Secretins revealed: structural insights into the giant gated outer membrane portals of bacteria
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:
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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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Structural lessons on bacterial secretins
2023, BiochimieCitation Excerpt :Secretins were discovered in the 80's during the study of Klebsiella oxytoca outer membrane (OM) constituents responsible for the export of folded pullulanase to the extracellular milieu [1]. Secretins have been intensively studied because of their unique ability to accommodate large folded exoproteins and protein complexes in several bacterial transport nanomachines [2]. Due to their function, secretins were thought for decades to form giant ring-shaped homo-oligomers in the OM [3–8], but their precise atomic organization was deciphered only recently thanks to spectacular advances in cryo-electron microscopy (cryo-EM) [9,10].
Recent structural advances towards understanding of the bacterial type III secretion injectisome
2022, Trends in Biochemical SciencesCitation Excerpt :These small α/β domains have been observed in multiple ring-forming proteins from different bacterial molecular machines [17,47–50] and frequently exploit a common interface during ring oligomerisation, with differentially extensive RBD interfaces providing the potential for variable stability, presumably needed during assembly. SctC belongs to the family of OM secretins also found in the type II secretion system (T2SS), type 4 pilus assembly system (T4PS), and filamentous phages [51]. Salmonella injectisome SctC was the first secretin structure to be determined, revealing that the family-defining secretin domain forms a massive doubled-walled β-barrel [25], and confirming the previously proposed 15mer stoichiometry [44].
Characterization of the Pilotin-Secretin Complex from the Salmonella enterica Type III Secretion System Using Hybrid Structural Methods
2021, StructureCitation Excerpt :The T3SS secretin is a single multi-domain polypeptide that oligomerizes at the OM into a massive (∼1 MDa) gated, double-walled β barrel with 15 subunits and 60 β strands (Hay et al., 2017a; Worrall et al., 2016; Yan et al., 2017; Yin et al., 2018). It is a member of the broadly conserved OM secretin family with significant sequence and structural conservation among other distinct bacterial secretion systems, including the type II secretion system (T2SS), the type IV pilus system (T4PS), and the filamentous phage extrusion channel (Majewski et al., 2018). Secretin monomers are transported into the periplasm by the general Sec secretion system and upon oligomerization undergo BAM-independent insertion into the OM (Dunstan et al., 2015).
Towards capture of dynamic assembly and action of the T3SS at near atomic resolution
2020, Current Opinion in Structural BiologyCitation Excerpt :The characteristic structural unit, stable on extraction from its native membranous environment, is the syringe-shaped needle complex (NC), spanning both bacterial membranes with an attached hollow needle-like projection that provides the delivery conduit for secreted effectors [4] (see Figure 1). Despite its size, the NC outer scaffold is surprisingly made up of just three highly oligomeric ring forming proteins: SctD and SctJ (universal nomenclature), which form concentric periplasmic rings tethered to the IM [5], and SctC, a member of the secretin family of OM pores broadly conserved in the type II secretion system (T2SS), type IV pilus system (T4PS) and filamentous phage [18]. Together, these three proteins form a stable housing, in which the helical needle filament – composed of homologous proteins the rod (SctI) and needle (SctF) – are polymerized onto the IM abutting ‘export apparatus’ complex assembled within the IM rings (SctRSTU).
Assembly mechanism of a Tad secretion system secretin-pilotin complex
2023, Nature Communications
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These authors contributed equally.