Istvan Botos

The Ton and Tol-Pal systems are molecular machines that are essential for survival of Gram-negative bacteria.Both use the energy derived from the proton gradient at the inner membrane to generate force on protein components at the outer membrane. Ton and Tol share extensive homology, but they fulfill different functions: Ton is involved in the active transport of essential nutrients from the extracellular media into the cell, while Tol maintains the outer membrane integrity and participates in the cell division process. Despite decades of biochemical and biophysical studies, the molecular mechanism coupling the proton gradient at the inner membrane with the propagation of force and movement to the outer membrane is not understood. In this review, we discuss the recent high-resolution structures obtained for both systems, and how these structures fit with existing mechanistic models.

The folding and insertion of β-barrel proteins into the mitochondrial outer membrane is facilitated by the s orting and a ssembly m achinery (SAM) complex. Here we report two 2.8 Å cryo-EM structures of the Thermothelomyces thermophilus SAM complex in the absence of substrate in which the Sam50 lateral gate adopts two different conformations: the first is a closed lateral gate as observed in previously published structures, while the second contains a Sam50 with the first four β-strands rotated outwards by approximately 45°, resulting in an open lateral gate. The observed monomeric open conformation contrasts our previous work where the open conformation was adopted by non-physiological up-down dimers. To understand how these lateral gate dynamics are influenced by substrate, we studied the interaction of the SAM complex with a β-signal peptide mimic, darobactin A. Darobactin A binds to the SAM complex with nanomolar affinity and inhibits the import and assembly of mitochondrial β-barrel proteins in vitro. Lastly, we solved a 3.0 Å cryo-EM structure of the Thermothelomyces thermophilus SAM complex bound to darobactin A, which reveals that darobactin A stabilizes the Sam50 lateral gate similar to the open conformation by binding to strand β1, therefore blocking β-barrel biogenesis.

The Ton and Tol motor proteins use the proton gradient at the inner membrane of Gram-negative bacteria as an energy source. The generated force is transmitted through the periplasmic space to protein components associated with the outer membrane, either to maintain the outer membrane integrity for the Tol system, or to allow essential nutrients to enter the cell for Ton. We have solved the high-resolution structures of the E. coli TonB-ExbB-ExbD and TolA-TolQ-TolR complexes, revealing the inner membrane embedded engine parts of the Ton and Tol systems, and showing how TonB and TolA interact with the ExbBD and TolQR subcomplexes. Structural similarities between the two motor complexes suggest a common mechanism for the opening of the proton channel and the propagation of the proton motive force into movement of the TonB and TolA subunits. Because TonB and TolA bind at preferential ExbB or TolQ subunits, we propose a new mechanism of assembly of TonB and TolA with their respective ExbBD and TolQR subcomplexes and discuss its impact on the mechanism of action for the Ton and Tol systems.

Drug-resistant bacterial infections have become a high-priority concern in the global healthcare landscape. Novel treatments for such infections are needed, but can be especially difficult to develop for Gram-negative species owing to the need to traverse the outer membrane to reach targets beneath. A promising solution is found in natural antibiotics such as albomycin, which can bind outer membrane siderophore receptors and co-opt them for import into the periplasm. Exploring this and similar mechanisms may open avenues for antibiotic development. An underappreciated class of natural antibiotics are the microcins, which are small antimicrobial proteins secreted by certain bacterial species as a means of inter-species competition. Once secreted, the microcins bind specific outer-membrane receptors of prey species and cross into the periplasm. Microcins have potent activity, bind specific targets, and have been shown to control pathobiont expansion and pathogen colonization in animal models. One such microcin, MccV, has previously been shown to utilize the E. coli colicin Ia receptor, Cir, for periplasmic import. Here, we report the first high-resolution structure of the Cir/MccV complex by Cryo-EM, revealing an interaction centered on an electropositive cavity within a usually occluded binding pocket in Cir. We also report the calculated affinity of MccV for Cir. We mutagenized putative interacting residues at this interface and identified key contacts that are critical to MccV binding and Cir-mediated import and bacteriolysis. Future efforts in this project will help us better understand the mechanisms of microcin killing, and will assess relationships between other microcins and their respective importers with the aim of understanding the potential for microcins to be used as treatment for drug-resistant pathogens.

