Infectious diseases are the second leading cause of death in the world. With novel classes of antibiotic drugs virtually nonexistent, and the resistance of pathogenic bacteria to current ones increasing rapidly, the develop- ment of new approaches is becoming an imperative for advancing human health efforts. Molecular modeling will play an essential role in these new approaches, due to the fundamentally atomic-scale nature of the critical structures, processes, and interactions underlying the function of both bacterial proteins and antibacterial agents. In order to illuminate these structures and processes, the PI will focus on a de?ning feature of Gram-negative bacteria, namely their second, outer membrane, and how integral membrane proteins are inserted there. These outer-membrane proteins (OMPs), practically all of which belong to a speci?c class known as -barrels, utilize two key systems for insertion: the BAM system, essential for viability, and the related TAM system, necessary for virulence. Exploiting these systems as antibacterial targets requires a comprehensive understanding of the relationship between structure, dynamics, and function. In the ?rst aim, a novel mechanism in which the key component of each system, BamA and TamA, respectively, catalyzes insertion through augmentation of its own -barrel will be evaluated. Intermediate states of insertion will be generated in molecular dynamics simulations and assayed experimentally through both disul?de cross-linking and electrophysiology measurements. In the second aim, how BamA and TamA perturb the membrane, itself an active participant in the insertion process, will be determined. Simulations have indicated the existence of a membrane defect that forms due to an unusually thin and unstable part of the -barrel of BamA; mutations to alter this perturbation, and presumably decrease OMP insertion ef?ciency, will be predicted in silico and tested in vivo. Finally, in the third aim, the dynamics of BamA and TamA as well as BamA's interactions with other BAM components will be characterized. Based on the conformational changes observed, small-molecule drug candidates will be selected that limit conformational ?exibility and/or inhibit binding of BamA or TamA to other complex members. These candidates will then be tested experimentally for antibacterial activity.

Public Health Relevance

The threat posed by bacterial infection is growing rapidly, despite efforts over nearly a century to keep it in check. This projects aims to limit its advance by revealing bacterial-speci?c structures and processes, namely the insertion of proteins to the Gram-negative bacterial outer membrane, at an unprecedented level of detail, thus enabling the next generation of rational drug design.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM123169-02
Application #
9552224
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Preusch, Peter
Project Start
2017-09-01
Project End
2022-06-30
Budget Start
2018-07-01
Budget End
2019-06-30
Support Year
2
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Georgia Institute of Technology
Department
Physics
Type
Schools of Arts and Sciences
DUNS #
097394084
City
Atlanta
State
GA
Country
United States
Zip Code
30318
Gumbart, James C; Ulmschneider, Martin B; Hazel, Anthony et al. (2018) Computed Free Energies of Peptide Insertion into Bilayers are Independent of Computational Method. J Membr Biol 251:345-356
Fu, Haohao; Gumbart, James C; Chen, Haochuan et al. (2018) BFEE: A User-Friendly Graphical Interface Facilitating Absolute Binding Free-Energy Calculations. J Chem Inf Model 58:556-560
Lundquist, Karl; Bakelar, Jeremy; Noinaj, Nicholas et al. (2018) C-terminal kink formation is required for lateral gating in BamA. Proc Natl Acad Sci U S A 115:E7942-E7949
Pavlova, Anna; Parks, Jerry M; Gumbart, James C (2018) Development of CHARMM-Compatible Force-Field Parameters for Cobalamin and Related Cofactors from Quantum Mechanical Calculations. J Chem Theory Comput 14:784-798
Gumbart, James C (2017) Producing membrane proteins one simulation at a time. J Biol Chem 292:19546-19547
Bamert, Rebecca S; Lundquist, Karl; Hwang, Hyea et al. (2017) Structural basis for substrate selection by the translocation and assembly module of the ?-barrel assembly machinery. Mol Microbiol 106:142-156