposed 12–15 copies oligomerize into a >1 MDa outer membrane (OM) pore [12, 13, 14]. The secretin domain is the most stringently conserved region, encompassing the C-terminal half of the protein and harboring a propensity for high β-sheet character [2]. T2SS and T3SS secretins from specific bacterial strains also encode a small C-terminal motif called the S domain, where cognate chaperone-like pilotin proteins can bind to facilitate assembly and/or OM localization of the pore [15, 16]. The most diverse sequence of the secretin protein is the periplasmic N-terminal region, which is comprised of a variable number of globular domains; these have evolved to suit the specific function unique to each system [17, 18, 19]. This review will provide a summary of the recently established structural architecture in secretin pores that now illuminate the role of these conserved sequences and the significance for potential mechanisms of pore assembly and function.

Section snippets

Recent first insights into secretin structure

The secretin family has historically been recalcitrant to high resolution structural study, with understanding of secretin architecture limited to low resolution molecular envelope EM reconstructions from various species and systems [13, 18, 20, 21, 22, 23, 24, 25] and the crystal structures of isolated, monomeric N-terminal modular domains [26, 27, 28, 29, 30]. However, enabled by the remarkable revolution in single-particle cryo-EM methodologies [31], and building on a two-decade foundation

The core secretin domain

The most conserved domain amongst the secretin family, the ‘secretin’ domain, encompasses the C-terminal region (40–70% sequence identity within and 20–40% between systems). Its architecture remained a mystery long after other domains of the various secretion systems, including the N-terminal N0–N2 modular domains, had been characterized by X-ray crystallography [26, 27, 28, 29, 30] (Figure 2); with advances in cryo-EM technology, the near-atomic resolution structures of T2SS and T3SS secretins

The peripheral N-terminal and C-terminal variable domains

The N-terminal half of the secretin protein is composed of a combination of distinct modular domains connected via short linkers, the number and identity of which varies between species; they are tuned to the function of their secretion system, the distinct inner membrane (IM) components they couple to and their specific periplasmic environments and span. These domains include a TonB-dependent transduction domain (N0), multiple KH-like DNA-binding domains (N1, N2, N3), and  in the case of the

Structural implications for localization, stable assembly and insertion into the OM

Specific T2SS and T3SS secretins  including those whose structures are discussed here  rely on a cognate, chaperone-like ‘pilotin’ protein for their efficient outer membrane localization and/or assembly [15, 45], potentially with aid of the LOL export pathway [46] (Figure 4). Pilotins are OM-targeted periplasmic lipoproteins which have been shown to bind tightly to their cognate secretin via the S domain (Figure 4.1, 4.2). Specifically, peptides encompassing the C-terminal-most helix of the S

Structural implications for periplasmic gate and opening mechanism

All isolated secretins characterized by EM to date have shown a large occlusion in the central lumen of the channel  termed the periplasmic gate  that serves to ensure secretion is limited to specific substrates, preventing the deleterious general passage of other molecules from entering or leaving the cell. The secondary structure elements comprising the gate are now defined by the recent cryo-EM structures and are shown to be well conserved: a β-hairpin from the inner secretin β-barrel bends

Concluding remarks

Bacterial secretion nanomachines still hide many mysteries at the molecular level, as their vast array of interacting components and membrane-spanning regions make biochemical characterization a challenging task. New advances in cryo-EM imaging provide hope for future analysis at the atomic level, as exemplified in the first derived structures of the major OM secretin gated portals. The structural characterization of multiple secretin pores at near-atomic resolution has provided the first

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

We would like to thank Dr. Seth Darst, for providing the EM map of the f1 phage secretin pore, pIV (Opalka 2003). This work was funded by graduate scholarship funding to DDM from the Natural Sciences and Engineering Research Council of Canada (NSERC), and operating grants to NCJS from Canadian Institutes of Health Research and the Howard Hughes International Senior Scholar program. NCJS is a Tier I Canada Research Chair in Antibiotic Discovery.

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