Insights into the Role of Conformational Change, Membrane Interactions and ATP Hydrolysis in the Min Protein Regulators of Bacterial Cell Division

Abstract

The bacterial cell division regulators MinD and MinE together with the division inhibitor MinC undergo coordinated pole-to-pole oscillation to help ensure that the cytokinetic division septum forms only at the mid-cell position. This dynamic localization is driven by MinD-catalyzed ATP hydrolysis, stimulated by interactions with the MinE anti-MinCD domain. This domain is buried in the 6-β–stranded MinE “closed” structure, but is liberated for interactions with MinD, giving rise to a 4-β–stranded “open” structure. Questions remain regarding how MinE undergoes this conformational change and whether the membrane plays a role in modulating MinD and MinE structure and function. In this thesis we investigated these questions using a combination of enzyme kinetics, circular dichroism spectroscopy and solution NMR spectroscopy. We showed that membrane binding induces a structural change in MinE that resembles the open conformation. However, MinE mutants lacking the MinE membrane-targeting sequence (ΔMTS) stimulated higher ATP hydrolysis rates than the full-length protein, indicating that direct interactions between MinE and the membrane are not required to trigger this conformational transition in MinE. These results led to an updated model where MinE is brought to the membrane through interactions with MinD and remains bound to the membrane in a state that does not catalyze additional rounds of ATP hydrolysis. Solution NMR was used to study the interaction between MinE and MinD, both in the presence and absence of lipid membranes. Our results suggest that conserved MinE residues in a loop region may interact weakly with MinD before conformational change is induced in MinE. In addition, we found that the membrane charge and fluidity, but not curvature, can modulate MinD activity, with faster rates being observed for membranes containing negatively charged headgroups and alkyl chains that promote higher membrane fluidity. In contrast to results with E. coli lipids, the MinE-membrane interaction is not rate limiting when lipids are used that promote the highest MinD-catalyzed ATP hydrolysis rates. Overall, our findings have general implications for Min protein oscillation cycles, including those that regulate cell division in bacterial pathogens that could potentially be targeted in for future development of new antimicrobial compounds.