Structural and Functional Characterization of the Escherichia coli MetNI Methionine Transporter

Abstract

Despite the ubiquitous role of ATP Binding Cassette (ABC) importers in nutrient uptake, only the E. coli maltose and vitamin B12 ABC transporters have been structurally characterized in multiple conformations relevant to the alternating access transport mechanism. To complement our previous structure determination of the E. coli MetNI methionine importer partner in the inward facing conformation (Kadaba et al. Science 321, 250-253, 2008), we have explored conditions stabilizing the outward facing conformation. Using two variants, the Walker B E166Q mutation with ATP and EDTA to stabilize MetNI in the ATP-bound conformation, and the N229A variant of the binding protein MetQ to disrupt methionine binding as shown in this work, a high affinity MetNIQ complex was formed with a dissociation constant measured to be 27 nM. We then solved a 2.95 A resolution crystal structure of the outward-facing conformation of the MetNI transporter, in complex with its binding protein, MetQ. The structure sheds light on how the C-regulatory domains regulate transport activity by rearrangement of a hydrogen bonding network between their interfaces in two different conformations. Structure of the substrate-free homologous MetQ from N. meningitides was also resolved using the N-to-A mutation (N238A). Superimposition of the substrate-bound, substrate-free (homologous model) MetQ and the binding protein MetQ in complex with its MetNI transporter (complexed MetQ) reveals unexpected structural features of the complexed MetQ, indicates a different substrate delivery mechanism for the MetNI transporter. These structural insights, coupled with thermodynamic binding constant and in vivo transport studies, support an unconventional transport mechanism for the Type-I methionine ABC importer.