In Vitro Reassembly of the Ribose ATP-binding Cassette Transporter Reveals a Distinct Set of Transport Complexes

Bacterial ATP-binding cassette (ABC) importers are primary active transporters that are critical for nutrient uptake. Based on structural and functional studies, ABC importers can be divided into two distinct classes, type I and type II. Type I importers follow a strict alternating access mechanism that is driven by the presence of the substrate. Type II importers accept substrates in a nucleotide-free state, with hydrolysis driving an inward facing conformation. The ribose transporter in Escherichia coli is a tripartite complex consisting of a cytoplasmic ATP-binding cassette protein, RbsA, with fused nucleotide binding domains; a transmembrane domain homodimer, RbsC₂; and a periplasmic substrate binding protein, RbsB. To investigate the transport mechanism of the complex RbsABC₂, we probed intersubunit interactions by varying the presence of the substrate ribose and the hydrolysis cofactors, ATP/ADP and Mg²⁺. We were able to purify a full complex, RbsABC₂, in the presence of stable, transition state mimics (ATP, Mg²⁺, and VO₄); a RbsAC complex in the presence of ADP and Mg²⁺; and a heretofore unobserved RbsBC complex in the absence of cofactors. The presence of excess ribose also destabilized complex formation between RbsB and RbsC. These observations suggest that RbsABC₂ shares functional traits with both type I and type II importers, as well as possessing unique features, and employs a distinct mechanism relative to other ABC transporters.     

Efficient Purification and Reconstitution of ATP Binding Cassette Transporter B6 (ABCB6) for Functional and Structural Studies

The mitochondrial ATP binding cassette transporter ABCB6 has been associated with a broad range of physiological functions, including growth and development, therapy-related drug resistance, and the new blood group system Langereis. ABCB6 has been proposed to regulate heme synthesis by shuttling coproporphyrinogen III from the cytoplasm into the mitochondria. However, direct functional information of the transport complex is not known. To understand the role of ABCB6 in mitochondrial transport, we developed an in vitro system with pure and active protein. ABCB6 overexpressed in HEK293 cells was solubilized from mitochondrial membranes and purified to homogeneity. Purified ABCB6 showed a high binding affinity for MgATP (K_d = 0.18 μm) and an ATPase activity with a K_m of 0.99 mm. Reconstitution of ABCB6 into liposomes allowed biochemical characterization of the ATPase including (i) substrate-stimulated ATPase activity, (ii) transport kinetics of its proposed endogenous substrate coproporphyrinogen III, and (iii) transport kinetics of substrates identified using a high throughput screening assay. Mutagenesis of the conserved lysine to alanine (K629A) in the Walker A motif abolished ATP hydrolysis and substrate transport. These results suggest a direct interaction between mitochondrial ABCB6 and its transport substrates that is critical for the activity of the transporter. Furthermore, the simple immunoaffinity purification of ABCB6 to near homogeneity and efficient reconstitution of ABCB6 into liposomes might provide the basis for future studies on the structure/function of ABCB6.     

Physicochemical Factors Controlling the Activity and Energy Coupling of an Ionic Strength-gated ATP-binding Cassette (ABC) Transporter

Cells control their volume through the accumulation of compatible solutes. The bacterial ATP-binding cassette transporter OpuA couples compatible solute uptake to ATP hydrolysis. Here, we study the gating mechanism and energy coupling of OpuA reconstituted in lipid nanodiscs. We show that anionic lipids are essential both for the gating and the energy coupling. The tight coupling between substrate binding on extracellular domains and ATP hydrolysis by cytoplasmic nucleotide-binding domains allows the study of transmembrane signaling in nanodiscs. From the tight coupling between processes at opposite sides of the membrane, we infer that the ATPase activity of OpuA in nanodiscs reflects solute translocation. Intriguingly, the substrate-dependent, ionic strength-gated ATPase activity of OpuA in nanodiscs is at least an order of magnitude higher than in lipid vesicles (i.e. with identical membrane lipid composition, ionic strength, and nucleotide and substrate concentrations). Even with the chemical components the same, the lateral pressure (profile) of the nanodiscs will differ from that of the vesicles. We thus propose that membrane tension limits translocation in vesicular systems. Increased macromolecular crowding does not activate OpuA but acts synergistically with ionic strength, presumably by favoring gating interactions of like-charged surfaces via excluded volume effects.     

