Nicholas Noinaj

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.

Structure of the agonist-bound neurotensin receptor

Neurotensin (NTS) is a 13-amino-acid peptide that functions as both a neurotransmitter and a hormone through the activation of the neurotensin receptor NTSR1, a G-protein-coupled receptor (GPCR). In the brain, NTS modulates the activity of dopaminergic systems, opioid-independent analgesia, and the inhibition of food intake; in the gut, NTS regulates a range of digestive processes. Here we present the structure at 2.8 Å resolution of Rattus norvegicus NTSR1 in an active-like state, bound to NTS8–13, the carboxy-terminal portion of NTS responsible for agonist-induced activation of the receptor. The peptide agonist binds to NTSR1 in an extended conformation nearly perpendicular to the membrane plane, with the C terminus oriented towards the receptor core. Our findings provide, to our knowledge, the first insight into the binding mode of a peptide agonist to a GPCR and may support the development of non-peptide ligands that could be useful in the treatment of neurological disorders, cancer and obesity.

Structural insight into the biogenesis of β-barrel membrane proteins

β-barrel membrane proteins are essential for nutrient import, signalling, motility and survival. In Gram-negative bacteria, the β-barrel assembly machinery (BAM) complex is responsible for the biogenesis of β-barrel membrane proteins, with homologous complexes found in mitochondria and chloroplasts. Here we describe the structure of BamA, the central and essential component of the BAM complex, from two species of bacteria: Neisseria gonorrhoeae and Haemophilus ducreyi. BamA consists of a large periplasmic domain attached to a 16-strand transmembrane β-barrel domain. Three structural features shed light on the mechanism by which BamA catalyses β-barrel assembly. First, the interior cavity is accessible in one BamA structure and conformationally closed in the other. Second, an exterior rim of the β-barrel has a distinctly narrowed hydrophobic surface, locally destabilizing the outer membrane. And third, the β-barrel can undergo lateral opening, suggesting a route from the interior cavity in BamA into the outer membrane.

Structural basis for iron piracy by pathogenic Neisseria

Neisseria are obligate human pathogens causing bacterial meningitis, septicaemia and gonorrhoea. Neisseria require iron for survival and can extract it directly from human transferrin for transport across the outer membrane. The transport system consists of TbpA, an integral outer membrane protein, and TbpB, a co-receptor attached to the cell surface; both proteins are potentially important vaccine and therapeutic targets. Two key questions driving Neisseria research are how human transferrin is specifically targeted, and how the bacteria liberate iron from transferrin at neutral pH. To address these questions, we solved crystal structures of the TbpA–transferrin complex and of the corresponding co-receptor TbpB. We characterized the TbpB–transferrin complex by small-angle X-ray scattering and the TbpA–TbpB–transferrin complex by electron microscopy. Our studies provide a rational basis for the specificity of TbpA for human transferrin, show how TbpA promotes iron release from transferrin, and elucidate how TbpB facilitates this process.

The structural biology of β-barrel membrane proteins: a summary of recent reports

The outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts all contain transmembrane β-barrel proteins. These β-barrel proteins serve essential functions in cargo transport and signaling and are also vital for membrane biogenesis. They have also been adapted to perform a diverse set of important cellular functions including acting as porins, transporters, enzymes, virulence factors and receptors. Recent structures of transmembrane β-barrels include that of a full length autotransporter (EstA), a bacterial heme transporter complex (HasR), a bacterial porin in complex with several ligands (PorB), and the mitochondrial voltage-dependent anion channel (VDAC) from both mouse and human. These represent only a few of the interesting structures of β-barrel membrane proteins recently elucidated. However, they demonstrate many of the advancements made within the field of transmembrane protein structure in the past few years.

Lateral opening and exit pore formation are required for BamA function.

