Travis J. Barnard

TonB-Dependent Transporters: Regulation, Structure, and Function

TonB-dependent transporters (TBDTs) are bacterial outer membrane proteins that bind and transport ferric chelates, called siderophores, as well as vitamin B(12), nickel complexes, and carbohydrates. The transport process requires energy in the form of proton motive force and a complex of three inner membrane proteins, TonB-ExbB-ExbD, to transduce this energy to the outer membrane. The siderophore substrates range in complexity from simple small molecules such as citrate to large proteins such as serum transferrin and hemoglobin. Because iron uptake is vital for almost all bacteria, expression of TBDTs is regulated in a number of ways that include metal-dependent regulators, σ/anti-σ factor systems, small RNAs, and even a riboswitch. In recent years, many new structures of TBDTs have been solved in various states, resulting in a more complete understanding of siderophore selectivity and binding, signal transduction across the outer membrane, and interaction with the TonB-ExbB-ExbD complex. However, the transport mechanism is still unclear. In this review, we summarize recent progress in understanding regulation, structure, and function in TBDTs and questions remaining to be answered.

Cleavage of a bacterial autotransporter by an evolutionarily convergent autocatalytic mechanism

Bacterial autotransporters are comprised of an N‐terminal ‘passenger domain’ and a C‐terminal β barrel (‘β domain’) that facilitates transport of the passenger domain across the outer membrane. Following translocation, the passenger domains of some autotransporters are cleaved by an unknown mechanism. Here we show that the passenger domain of the Escherichia coli O157:H7 autotransporter EspP is released in a novel autoproteolytic reaction. After purification, the uncleaved EspP precursor underwent proteolytic processing in vitro. An analysis of protein topology together with mutational studies strongly suggested that the reaction occurs inside the β barrel and revealed that two conserved residues, an aspartate within the β domain (Asp1120) and an asparagine (Asn1023) at the P1 position of the cleavage junction, are essential for passenger domain cleavage. Interestingly, these residues were also essential for the proteolytic processing of two distantly related autotransporters. The data strongly suggest that Asp1120 and Asn1023 form an unusual catalytic dyad that mediates self‐cleavage through the cyclization of the asparagine. Remarkably, a very similar mechanism has been proposed for the maturation of eukaryotic viral capsids.

Efficient secretion of a folded protein domain by a monomeric bacterial autotransporter

Bacterial autotransporters are proteins that contain a small C‐terminal ‘β domain’ that facilitates translocation of a large N‐terminal ‘passenger domain’ across the outer membrane (OM) by an unknown mechanism. Here we used EspP, an autotransporter produced by Escherichia coli 0157:H7, as a model protein to gain insight into the transport reaction. Initially we found that the passenger domain of a truncated version of EspP (EspPΔ1‐851) was translocated efficiently across the OM. Blue Native polyacrylamide gel electrophoresis, analytical ultracentrifugation and other biochemical methods showed that EspPΔ1‐851 behaves as a compact monomer and strongly suggest that the channel formed by the β domain is too narrow to accommodate folded polypeptides. Surprisingly, we found that a folded protein domain fused to the N‐terminus of EspPΔ1‐851 was efficiently translocated across the OM. Further analysis revealed that the passenger domain of wild‐type EspP also folds at least partially in the periplasm. To reconcile these data, we propose that the EspP β domain functions primarily to target and anchor the protein and that an external factor transports the passenger domain across the OM.

Structure of colicin I receptor bound to the R-domain of colicin Ia: implications for protein import

Colicin Ia is a 69 kDa protein that kills susceptible Escherichia coli cells by binding to a specific receptor in the outer membrane, colicin I receptor (70 kDa), and subsequently translocating its channel forming domain across the periplasmic space, where it inserts into the inner membrane and forms a voltage‐dependent ion channel. We determined crystal structures of colicin I receptor alone and in complex with the receptor binding domain of colicin Ia. The receptor undergoes large and unusual conformational changes upon colicin binding, opening at the cell surface and positioning the receptor binding domain of colicin Ia directly above it. We modelled the interaction with full‐length colicin Ia to show that the channel forming domain is initially positioned 150 Å above the cell surface. Functional data using full‐length colicin Ia show that colicin I receptor is necessary for cell surface binding, and suggest that the receptor participates in translocation of colicin Ia across the outer membrane.

Structural engineering of a phage lysin that targets Gram-negative pathogens

Bacterial pathogens are becoming increasingly resistant to antibiotics. As an alternative therapeutic strategy, phage therapy reagents containing purified viral lysins have been developed against Gram-positive organisms but not against Gram-negative organisms due to the inability of these types of drugs to cross the bacterial outer membrane. We solved the crystal structures of a Yersinia pestis outer membrane transporter called FyuA and a bacterial toxin called pesticin that targets this transporter. FyuA is a β-barrel membrane protein belonging to the family of TonB dependent transporters, whereas pesticin is a soluble protein with two domains, one that binds to FyuA and another that is structurally similar to phage T4 lysozyme. The structure of pesticin allowed us to design a phage therapy reagent comprised of the FyuA binding domain of pesticin fused to the N-terminus of T4 lysozyme. This hybrid toxin kills specific Yersinia and pathogenic E. coli strains and, importantly, can evade the pesticin immunity protein (Pim) giving it a distinct advantage over pesticin. Furthermore, because FyuA is required for virulence and is more common in pathogenic bacteria, the hybrid toxin also has the advantage of targeting primarily disease-causing bacteria rather than indiscriminately eliminating natural gut flora.