Structure of the Type IVa Major Pilin from the Electrically Conductive Bacterial Nanowires of Geobacter sulfurreducens

Several species of δ proteobacteria are capable of reducing insoluble metal oxides as well as other extracellular electron acceptors. These bacteria play a critical role in the cycling of minerals in subsurface environments, sediments, and groundwater. In some species of bacteria such as Geobacter sulfurreducens, the transport of electrons is proposed to be facilitated by filamentous fibers that are referred to as bacterial nanowires. These nanowires are polymeric assemblies of proteins belonging to the type IVa family of pilin proteins and are mainly comprised of one subunit protein, PilA. Here, we report the high resolution solution NMR structure of the PilA protein from G. sulfurreducens determined in detergent micelles. The protein is >85% α-helical and exhibits similar architecture to the N-terminal regions of other non-conductive type IVa pilins. The detergent micelle interacts with the first 21 amino acids of the protein, indicating that this region likely associates with the bacterial inner membrane prior to fiber formation. A model of the G. sulfurreducens pilus fiber is proposed based on docking of this structure into the fiber model of the type IVa pilin from Neisseria gonorrhoeae. This model provides insight into the organization of aromatic amino acids that are important for electrical conduction.     

An Annular Lipid Belt Is Essential for Allosteric Coupling and Viral Inhibition of the Antigen Translocation Complex TAP (Transporter Associated with Antigen Processing)

The transporter associated with antigen processing (TAP) constitutes a focal element in the adaptive immune response against infected or malignantly transformed cells. TAP shuttles proteasomal degradation products into the lumen of the endoplasmic reticulum for loading of major histocompatibility complex (MHC) class I molecules. Here, the heterodimeric TAP complex was purified and reconstituted in nanodiscs in defined stoichiometry. We demonstrate that a single heterodimeric core-TAP complex is active in peptide binding, which is tightly coupled to ATP hydrolysis. Notably, with increasing peptide length, the ATP turnover was gradually decreased, revealing that ATP hydrolysis is coupled to the movement of peptide through the ATP-binding cassette transporter. In addition, all-atom molecular dynamics simulations show that the observed 22 lipids are sufficient to form an annular belt surrounding the TAP complex. This lipid belt is essential for high affinity inhibition by the herpesvirus immune evasin ICP47. In conclusion, nanodiscs are a powerful approach to study the important role of lipids as well as the function, interaction, and modulation of the antigen translocation machinery.     

Quaternary structure and functional unit of energy coupling factor (ECF)-type transporters

ATP-binding cassette (ABC) transporters mediate transport of diverse substrates across membranes. We have determined the quaternary structure and functional unit of the recently discovered ECF-type (energy coupling factor) of ABC transporters, which is widespread among prokaryotes. ECF transporters are protein complexes consisting of a conserved energizing module (two peripheral ATPases and the integral membrane protein EcfT) and a non-conserved integral membrane protein responsible for substrate specificity (S-component). S-components for different substrates are often unrelated in amino acid sequence but may associate with the same energizing module. Here, the energizing module from Lactococcus lactis was shown to form stable complexes with each of the eight predicted S-components found in the organism. The quaternary structures of three of these complexes were determined by light scattering. EcfT, the two ATPases (EcfA and EcfA'), and the S-components were found to be present in a 1:1:1:1 ratio. The complexes were reconstituted in proteoliposomes and shown to mediate ATP-dependent transport. ECF-type transporters are the smallest known ABC transporters.     

Dynamic Structure of the Translocon SecYEG in Membrane: DIRECT SINGLE MOLECULE OBSERVATIONS*

Purified SecYEG was reconstituted into liposomes and studied in near-native conditions using atomic force microscopy. These SecYEG proteoliposomes were active in translocation assays. Changes in the structure of SecYEG as a function of time were directly visualized. The dynamics observed were significant in magnitude (∼1–10 Å) and were attributed to the two large loops of SecY linking transmembrane helices 6–7 and 8–9. In addition, we identified a distribution between monomers and dimers of SecYEG as well as a smaller population of higher order oligomers. This work provides a new vista of the flexible and dynamic structure of SecYEG, an intricate and vital membrane protein.     

