Erik Thiede, Ph.D.

Tuberculosis (TB) remains a major global health problem. Mtb Phosphopantetheinyl Transferase (PptT), is an essential enzyme for Mtb growth and survival, making it an attractive candidate for inhibitor design. However, inhibitor design is complicated by our lack of understanding of PptT’s structural biology. PptT exhibits significant structural heterogeneity. While crystal structures of PptT with Coenzyme A (CoA) bound exist, researchers have been unable to crystallize the protein in its apo state, potentially suggesting a different conformational ensemble. Moreover, our preliminary molecular dynamics (MD) simulations have shown considerable domain motions. These findings suggest the existence of multiple conformational states that could influence inhibitor binding, either by acting as new targets for rational inhibitor design or by affecting the entropic contribution to the free energy of inhibitor binding. We will perform a detailed investigation into the conformational heterogeneity of PptT. Using MD simulations, we can observe possible conformations and predict their relative free energies. First, we will study domain motion in the N-terminal domain of PptT, with a specific focus on the phosphoadenosine phosphate (PAP) binding loop. Second, we will study conformational change in the phosphopantetheine (ppt) binding tunnel as well as the energetics of ppt binding and unbinding. To quantitatively characterize the free energies associated with the PptT’s conformational changes, we will employ an enhanced sampling algorithm known as Replica Exchange Umbrella Sampling (REUS). We will then confirm predictions from simulation using in vitro studies of rPptT, as well as experiments comparing wild-type and mutant Mtb strains. Together, this research will deepen our understanding of the chemical biology of PptT and provide a foundation for future drug discovery efforts targeting Mtb.