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1.B.20 The Two-partner Secretion (TPS) Family

The first member of the TPS family to be characterized was the ShlB (HlyB) protein of Serratia marcescens which exports the ShlA hemolysin from the periplasm of the Gram-negative bacterial envelope into the external medium (Poole et al., 1988). ShlA reaches the periplasm by export from the cytoplasm via the general secretory pathway (GSP or IISP; TC #3.A.5). ShlB and some, but not all TPS homologues, include domains with both an outer membrane export channel and a 'hemolysin activator protein' which activates ShlA by derivatization with phosphatidyl ethanolamine (Hertle et al., 1997). Several ShlB homologues have been functionally characterized (Hirono et al, 1997; Jacob-Debuisson et al., 1997, Jacob-Debuisson et al., 2000; Palmer and Munson, 1995). The channel activities of some of these homologues have been demonstrated (Jacob-Debuisson et al., 1999; Kànninger et al., 1999), and topological features of these β-barrel porins have been studied (Guédin et al., 2000; Kànninger et al., 1999). Specificity for particiular protein substrates has been demonstrated (Jacob-Debuisson et al., 1997). One such protein, FhaC of B. pertussis, exhibits a surface exposed N-terminus and an odd number of β-strands with large surface loops and small periplasmic loops (Guédin et al., 2000; Kànninger et al., 1999; Méli et al., 2006).

Substrates of TPS family secretins include Ca+-independent cytolysins, an ion acquisition protein and several adhesins. The hallmarks of TPS systems are the presence of (1) an N-proximal module where specific secretion signals in the substrate protein are found, and (2) a β-barrel channel (TpsB) homologue (Jacob-Debuisson et al., 2000). Usually, the genes encoding these two proteins occur within an operon. While transport via the GSP occurs in the unfolded state, the substrate protein probably folds in the periplasm and on the periplasmic surface of the outer membrane before it is exported via the TPS porin (Jacob-Debuisson et al., 2000). Evidence for secretion-dependent folding of mature exoproteins has also been obtained (Mazar and Cotter, 2007).

FhaC of B. pertussis, the TpsB protein that transports the TpsA partner, FHA, exhibits a surface-exposed N-terminus and 16 β-strands with large surface loops and small periplasmic turns (Méli et al., 2006; Clantin et al., 2007). These features may be characteristic of the family. Surface exposed residues and pore formation in artificial membranes have been characterized with FhaC and the ShlB outer membrane porins. Méli et al. (2006) reported that FhaC (TpsB) exhibits ion channel properties, and mutants altered for FHA (TpsA) transport affected ion channel activity. The N-terminal 200 residues probably form a functionally distinct domain that modulates the pore properties and may participate in FHA recognition. The C-terminal two-thirds of TpsB forms the transmembrane channel-forming β-barrel domain. A C-proximal motif (the family signature sequence) appeared to be essential for pore formation (Méli et al., 2006). FHA moves through the periplasm in an extended conformation maintained by the DegP chaparone before it is exported and folds into a β-helical structure (Baud et al., 2009).

In Gram-negative bacteria and eukaryotic organelles, ß-barrel proteins of the outer membrane protein YaeT (TC#1.B.33) / two-partner secretion B (TC#1.B.20) (Omp85-TpsB) superfamily are essential components of protein transport machineries. The TpsB transporter, FhaC (TC# 1.B.20.2.1) mediates the secretion of Bordetella pertussis filamentous hemagglutinin (FHA). The 3.15 Å crystal structure of FhaC has been reported (Clantin et al., 2007). The transporter comprises a 16-stranded ß barrel that is occluded by an N-terminal {alpha} helix and an extracellular loop and a periplasmic module composed of two aligned polypeptide-transport–associated (POTRA) domains. Functional data reveal that FHA binds to the POTRA 1 domain via its N-terminal domain and likely translocates the adhesin-repeated motifs in an extended hairpin conformation, with folding occurring at the cell surface. General features of the mechanism are likely to apply throughout the superfamily.