The outer membrane of Gram-negative bacteria is replete with a host of β-barrel outer membrane proteins (OMPs). Despite serving a variety of essential functions, including immune response evasion, the exact mechanism of OMP folding and membrane insertion remains largely unclear. The β-barrel assembly machinery complex is required for OMP biogenesis. Crystal structures and molecular dynamics (MD) simulations of the central and essential component, BamA, suggest a mechanism involving lateral opening of its barrel domain. MD simulations reported here reveal an additional feature of BamA: a substrate exit pore positioned above the lateral opening site. Disulfide crosslinks that prevent lateral opening and exit pore formation result in a loss of BamA function, which can be fully rescued by the reductant tris(2-carboxyethyl)phosphine. These data provide strong evidence that lateral opening and exit pore formation are required for BamA function.

The structure of the β-barrel assembly machinery complex

Going in with a BAM Integral membrane proteins in bacterial outer membranes play roles in nutrient import and infectivity. These proteins are folded into a barrel shape composed of β-strands and inserted into the outer membrane by the β-barrel assembly machinery (BAM) complex. Bakelar et al. determined the crystal structure of a four-component BAM subcomplex. The structure of a central β barrel in BAM changes in the presence of the accessory components to create a lateral opening that may be involved in how BAM inserts proteins into the outer membrane. Science, this issue p. 180 A complex in the outer membrane of Gram-negative bacteria inserts other outer membrane proteins sideways. β-Barrel outer membrane proteins (OMPs) are found in the outer membranes of Gram-negative bacteria and are essential for nutrient import, signaling, and adhesion. A 200-kilodalton five-component complex called the β-barrel assembly machinery (BAM) complex has been implicated in the biogenesis of OMPs. We report the structure of the BAM complex from Escherichia coli, revealing that binding of BamCDE modulates the conformation of BamA, the central component, which may serve to regulate the BAM complex. The periplasmic domain of BamA was in a closed state that prevents access to the barrel lumen, which indicates substrate OMPs may not be threaded through the barrel during biogenesis. Further, conformational shifts in the barrel domain lead to opening of the exit pore and rearrangement at the lateral gate.

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.

Crystal Structure of Baeyer−Villiger Monooxygenase MtmOIV, the Key Enzyme of the Mithramycin Biosynthetic Pathway,

Baeyer-Villiger monooxygenases (BVMOs), mostly flavoproteins, were shown to be powerful biocatalysts for synthetic organic chemistry applications and were also suggested to play key roles for the biosyntheses of various natural products. Here we present the three-dimensional structure of MtmOIV, a 56 kDa homodimeric FAD- and NADPH-dependent monooxygenase, which catalyzes the key frame-modifying step of the mithramycin biosynthetic pathway and currently the only BVMO proven to react with its natural substrate via a Baeyer-Villiger reaction. MtmOIVs structure was determined by X-ray crystallography using molecular replacement to a resolution of 2.9 A. MtmOIV cleaves a C-C bond, essential for the conversion of the biologically inactive precursor, premithramycin B, into the active drug mithramycin. The MtmOIV structure combined with substrate docking calculations and site-directed mutagenesis experiments identifies several residues that participate in cofactor and substrate binding. Future experimentation aimed at broadening the substrate specificity of the enzyme could facilitate the generation of chemically diverse mithramycin analogues through combinatorial biosynthesis.

The β-barrel membrane protein insertase machinery from Gram-negative bacteria

The outer membranes (OM) of Gram-negative bacteria contain a host of β-barrel outer membrane proteins (OMPs) which serve many functions for cell survival and virulence. The biogenesis of these OMPs is mediated by the β-barrel assembly machinery (BAM) complex which is composed of five components including the essential core component called BamA that mediates the insertase function within the OM. The crystal structure of BamA has recently been reported from three different species, including a full-length structure from Neisseria gonorrhoeae. Mutagenesis and functional studies identified several conformational changes within BamA that are required for function, providing a significant advancement towards unraveling exactly how BamA and the BAM complex are able to fold and insert new OMPs in the OM.