Discovery of the Membrane Binding Domain in Trifunctional Proline Utilization A

<i>Escherichia coli</i> proline utilization A (<i>Ec</i>PutA) is the archetype of trifunctional PutA flavoproteins, which function both as regulators of the proline utilization operon and bifunctional enzymes that catalyze the four-electron oxidation of proline to glutamate. <i>Ec</i>PutA shifts from a self-regulating transcriptional repressor to a bifunctional enzyme in a process known as functional switching. The flavin redox state dictates the function of <i>Ec</i>PutA. Upon proline oxidation, the flavin becomes reduced, triggering a conformational change that causes <i>Ec</i>PutA to dissociate from the <i>put</i> regulon and bind to the cellular membrane. Major structure/function domains of <i>Ec</i>PutA have been characterized, including the DNA-binding domain, proline dehydrogenase (PRODH) and l-glutamate-γ-semialdehyde dehydrogenase catalytic domains, and an aldehyde dehydrogenase superfamily fold domain. Still lacking is an understanding of the membrane-binding domain, which is essential for <i>Ec</i>PutA catalytic turnover and functional switching. Here, we provide evidence for a conserved C-terminal motif (CCM) in <i>Ec</i>PutA having a critical role in membrane binding. Deletion of the CCM or replacement of hydrophobic residues with negatively charged residues within the CCM impairs <i>Ec</i>PutA functional and physical membrane association. Furthermore, cell-based transcription assays and limited proteolysis indicate that the CCM is essential for functional switching. Using fluorescence resonance energy transfer involving dansyl-labeled liposomes, residues in the α-domain are also implicated in membrane binding. Taken together, these experiments suggest that the CCM and α-domain converge to form a membrane-binding interface near the PRODH domain. The discovery of the membrane-binding region will assist efforts to define flavin redox signaling pathways responsible for <i>Ec</i>PutA functional switching