Proteins showing large regions of sequence similarity to established members of the TPS family have been identified in Gram-positive bacteria, yeast, plants and animals. They clearly share homologous domains. In Gram-negative bacteria, the two-partner secretion (TPS) pathway is dedicated to the secretion of large, mostly virulence-related proteins. The secreted TpsA proteins carry a characteristic 250-residue-long N-terminal 'TPS domain' essential for secretion, while their TpsB transporters are pore-forming proteins that specifically recognize their respective TpsA partners and mediate their translocation across the outer membrane. Bordetella pertussis secretes its major adhesin filamentous haemagglutinin (FHA) via the TpsB transporter, FhaC (TC# 1.B.20.2.1). Specific interactions between an N-terminal fragment of FHA containing the TPS domain and FhaC occur (Hodak et al., 2006). FhaC recognizes only non-native conformations of the TPS domain, and in vivo, periplasmic FHA is not folded. Interaction determinants forming the secretion signal have been identified (Hodak et al., 2006). They are found far into the TPS domain and include both conserved and variable residues, which most likely explains the specificity of the TpsA-TpsB interaction. The N-terminal domain of FhaC is involved in the FHA-FhaC interaction, in agreement with its proposed function and periplasmic localization.

In Gram-negative bacteria, most surface-associated proteins are present as integral outer-membrane proteins. Exceptions include the Haemophilus influenzae HMW1 and HMW2 adhesins and a subset of other proteins secreted by the two-partner secretion system. HMW1 forms hair-like fibres on the bacterial surface and is usually present as pairs that appear to be joined together at one end. HMW1 is anchored to the multimeric HMW1B outer membrane translocator, resulting in a direct correlation between the level of surface-associated HMW1 and the quantity of HMW1B in the outer membrane. Anchoring of HMW1 requires the C-terminal 20 amino acids of the protein and is dependent upon disulphide bond formation between two conserved cysteine residues in this region (Buscher et al., 2006). The immediate C-terminus of HMW1 is inaccessible to surface labelling, suggesting that it remains buried in HMW1B. These observations may have broad relevance to many proteins secreted by the two-partner secretion system, especially given the conservation of C-terminal cysteine residues among surface-associated proteins in this family.

Bacterial contact-dependent growth inhibition (CDI) systems use a type Vb secretion mechanism to export large CdiA toxins across the outer membrane by dedicated outer membrane transporters called CdiB. Guerin et al. 2020 reported the first crystal structures of two CdiB transporters from Acinetobacter baumannii and E. coli. CdiB transporters adopt a TpsB fold, containing a 16-stranded transmembrane beta-barrel connected to two periplasmic domains. The lumen of the CdiB pore is occluded by an N-terminal alpha-helix and the conserved extracellular loop 6; these two elements adopt different conformations in the structures. A conserved DxxG motif is located on strand beta1 that connects loop 6 through different networks of interactions. Structural modifications of DxxG induce rearrangement of extracellular loops and alter interactions with the N-terminal alpha-helix, preparing the system for alpha-helix ejection. The barrel pore is probably primed for CdiA toxin secretion (Guerin et al. 2020).

Omp85 transporters and Two Partner Secretion (TPS) systems have a single conserved architecture, with POTRA domains that interact with substrate proteins, a 16-stranded transmembrane beta barrel, and an extracellular loop, L6, folded back in the barrel pore. Guérin et al. 2015 showed that the L6 loop of  FhaC changes conformation and modulates channel opening. Those conformational changes involve breaking the conserved interaction between the tip of L6 and the inner beta-barrel wall. The membrane-proximal POTRA domain also exchanges between several conformations, and the binding of FHA displaces this equilibrium. There is dynamic, physical communication between the POTRA domains and L6 within the beta barrel (Guérin et al. 2015).

The transport reaction catalyzed by bacterial members of the TPS family is:

Partially folded protein (periplasm) → Folded protein (external milieu)

References associated with 1.B.20 family:

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