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The translocon at the outer membrane of chloroplasts (TOC)

Protein trafficking across membranes is an essential function in cells; however, the exact mechanism for how this occurs is not well understood. In the endosymbionts, mitochondria and chloroplasts, the vast majority of proteins are synthesized in the cytoplasm as preproteins and then imported into the organelles via specialized machineries. In chloroplasts, protein import is accomplished by the TOC(translocon on the outer chloroplast membrane) and TIC (translocon on the inner chloroplast membrane) machineries in the outer and inner envelope membranes, respectively. TOC mediates initial recognition of preproteins at the outer membrane and includes a core membrane channel, Toc75, and two receptor proteins, Toc33/34 and Toc159, each containing GTPase domains that control preprotein binding and translocation. Toc75 is predicted to have a β-barrel fold consisting of an N-terminal intermembrane space (IMS) domain and a C-terminal 16-stranded β-barrel domain. Progress in the field has been hindered by the lack of structural information on the Toc proteins. Our goal is to use X-ray crystallography and cryoEM to structurally characterize the TOC complex.

The β-barrel assembly machinery (BAM) complex

The β-barrel assembly machinery (BAM) is a multicomponent complex responsible for the biogenesis of β-barrel outer membrane proteins (OMPs) in Gram-negative bacteria, with conserved systems in both mitochondria and chloroplasts. Given its importance in the integrity of the outer membrane and in the assembly of surface exposed virulence factors, BAM is an attractive therapeutic target against pathogenic bacteria, particularly multidrug-resistant strains. While the mechanism for how BAM functions remains elusive, previous structural studies have described each of the individual components of BAM, offering only a few clues to how the complex functions. Recently, a number of structures have been reported of complexes, including that of fully assembled BAM in differing conformational states. These studies have provided the molecular blueprint detailing the atomic interactions between the components and have revealed new details about BAM, which suggest a dynamic mechanism that may use conformational changes to assist in the biogenesis of new OMPs. Our goal is to characterize the mechanism by which BAM mediate the biogenesis of new OMPs and to use this information to target BAM for antibiotic discovery against pathogenic bacteria.

Targeting metal import systems in pathogenic bacteria


Neisseria are Gram-negative bacteria with two species, Neisseria meningitidis and Neisseria gonorrhoeae, being obligate human pathogens that are serious threats to human health both in the US and worldwide. N. meningitidis infections lead to septicemia and meningitis, while N. gonorrhoeae leads to the sexually transmitted disease gonorrhea, the second most reported infectious disease (820,000 new cases each year). While vaccines are currently available for all five pathogenic serogroups of N. meningitidis, no vaccine has yet been developed against N. gonorrhoeae. And while antibiotics such as ceftriaxone and azithromycin are most often successfully used to treat infections, the recent discovery of multidrug and cephalosporin-resistant strains has led to the CDC now assigning N. gonorrhoeae its highest threat level of ‘Urgent’. To combat multidrug resistance in Neisseria, there is an immediate need to discover and develop new and improved antibiotics. A promising approach for antibiotic discovery is to target important surface proteins that assist in mediating Neisserial pathogenesis. Like all life, pathogenic Neisseria require iron for survival. And since free iron in the human host is essential non-existent, Neisseria have evolved systems to pirate iron from human iron-carrying proteins such as transferrin and lactoferrin. Two surface protein systems that have been recently targeted in Neisseria for both antibiotic discovery and vaccine development are the transferrin binding proteins (Tbp) and the lactoferrin binding proteins (Lbp). Our goal is to structurally and functionally characterize these systems, and others, in order to target them for novel antibiotic discovery efforts.

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