In mitochondria, β-barrel outer membrane proteins mediate protein import, metabolite transport, lipid transport, and biogenesis. The Sorting and Assembly Machinery (SAM) complex consists of three proteins that assemble as a 1:1:1 complex to fold β-barrel proteins and insert them into the mitochondrial outer membrane. We report cryoEM structures of the SAM complex from Myceliophthora thermophila, which show that Sam50 forms a 16-stranded transmembrane β-barrel with a single polypeptide-transport-associated (POTRA) domain extending into the intermembrane space. Sam35 and Sam37 are located on the cytosolic side of the outer membrane, with Sam35 capping Sam50, and Sam37 interacting extensively with Sam35. Sam35 and Sam37 each adopt a GST-like fold, with no functional, structural, or sequence similarity to their bacterial counterparts. Structural analysis shows how the Sam50 β-barrel opens a lateral gate to accommodate its substrates.

The TonB–ExbB–ExbD molecular motor harnesses the proton motive force across the bacterial inner membrane to couple energy to transporters at the outer membrane, facilitating uptake of essential nutrients such as iron and cobalamine. TonB physically interacts with the nutrient-loaded transporter to exert a force that opens an import pathway across the outer membrane. Until recently, no high-resolution structural information was available for this unique molecular motor. We published the first crystal structure of ExbB–ExbD in 2016 and showed that five copies of ExbB are arranged as a pentamer around a single copy of ExbD. However, our spectroscopic experiments clearly indicated that two copies of ExbD are present in the complex. To resolve this ambiguity, we used single-particle cryo-electron microscopy to show that the ExbB pentamer encloses a dimer of ExbD in its transmembrane pore, and not a monomer as previously reported. The revised stoichiometry has implications for motor function.

LonA proteases and ClpB chaperones are key components of the protein quality control system in bacterial cells. LonA proteases form a unique family of ATPases associated with diverse cellular activities (AAA⁺) proteins due to the presence of an unusual N‐terminal region comprised of two domains: a β‐structured N domain and an α‐helical domain, including the coiled‐coil fragment, which is referred to as HI(CC). The arrangement of helices in the HI(CC) domain is reminiscent of the structure of the H1 domain of the first AAA⁺ module of ClpB chaperones. It has been hypothesized that LonA proteases with a single AAA⁺ module may also contain a part of another AAA⁺ module, the full version of which is present in ClpB. Here, we established and tested the structural basis of this hypothesis using the known crystal structures of various fragments of LonA proteases and ClpB chaperones, as well as the newly determined structure of the Escherichia coli LonA fragment (235–584). The similarities and differences in the corresponding domains of LonA proteases and ClpB chaperones were examined in structural terms. The results of our analysis, complemented by the finding of a singular match in the location of the most conserved axial pore‐1 loop between the LonA NB domain and the NB2 domain of ClpB, support our hypothesis that there is a structural and functional relationship between two coiled–coil fragments and implies a similar mechanism of engagement of the pore‐1 loops in the AAA⁺ modules of LonAs and ClpBs.

The bacterial outer membrane contains phospholipids in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet. Both proteins and LPS must be frequently inserted into the outer membrane to preserve its integrity. The protein complex that inserts LPS into the outer membrane is called LptDE, and consists of an integral membrane protein, LptD, with a separate globular lipoprotein, LptE, inserted in the barrel lumen. The protein complex that inserts newly synthesized outer-membrane proteins (OMPs) into the outer membrane is called the BAM complex, and consists of an integral membrane protein, BamA, plus four lipoproteins, BamB, C, D and E. Recent structural and functional analyses illustrate how these two complexes insert their substrates into the outer membrane by distorting the membrane component (BamA or LptD) to directly access the lipid bilayer. This article is part of the themed issue ‘Membrane pores: from structure and assembly, to medicine and technology’.

Many G protein-coupled receptors show constitutive activity, resulting in the production of a second messenger in the absence of an agonist; and naturally occurring constitutively active mutations in receptors have been implicated in diseases. To gain insight into mechanistic aspects of constitutive activity, we report here the 3.3 Å crystal structure of a constitutively active, agonist-bound neurotensin receptor (NTSR1) and molecular dynamics simulations of agonist-occupied and ligand-free receptor. Comparison with the structure of a NTSR1 variant that has little constitutive activity reveals uncoupling of the ligand-binding domain from conserved connector residues, that effect conformational changes during GPCR activation. Furthermore, molecular dynamics simulations show strong contacts between connector residue side chains and increased flexibility at the intracellular receptor face as features that coincide with robust signalling in cells. The loss of correlation between the binding pocket and conserved connector residues, combined with altered receptor dynamics, possibly explains the reduced neurotensin efficacy in the constitutively active NTSR1 and a facilitated initial engagement with G protein in the absence of agonist.