The Extreme C Terminus of the ABC Protein DrrA Contains Unique Motifs Involved in Function and Assembly of the DrrAB Complex

Two novel regulatory motifs, LDEVFL and C-terminal regulatory Glu (E)-rich motif (CREEM), are identified in the extreme C terminus of the ABC protein DrrA, which is involved in direct interaction with the N-terminal cytoplasmic tail of the membrane protein DrrB and in homodimerization of DrrA. Disulfide cross-linking analysis showed that the CREEM and the region immediately upstream of CREEM participate directly in forming an interaction interface with the N terminus of DrrB. A series of mutations created in the LDEVFL and CREEM motifs drastically affected overall function of the DrrAB transporter. Mutations in the LDEVFL motif also significantly impaired interaction between the C terminus of DrrA and the N terminus of DrrB as well as the ability of DrrA and DrrB to co-purify, therefore suggesting that the LDEVFL motif regulates CREEM-mediated interaction between DrrA and DrrB and plays a key role in biogenesis of the DrrAB complex. Modeling analysis indicated that the LDEVFL motif is critical for conformational integrity of the C-terminal domain of DrrA and confirmed that the C terminus of DrrA forms an independent domain. This is the first report which describes the presence of an assembly domain in an ABC protein and uncovers a novel mechanism whereby the ABC component facilitates the assembly of the membrane component. Homology sequence comparisons showed the presence of the LDEVFL and CREEM motifs in close prokaryotic and eukaryotic homologs of DrrA, suggesting that these motifs may play a similar role in other homologous drug and lipid export systems.     

Transmembrane Topology and Oligomeric Structure of the High-affinity Choline Transporter

The high-affinity choline transporter CHT1 mediates choline uptake essential for acetylcholine synthesis in cholinergic nerve terminals. CHT1 belongs to the Na⁺/glucose cotransporter family (SLC5), which is postulated to have a common 13-transmembrane domain core; however, no direct experimental evidence for CHT1 transmembrane topology has yet been reported. We examined the transmembrane topology of human CHT1 using cysteine-scanning analysis. Single cysteine residues were introduced into the putative extra- and intracellular loops and probed for external accessibility for labeling with a membrane-impermeable, sulfhydryl-specific biotinylating reagent in intact cells expressing these mutants. The results provide experimental evidence for a topological model of a 13-transmembrane domain protein with an extracellular amino terminus and an intracellular carboxyl terminus. We also constructed a three-dimensional homology model of CHT1 based on the crystal structure of the bacterial Na⁺/galactose cotransporter, which supports our conclusion of CHT1 transmembrane topology. Furthermore, we examined whether CHT1 exists as a monomer or oligomer. Chemical cross-linking induces the formation of a higher molecular weight form of CHT1 on the cell surface in HEK293 cells. Two different epitope-tagged CHT1 proteins expressed in the same cells can be co-immunoprecipitated. Moreover, co-expression of an inactive mutant I89A with the wild type induces a dominant-negative effect on the overall choline uptake activity. These results indicate that CHT1 forms a homo-oligomer on the cell surface in cultured cells.     

Structure of Pneumococcal Peptidoglycan Hydrolase LytB Reveals Insights into the Bacterial Cell Wall Remodeling and Pathogenesis

Streptococcus pneumoniae causes a series of devastating infections in humans. Previous studies have shown that the endo-β-N-acetylglucosaminidase LytB is critical for pneumococcal cell division and nasal colonization, but the biochemical mechanism of LytB action remains unknown. Here we report the 1.65 Å crystal structure of the catalytic domain (residues Lys-375–Asp-658) of LytB (termed LytB_CAT), excluding the choline binding domain. LytB_CAT consists of three structurally independent modules: SH3b, WW, and GH73. These modules form a “T-shaped” pocket that accommodates a putative tetrasaccharide-pentapeptide substrate of peptidoglycan. Structural comparison and simulation revealed that the GH73 module of LytB harbors the active site, including the catalytic residue Glu-564. In vitro assays of hydrolytic activity indicated that LytB prefers the peptidoglycan from the lytB-deficient pneumococci, suggesting the existence of a specific substrate of LytB in the immature peptidoglycan. Combined with in vitro cell-dispersing and in vivo cell separation assays, we demonstrated that all three modules are necessary for the optimal activity of LytB. Further functional analysis showed that the full catalytic activity of LytB is required for pneumococcal adhesion to and invasion into human lung epithelial cells. Structure-based alignment indicated that the unique modular organization of LytB is highly conserved in its orthologs from Streptococcus mitis group and Gemella species. These findings provided structural insights into the pneumococcal cell wall remodeling and novel hints for the rational design of therapeutic agents against pneumococcal growth and thereby the related diseases.     

