Mechanisms of Substrate Selectivity and Transport by a Bacterial Methionine ABC Importer

Abstract

ABC transporters are central in many cellular functions including nutrient uptake, signal transduction membrane assembly, and cellular detoxification. Both structural and functional studies have revealed insights into the high‐affinity uptake mechanism of the MetNI methionine ABC transporter. Using the energy from ATP binding and hydrolysis, the MetNI‐Q system can import L‐Met, D‐Met, and other methionine derivatives against concentration gradients. Many mechanistic studies of ABC transporter propose a model in which cognate binding proteins sequester substrates in the periplasm and deliver them to the transporter. In contrast, recent in vivo and crystallographic studies of MetNI‐Q suggest that some substrates may be able to access the transporter through a solvent accessible tunnel in the cognate binding protein‐transporter complex. These studies suggest that the substrate delivery to MetNI transporter may be more flexible than other ABC transport systems.To test if the MetNI‐Q system transport system uses two mechanisms of substrate delivery, we designed a series of experiments to dissect the individual steps in the transport cycle. As the starting point, we have developed a fluorescent anisotropy assay to measure the binding affinity between the MetQ substrate‐binding protein and the MetNI transporter in the presence of different methionine derivatives. Our preliminary data show that MetNI binds to apo‐MetQ ~10 fold tighter than L‐Met liganded MetQ, and additionally may differ in the presence of D‐Met. This suggests that different methionine derivatives may utilize discrete transport models.