Structural Determinants of the Interaction between the TpsA and TpsB Proteins in the Haemophilus influenzae HMW1 Two-Partner Secretion System

Abstract

The two-partner secretion (TPS) pathway in Gram-negative bacteria consists of a TpsA exoprotein and a cognate TpsB outer membrane pore-forming translocator protein. Previous work has demonstrated that the TpsA protein contains an N-terminal TPS domain that plays an important role in targeting the TpsB protein and is required for secretion. The nontypeable Haemophilus influenzae HMW1 and HMW2 adhesins are homologous proteins that are prototype TpsA proteins and are secreted by the HMW1B and HMW2B TpsB proteins. In the present study, we sought to define the structural determinants of HMW1 interaction with HMW1B during the transport process and while anchored to the bacterial surface. Modeling of HMW1B revealed an N-terminal periplasmic region that contains two polypeptide transport-associated (POTRA) domains and a C-terminal membrane-localized region that forms a pore. Biochemical studies demonstrated that HMW1 engages HMW1B via interaction between the HMW1 TPS domain and the HMW1B periplasmic region, specifically, the predicted POTRA1 and POTRA2 domains. Subsequently, HMW1 is shuttled to the HMW1B pore, facilitated by the N-terminal region, the middle region, and the NPNG motif in the HMW1 TPS domain. Additional analysis revealed that the interaction between HMW1 and HMW1B is highly specific and is dependent upon the POTRA domains and the pore-forming domain of HMW1B. Further studies established that tethering of HMW1 to the surface-exposed region of HMW1B is dependent upon the external loops of HMW1B formed by residues 267 to 283 and residues 324 to 330. These observations may have broad relevance to proteins secreted by the TPS pathway. IMPORTANCESecretion of HMW1 involves a recognition event between the extended form of the HMW1 propiece and the HMW1B POTRA domains. Our results identify specific interactions between the HMW1 propiece and the periplasmic HMW1B POTRA domains. The results also suggest that the process of HMW1 translocation involves at least two discrete steps, including initial interaction between the HMW1 propiece and the HMW1B POTRA domains and then a separate translocation event. We have also discovered that the HMW1B pore itself appears to influence the translocation process. These observations extend our knowledge of the two-partner secretion system and may be broadly relevant to other proteins secreted by the TPS pathway. G ram-negative bacteria have developed a number of pathways for targeting proteins to an extracellular location (1). Among the most common of these pathways is the two-partner secretion (TPS) system. TPS systems typically consist of a large secreted protein called a TpsA protein (encoded by a tpsA gene) and a cognate outer membrane pore-forming translocator protein called a TpsB protein (encoded by a tpsB gene) (2). In addition, some TPS systems include a protein that has glycosyltransferase activity and modifies the TpsA protein (3).TpsA proteins are rich in ␤ structure and are involved in a variety of functions on the bacterial surface or in the extracellular mil...

Two-Partner Secretion: Combining Efficiency and Simplicity in the Secretion of Large Proteins for Bacteria-Host and Bacteria-Bacteria Interactions

“…The POTRA domains might thus contribute to addressing the TpsA protein to the pore of the TpsB transporter. Similar suggestions were made for the POTRA domains of HMW1B and LepB (Garnett et al, 2015 ; Grass et al, 2015 ). Their role in shuttling the passenger to the barrel might explain why PlpD, the prototypic Vd protein, harbors a POTRA-like domain.…”

“…The TPS domain of the TpsA protein must be in an extended conformation for interaction with the POTRA domains of the TpsB transporter, as shown for the FHA/FhaC and HMW1/HMW1B pairs (Hodak et al, 2006; Grass et al, 2015). Surface plasmon resonance experiments with the FHA/FhaC pair have indicated interactions of micromolar affinity between the two proteins and suggested that association and dissociation occur very fast (Delattre et al, 2011).…”

“…Molecular recognition between the two partners is mediated by the TPS domain and the POTRA domains of the TpsA and TpsB proteins, respectively (Surana et al, 2004 ; Hodak et al, 2006 ; Delattre et al, 2011 ; Garnett et al, 2015 ). The TPS domain of the TpsA protein must be in an extended conformation for interaction with the POTRA domains of the TpsB transporter, as shown for the FHA/FhaC and HMW1/HMW1B pairs (Hodak et al, 2006 ; Grass et al, 2015 ). Surface plasmon resonance experiments with the FHA/FhaC pair have indicated interactions of micromolar affinity between the two proteins and suggested that association and dissociation occur very fast (Delattre et al, 2011 ).…”

“…POTRA domains have been proposed to mediate protein-protein interactions, probably by β augmentation (Kim et al, 2007 ). The POTRA domains of several TpsB proteins have been shown to recognize the TPS domains of their respective TpsA partners (Surana et al, 2004 ; Hodak et al, 2006 ; Delattre et al, 2011 ; ur Rahman et al, 2014 ; Garnett et al, 2015 ; Grass et al, 2015 ). A major binding site for the FHA TPS domain in FhaC is a hydrophobic groove formed by the edge of the β sheet and the flanking α helix of the barrel-proximal POTRA2 domain (Delattre et al, 2011 ).…”

“…The edge of this small surface-exposed β sheet of FhaC might guide the secretion of the TPS domain and template its initial folding at the cell surface by β augmentation (Baud et al, 2014 ). Interestingly, the corresponding loops of HMW1B appear to tether the HMW1 adhesin at the cell surface of H. influenzae (Grass et al, 2015 ). Thus, specific structural features at the extracellular surface of the TpsB transporter might be involved in the secretion of the cognate TpsA partner and possibly also retain its N terminus close to the cell surface.…”