title: The contribution of methionine to the stability of the Escherichia coli MetNIQ ABC transporter-substrate binding protein complex.
abstract: Despite the ubiquitous role of ATP-binding cassette (ABC) importers in nutrient uptake, only the Escherichia 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 in the inward facing conformation (Kadaba et al. (2008) Science 321, 250-253), we have explored conditions stabilizing the outward facing conformation. Using two variants, the Walker B E166Q mutation with ATP+EDTA to stabilize MetNI in the ATP-bound conformation and the N229A variant of the binding protein MetQ, shown in this work to disrupt methionine binding, a high affinity MetNIQ complex was formed with a dissociation constant measured to be 27 nm. Using wild type MetQ containing a co-purified methionine (for which the crystal structure is reported at 1.6 Å resolution), the dissociation constant for complex formation with MetNI is measured to be ∼40-fold weaker, indicating that complex formation lowers the affinity of MetQ for methionine by this amount. Preparation of a stable MetNIQ complex is an essential step towards the crystallographic analysis of the outward facing conformation, a key intermediate in the uptake of methionine by this transport system.
authors: Phong T
authors: PT
authors: Nguyen
authors: Qi Wen
authors: QW
authors: Li
authors: Neena S
authors: NS
authors: Kadaba
authors: Jeffrey Y
authors: JY
authors: Lai
authors: Janet G
authors: JG
authors: Yang
authors: Douglas C
authors: DC
authors: Rees
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D008715:Methionine / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D055550:Protein Stability
chemical_list: D018528:ATP-Binding Cassette Transporters; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; C466429:MetI protein, E coli; C466430:MetQ protein, E coli; D046912:Multiprotein Complexes; D008715:Methionine; D000251:Adenosine Triphosphatases; C466428:MetN protein, E coli