Crystal Structure of the Bacillus subtilis Phosphodiesterase PhoD Reveals an Iron and Calcium-containing Active Site

The PhoD family of extra-cytoplasmic phosphodiesterases are among the most commonly occurring bacterial phosphatases. The exemplars for this family are the PhoD protein of Bacillus subtilis and the phospholipase D of Streptomyces chromofuscus. We present the crystal structure of B. subtilis PhoD. PhoD is most closely related to purple acid phosphatases (PAPs) with both types of enzyme containing a tyrosinate-ligated Fe³⁺ ion. However, the PhoD active site diverges from that found in PAPs and uses two Ca²⁺ ions instead of the single extra Fe²⁺, Mn²⁺, or Zn²⁺ ion present in PAPs. The PhoD crystals contain a phosphate molecule that coordinates all three active site metal ions and that is proposed to represent a product complex. A C-terminal helix lies over the active site and controls access to the catalytic center. The structure of PhoD defines a new phosphatase active site architecture based on Fe³⁺ and Ca²⁺ ions.     

Interactions between the conserved hydrophobic region of the prion protein and dodecylphosphocholine micelles

The three-dimensional structure of PrP110-136, a peptide encompassing the conserved hydrophobic region of the human prion protein, has been determined at high resolution in dodecylphosphocholine micelles by NMR. The results support the conclusion that the (Ctm)PrP, a transmembrane form of the prion protein, adopts a different conformation than the reported structures of the normal prion protein determined in solution. Paramagnetic relaxation enhancement studies with gadolinium-diethylenetriaminepentaacetic acid indicated that the conserved hydrophobic region peptide is not inserted symmetrically in the micelle, thus suggesting the presence of a guanidium-phosphate ion pair involving the side chain of the terminal arginine and the detergent headgroup. Titration of dodecylphosphocholine into a solution of PrP110-136 revealed the presence of a surface-bound species. In addition, paramagnetic probes located the surface-bound peptide somewhere below the micelle-water interface when using the inserted helix as a positional reference. This localization of the unknown population would allow a similar ion pair interaction.     

Solution Structure of Proinsulin: CONNECTING DOMAIN FLEXIBILITY AND PROHORMONE PROCESSING*

The folding of proinsulin, the single-chain precursor of insulin, ensures native disulfide pairing in pancreatic β-cells. Mutations that impair folding cause neonatal diabetes mellitus. Although the classical structure of insulin is well established, proinsulin is refractory to crystallization. Here, we employ heteronuclear NMR spectroscopy to characterize a monomeric analogue. Proinsulin contains a native-like insulin moiety (A- and B-domains); the tethered connecting (C) domain (as probed by {¹H}-¹⁵N nuclear Overhauser enhancements) is progressively less ordered. Although the BC junction is flexible, residues near the CA junction exhibit α-helical-like features. Relative to canonical α-helices, however, segmental ¹³C_α/β chemical shifts are attenuated, suggesting that this junction and contiguous A-chain residues are molten. We propose that flexibility at each C-domain junction facilitates prohormone processing. Studies of protease SPC3 (PC1/3) suggest that C-domain sequences contribute to cleavage site selection. The structure of proinsulin provides a foundation for studies of insulin biosynthesis and its impairment in monogenic forms of diabetes mellitus.     

Assembly and Function of the Major Histocompatibility Complex (MHC) I Peptide-loading Complex Are Conserved Across Higher Vertebrates

Antigen presentation to cytotoxic T lymphocytes via major histocompatibility complex class I (MHC I) molecules depends on the heterodimeric transporter associated with antigen processing (TAP). For efficient antigen supply to MHC I molecules in the ER, TAP assembles a macromolecular peptide-loading complex (PLC) by recruiting tapasin. In evolution, TAP appeared together with effector cells of adaptive immunity at the transition from jawless to jawed vertebrates and diversified further within the jawed vertebrates. Here, we compared TAP function and interaction with tapasin of a range of species within two classes of jawed vertebrates. We found that avian and mammalian TAP1 and TAP2 form heterodimeric complexes across taxa. Moreover, the extra N-terminal domain TMD0 of mammalian TAP1 and TAP2 as well as avian TAP2 recruits tapasin. Strikingly, however, only TAP1 and TAP2 from the same taxon can form a functional heterodimeric translocation complex. These data demonstrate that the dimerization interface between TAP1 and TAP2 and the tapasin docking sites for PLC assembly are conserved in evolution, whereas elements of antigen translocation diverged later in evolution and are thus taxon specific.     