Structural insights into Ail-mediated adhesion in Yersinia pestis

Ail is an outer membrane protein from Yersinia pestis that is highly expressed in a rodent model of bubonic plague, making it a good candidate for vaccine development. Ail is important for attaching to host cells and evading host immune responses, facilitating rapid progression of a plague infection. Binding to host cells is important for injection of cytotoxic Yersinia outer proteins. To learn more about how Ail mediates adhesion, we solved two high-resolution crystal structures of Ail, with no ligand bound and in complex with a heparin analog called sucrose octasulfate. We identified multiple adhesion targets, including laminin and heparin, and showed that a 40 kDa domain of laminin called LG4-5 specifically binds to Ail. We also evaluated the contribution of laminin to delivery of Yops to HEp-2 cells. This work constitutes a structural description of how a bacterial outer membrane protein uses a multivalent approach to bind host cells.

Molecular basis for the activation of a catalytic asparagine residue in a self-cleaving bacterial autotransporter.

Autotransporters are secreted proteins produced by pathogenic Gram-negative bacteria. They consist of a membrane-embedded β-domain and an extracellular passenger domain that is sometimes cleaved and released from the cell surface. We solved the structures of three noncleavable mutants of the autotransporter EspP to examine how it promotes asparagine cyclization to cleave its passenger. We found that cyclization is facilitated by multiple factors. The active-site asparagine is sterically constrained to conformations favorable for cyclization, while electrostatic interactions correctly orient the carboxamide group for nucleophilic attack. During molecular dynamics simulations, water molecules were observed to enter the active site and to form hydrogen bonds favorable for increasing the nucleophilicity of the active-site asparagine. When the activated asparagine attacks its main-chain carbonyl carbon, the resulting oxyanion is stabilized by a protonated glutamate. Upon cleavage, this proton could be transferred to the leaving amine group, helping overcome a significant energy barrier. Together, these findings provide insight into factors important for asparagine cyclization, a mechanism broadly used for protein cleavage.

Structural insight into the role of the Ton complex in energy transduction

In Gram-negative bacteria, outer membrane transporters import nutrients by coupling to an inner membrane protein complex called the Ton complex. The Ton complex consists of TonB, ExbB, and ExbD, and uses the proton motive force at the inner membrane to transduce energy to the outer membrane via TonB. Here, we structurally characterize the Ton complex from Escherichia coli using X-ray crystallography, electron microscopy, double electron–electron resonance (DEER) spectroscopy, and crosslinking. Our results reveal a stoichiometry consisting of a pentamer of ExbB, a dimer of ExbD, and at least one TonB. Electrophysiology studies show that the Ton subcomplex forms pH-sensitive cation-selective channels and provide insight into the mechanism by which it may harness the proton motive force to produce energy.

Using a bacteriocin structure to engineer a phage lysin that targets Yersinia pestis

Purified phage lysins present an alternative to traditional antibiotics and work by hydrolysing peptidoglycan. Phage lysins have been developed against Gram-positive pathogens such as Bacillus anthracis and Streptococcus pneumoniae, where the peptidoglycan layer is exposed on the cell surface. Addition of the lysin to a bacterial culture results in rapid death of the organism. Gram-negative bacteria are resistant to phage lysins because they contain an outer membrane that protects the peptidoglycan from degradation. We solved crystal structures of a Yersinia pestis outer-membrane protein and the bacteriocin that targets it, which informed engineering of a bacterial-phage hybrid lysin that can be transported across the outer membrane to kill specific Gram-negative bacteria. This work provides a template for engineering phage lysins against a wide variety of bacterial pathogens.

Specific targeting and killing of Gram-negative pathogens with an engineered phage lytic enzyme.

Phage lytic enzymes have potential as new inroads toward novel antibiotics. Until now this approach has only been promising for Gram-positive bacteria because in Gram-negatives the target of lytic action is protected by an outer envelope. Information gleaned from the structural studies of two plague proteins—pesticin and FyuA—allowed us to engineer a “hybrid” protein to address this problem. This hybrid consisted of T4 phage lysozyme linked to a FyuA targeting domain and was capable of killing Gram-negative cells. This work therefore presents a proof of principle that phage lytic enzymes can be engineered to cross the outer envelope. Furthermore, hybrid engineering handed us a tool for the mechanistic investigation of TonB mediated membrane transport. This commentary describes our recent efforts to test the efficacy of the hybrid in a mouse infection model and the directions this work might take in the future.