of the original project Antibiotic resistance is a major threat to public health. An important mechanism of resistance to antibiotics such as tetracyclines is efflux mediated by membrane transporter proteins. The structural basis for substrate recognition by tetracycline efflux pumps, Tet proteins, is unknown. The efflux pump TetL from Bacillus subtilis exports tetracycline (Tc) in the form of a tetracycline-magnesium [Tc.Mg2+]+ complex, and is responsible for this bacteriums resistance to the once widely efficacious antibiotic. TetL, with 14 transmembrane ?-helices, is a member of the family of Tet efflux proteins in Gram-positive bacterial pathogens, including Bacillus anthracis, Bacillus cereus, Streptococcus pneumoniae, Staphylococcus aureus. Clostridium spp., Enterococcus spp. and Listeria spp. All Tet transporters belong to the major facilitator superfamily (MFS). No crystal structure is available for any Tet protein or any MFS protein with 14 helices. A crystal structure of TetL, in combination with biochemical and biophysical studies, will not only greatly advance our understanding of the molecular mechanism of efflux-mediated antibiotic resistance, it will also suggest new ways to modify tetracycline to reverse resistance. 2. Major setback due to Sandy We will use nanobodies to improve diffraction resolution. Our experience with antibodies allowed us to rapidly progress in the evaluation of nanobodies produced through a collaboration with the Steyaert Lab in Brussels, Belgium. We have generated a panel of 18 nanobodies that recognize purified TetL. These nanobodies can be readily made and purified from bacterial hosts, providing a continuous supply of nanobodies for structural and functional studies. Of these, 12 produce a stable complex that has been used in co-crystallization studies. Crystals have been grown for 9 of these, with the best resolution reaching 4-5 . These TetL/nanobody complexes are being further optimized to improve diffraction resolution. Additionally, the nanobodies are being combined to form heteromeric complexes to increase the available crystallization space in order to facilitate higher resolution diffraction. These nanobodies may also bind to preferential conformations of TetL, which may structurally shed light on the conformational changes during the tetracycline transport cycle.

Public Health Relevance

Antibiotic resistance is a major threat to public health. The major mechanism of resistance to antibiotics like tetracyclines is efflux mediated by membrane transporter proteins. The structural basis for substrate recognition by such efflux proteins is lacking. The efflux pump TetL from Bacillus subtilis exports tetracycline in the form of tetracycline-magnesium complex; and is responsible for this bacterium's resistance to the once widely efficacious antibiotic. A crystal structure of TetL; in combination with biochemical and biophysical studies; not only will greatly advance our understanding of the molecular mechanism of antibiotic resistance; it will also suggest new ways to modify tetracycline to reverse resistance.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM093825-04S1
Application #
8663548
Study Section
Special Emphasis Panel (ZRG1 (03))
Program Officer
Chin, Jean
Project Start
2014-01-01
Project End
2015-12-31
Budget Start
2014-01-01
Budget End
2015-12-31
Support Year
4
Fiscal Year
2014
Total Cost
$101,700
Indirect Cost
$41,700
Name
New York University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
121911077
City
New York
State
NY
Country
United States
Zip Code
10016
Mulligan, Christopher; Fitzgerald, Gabriel A; Wang, Da-Neng et al. (2014) Functional characterization of a Na+-dependent dicarboxylate transporter from Vibrio cholerae. J Gen Physiol 143:745-59
Morino, Masato; Suzuki, Toshiharu; Ito, Masahiro et al. (2014) Purification and functional reconstitution of a seven-subunit mrp-type na+/h+ antiporter. J Bacteriol 196:28-35
Waight, Andrew B; Czyzewski, Bryan K; Wang, Da-Neng (2013) Ion selectivity and gating mechanisms of FNT channels. Curr Opin Struct Biol 23:499-506
Wang, Da-Neng; Stieglitz, Heather; Marden, Jennifer et al. (2013) Benjamin Franklin, Philadelphia's favorite son, was a membrane biophysicist. Biophys J 104:287-91
Karpowich, Nathan K; Wang, Da-Neng (2013) Assembly and mechanism of a group II ECF transporter. Proc Natl Acad Sci U S A 110:2534-9
Czyzewski, Bryan K; Wang, Da-Neng (2012) Identification and characterization of a bacterial hydrosulphide ion channel. Nature 483:494-7
Mancusso, Romina; Karpowich, Nathan K; Czyzewski, Bryan K et al. (2011) Simple screening method for improving membrane protein thermostability. Methods 55:324-9
Karpowich, Nathan K; Wang, Da-Neng (2010) Biophysics: Transporter in the spotlight. Nature 465:171-2