Structural Insights into Higher Order Assembly and Function of the Bacterial Microcompartment Protein PduA

Bacterial microcompartments are large proteinaceous assemblies that are found in the cytoplasm of some bacteria. These structures consist of proteins constituting a shell that houses a number of enzymes involved in specific metabolic processes. The 1,2-propanediol-utilizing microcompartment is assembled from seven different types of shell proteins, one of which is PduA. It is one of the more abundant components of the shell and intriguingly can form nanotubule-like structures when expressed on its own in the cytoplasm of Escherichia coli. We propose a model that accounts for the size and appearance of these PduA structures and underpin our model using a combinatorial approach. Making strategic mutations at Lys-26, Val-51, and Arg-79, we targeted residues predicted to be important for PduA assembly. We present the effect of the amino acid residue substitution on the phenotype of the PduA higher order assemblies (transmission electron microscopy) and the crystal structure of the K26D mutant with one glycerol molecule bound to the central pore. Our results support the view that the hexamer-hexamer interactions seen in PduA crystals persist in the cytoplasmic structures and reveal the profound influence of the two key amino acids, Lys-26 and Arg-79, on tiling, not only in the crystal lattice but also in the bacterial cytoplasm. Understanding and controlling PduA assemblies is valuable in order to inform manipulation for synthetic biology and biotechnological applications.     

Functional Equilibrium of the KcsA Structure Revealed by NMR

KcsA is a tetrameric K⁺ channel that is activated by acidic pH. Under open conditions of the helix bundle crossing, the selectivity filter undergoes an equilibrium between permeable and impermeable conformations. Here we report that the population of the permeable conformation (p_perm) positively correlates with the tetrameric stability and that the population in reconstituted high density lipoprotein, where KcsA is surrounded by the lipid bilayer, is lower than that in detergent micelles, indicating that dynamic properties of KcsA are different in these two media. Perturbation of the membrane environment by the addition of 1–3% 2,2,2-trifluoroethanol increases p_perm and the open probability, revealed by NMR and single-channel recording analyses. These results demonstrate that KcsA inactivation is determined not only by the protein itself but also by the surrounding membrane environments.     

Palmitoylation of the SNAP25 Protein Family: SPECIFICITY AND REGULATION BY DHHC PALMITOYL TRANSFERASES*

SNAP25 plays an essential role in neuronal exocytosis pathways. SNAP25a and SNAP25b are alternatively spliced isoforms differing by only nine amino acids, three of which occur within the palmitoylated cysteine-rich domain. SNAP23 is 60% identical to SNAP25 and has a distinct cysteine-rich domain to both SNAP25a and SNAP25b. Despite the conspicuous differences within the palmitoylated domains of these secretory proteins, there is no information on their comparative interactions with palmitoyl transferases. We report that membrane association of all SNAP25/23 proteins is enhanced by Golgi-localized DHHC3, DHHC7, and DHHC17. In contrast, DHHC15 promoted a statistically significant increase in membrane association of only SNAP25b. To investigate the underlying cause of this differential specificity, we examined a SNAP23 point mutant (C79F) designed to mimic the cysteine-rich domain of SNAP25b. DHHC15 promoted a marked increase in membrane binding and palmitoylation of this SNAP23 mutant, demonstrating that the distinct cysteine-rich domains of SNAP25/23 contribute to differential interactions with DHHC15. The lack of activity of DHHC15 toward wild-type SNAP23 was not overcome by replacing its DHHC domain with that from DHHC3, suggesting that substrate specificity is not determined by the DHHC domain alone. Interestingly, DHHC2, which is closely related to DHHC15, associates with the plasma membrane in PC12 cells and can palmitoylate all SNAP25 isoforms. DHHC2 is, thus, a candidate enzyme to regulate SNAP25/23 palmitoylation dynamics at the plasma membrane. Finally, we demonstrate that overexpression of specific Golgi-localized DHHC proteins active against SNAP25/23 proteins perturbs the normal secretion of human growth hormone from PC12 cells.     

