| dc.description.abstract | Many biological processes are characterized by transient and weak interactions. Nuclear Magnetic Resonance (NMR) is a powerful tool to characterize these interactions on a per-residue basis. In this dissertation, I have utilized NMR spectroscopy to characterize protein-protein and protein-small molecule interactions of different proteins of the type III secretion system (T3SS) in pathogenic Gram-negative bacteria. Pathogenic Gram-negative bacteria such as Salmonella, Shigella, Burkholderia, Pseudomonas and Yersinia assemble the T3SS to inject virulence proteins into eukaryotic cells, causing millions of deaths worldwide. A major public health concern is the development of antibiotic resistance among the Gram-negative pathogens. This problem is exacerbated by the low number of new antibiotics in the pipeline. Because of its essential role in virulence, understanding the assembly of the T3SS can lead to the development of new antimicrobials. The structural component of the T3SS is the needle complex, which consists of a base, an extracellular needle, a tip, and a translocon. The translocon forms a pore on the host cell membrane to allow the injection of virulence proteins into the host cell. The two membrane proteins that assemble the translocon – the major translocon protein and the minor translocon protein – are designated based on their relative sizes. The minor translocon proteins from various bacteria are predicted to contain one transmembrane domain. IpaC is the minor translocon protein of Shigella, and plays an essential role in pathogenesis. The atomic structure for any minor translocon protein and its conformational changes in membrane mimetics and micelles are currently unknown. The protein-protein interactions of IpaC are also not well understood. In this dissertation, I show by NMR that full-length IpaC, as well as its N-terminal domain (NTD) and C terminal domain (CTD), are intrinsically disordered. NMR titrations show that IpaC and its NTD and CTD bind to the chaperone protein IpgC and the tip protein IpaD. Further, the IpaC CTD interacts with the N-terminal domain of the major translocon protein IpaB. IpaC is known to oligomerize, and NMR detected homotypic protein-protein interactions of the IpaC CTD. My results show the first NMR spectrum for any minor translocon protein, identify previously unknown binding partners of IpaC, and provide possible insights into the mechanism of oligomerization of IpaC. Circular dichroism (CD) spectroscopy and NMR also show that full-length IpaC and its domains undergo conformational changes in DPC, LMPG and SDS micelles. This knowledge is important in the biophysical characterization of IpaC. The tip protein in Salmonella is SipD, which is exposed on the cell surface and is essential in virulence. This makes SipD an attractive target for developing new antimicrobials. Currently, the only known small molecules that bind to SipD are bile salts and small molecule compounds based on hydroxyindole, morpholinoaniline and indole-acetic acid scaffolds. Results of the computational screening of compound libraries for binding to tip proteins is also currently unknown. In this dissertation, I report the results of our computational screening of a compound library containing ~ 8.33 million compounds using Rosetta and ROCS/FastROCS for binding to SipD. The screening predicted the binding of three compounds to SipD, based on piperidine, piperazine and phenyl scaffolds. Through a collaboration, we also used surface plasmon resonance (SPR) to screen one small molecule library containing 104 compounds with structures similar to known antibiotics, and another library consisting of 440 compounds with structures similar to intermediates in the synthesis of larger drug-like candidates. We identified the binding of three small molecules based on quinoline and phenyl scaffolds using the SPR screen. I then used NMR to identify the residues and surfaces of SipD that interact with these small molecules. This knowledge increases the number of known fragments that can bind to SipD, and can be used to screen other virulence proteins from the T3SS, aiding in designing novel therapeutics to combat the growing threat posed by multidrug resistance in bacteria. My dissertation illustrates the strength of NMR spectroscopy to characterize weak and transient protein-ligand interactions in the T3SS. | |
