Erika A. Taylor
The ability of antibiotics to kill Gram-negative bacteria is greatly enhanced if a single heptose moiety is excluded from the biosynthesis of lipopolysaccharide (LPS), a critical component of the outer membrane. Knockouts of Heptosyltransferase I (HepI) – the enzyme that catalyzes the nucleophilic addition of heptose to the growing LPS chain – in Escherichia. coli have reduced virulence, biofilm formation, and intestinal colonization, as well as hypersensitivity to hydrophobic antibiotics, making HepI a viable target for combatting Gram-negative bacterial infection. Previous characterization of HepI demonstrated the importance of protein dynamics to catalysis, with large-scale conformational changes being partially rate-limiting. Tryptophan fluorescence studies and MD simulations suggest that the enzyme undergoes a closing motion upon substrate binding, where one or more loop rearrangements or domain movements bring the HepI substrates into proximity for reaction. However, in the absence of a fully-liganded HepI crystal structure, it is somewhat unknown what these dynamics may be and what the HepI Michaelis complex looks like. This structural information is critical to the design of allosteric inhibitors that prevent conformational dynamics and inhibit HepI.
Here, we probe HepI dynamics with two distance-sensitive fluorescence methods: tryptophan-induced quenching (TrIQ) and pyrene excimer fluorescence. Both techniques are extremely sensitive to the intramolecular distance of fluorophore-quencher (TrIQ) or fluorophore-fluorophore (excimer fluorescence) pairs. Therefore, as the distance between these molecules change, distinct spectral differences can be observed and information about conformational change(s) can be elucidated. Installation of probes at various locations within HepI can enable a more complete understanding of HepI dynamics.
To functionalize HepI with fluorophores, cysteine substitutions were made in locations hypothesized to be key for dynamics. Characterization of these mutants with kinetics and circular dichroism show WT-like activity and no major perturbations of the protein fold, suggesting minimal impacts of cysteine mutagenesis on HepI function. Initial excimer results suggest successful functionalization of HepI with pyrene, as well as the slight excimer formation for mutant C297S, H221C, R61C; however, these results need to be verified. Initial TrIQ mutants with bimane-labeled C297S, L286C suggest modest bimane quenching by W116 with ODLA (O-deacylated Lipid-A) present, consistent with a ~5 Å movement of the N-9 loop towards the C-terminal domain. This also needs to be validated using a C297S, L286C HepI mutant that is deficient in W116 to ensure that this is the residue responsible for quenching. Additionally, a series of mutants were made based upon predictions of residues anticipated to be involved in ODLA induced conformational changes. A series of arginine and lysine residues found in the N-terminal domain were mutated to alanine residues to test their impact, and the mutants were subjected to kinetic, thermodynamic and spectrophotometric analyses.
Small angle X-ray scattering (SAXS) was also explored to attempt to model the “closed” structure of HepI and experimentally verify closed models developed by MD simulations. Initial results reveal substantial scattering differences between apo and ligand-bound HepI, suggesting that SAXS is a powerful method for monitoring HepI conformational change. However, this data showed significant oligomerization of HepI which prevented the modeling of an accurate, monomeric closed structure. Future experiments using different buffer systems or size-exclusion chromatography will hopefully ameliorate these problems.
Overall these techniques serve a dual purpose. First, they are capable of monitoring conformational change with high precision, thereby localizing regions involved in HepI dynamics to enhance our understanding of the catalytically active HepI structure. Second, these techniques can be used to observe the change (or lack thereof) in dynamics due to inhibitor binding. In other words, they can assess the ability of inhibitors to perturb dynamics. Ultimately, creating a full picture of dynamics is critical to our HepI inhibition efforts. Inhibition of dynamics could prove an effective and novel inhibition method to be used in therapeutics for Gram-negative bacterial infection in an age of rampant antibiotic resistance.
Hecht, Cody J.S., "Quenchers and Excimers: Distance Mapping of Heptosyltransferase I Protein Dynamics" (2019). Masters Theses. 252.
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