Membrane Region M2C2 in Subunit KtrB of the K+ Uptake System KtrAB from Vibrio alginolyticus Forms a Flexible Gate Controlling K+ Flux: AN ELECTRON PARAMAGNETIC RESONANCE STUDY*

Transmembrane stretch M_2C from the bacterial K⁺-translocating protein KtrB is unusually long. In its middle part, termed M_2C2, it contains several small and polar amino acids. This region is flanked by the two α-helices M_2C1 and M_2C3 and may form a flexible gate at the cytoplasmic side of the membrane controlling K⁺ translocation. In this study, we provide experimental evidence for this notion by using continuous wave and pulse EPR measurements of single and double spin-labeled cysteine variants of KtrB. Most of the spin-labeled residues in M_2C2 were shown to be immobile, pointing to a compact structure. However, the high polarity revealed for the microenvironment of residue positions 317, 318, and 327 indicated the existence of a water-accessible cavity. Upon the addition of K⁺ ions, M_2C2 residue Thr-318R1 (R1 indicates the bound spin label) moved with respect to M_2B residue Asp-222R1 and M_2C3 residue Val-331R1 but not with respect to M_2C1 residue Met-311R1. Based on distances determined between spin-labeled residues of double-labeled variants of KtrB in the presence and absence of K⁺ ions, structural models of the open and closed conformations were developed.     

Solution NMR Structure and Functional Analysis of the Integral Membrane Protein YgaP from Escherichia coli

The solution NMR structure of the α-helical integral membrane protein YgaP from Escherichia coli in mixed 1,2-diheptanoyl-sn-glycerol-3-phosphocholine/1-myristoyl-2-hydroxy-sn-glycero-3-phospho-(1′-rac-glycerol) micelles is presented. In these micelles, YgaP forms a homodimer with the two transmembrane helices being the dimer interface, whereas the N-terminal cytoplasmic domain includes a rhodanese-fold in accordance to its sequence homology to the rhodanese family of sulfurtransferases. The enzymatic sulfur transfer activity of full-length YgaP as well as of the N-terminal rhodanese domain only was investigated performing a series of titrations with sodium thiosulfate and potassium cyanide monitored by NMR and EPR. The data indicate the thiosulfate concentration-dependent addition of several sulfur atoms to the catalytic Cys-63, which process can be reversed by the addition of potassium cyanide. The catalytic reaction induces thereby conformational changes within the rhodanese domain, as well as on the transmembrane α-helices of YgaP. These results provide insights into a potential mechanism of YgaP during the catalytic thiosulfate activity in vivo.     

New Insights into the Assembly of Bacterial Secretins: STRUCTURAL STUDIES OF THE PERIPLASMIC DOMAIN OF XcpQ FROM PSEUDOMONAS AERUGINOSA*

The type II secretion system is a multiprotein assembly spanning the inner and outer membranes in Gram-negative bacteria. It is found in almost all pathogenic bacteria where it contributes to virulence, host tissue colonization, and infection. The exoproteins are secreted across the outer membrane via a large translocation channel, the secretin, which typically adopts a dodecameric structure. These secretin channels have large periplasmic N-terminal domains that reach out into the periplasm for communication with the inner membrane platform and with a pseudopilus structure that spans the periplasm. Here we report the crystal structure of the N-terminal periplasmic domain of the secretin XcpQ from Pseudomonas aeruginosa, revealing a two-lobe dimeric assembly featuring parallel subunits engaging in well defined interactions at the tips of each lobe. We have employed structure-based engineering of disulfide bridges and native mass spectrometry to show that the periplasmic domain of XcpQ dimerizes in a concentration-dependent manner. Validation of these insights in the context of cellular full-length XcpQ and further evaluation of the functionality of disulfide-linked XcpQ establishes that the basic oligomerization unit of XcpQ is a dimer. This is consistent with the notion that the dodecameric secretin assembles as a hexamer of dimers to ensure correct projection of the N-terminal domains into the periplasm. Therefore, our studies provide a key conceptual advancement in understanding the assembly principles and dynamic function of type II secretion system secretins and challenge recent studies reporting monomers as the basic subunit of the secretin oligomer.