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Structural and Functional Characterization of the Escherichia coli MetNI Methionine Transporter


Nguyen, Phong Thanh (2018) Structural and Functional Characterization of the Escherichia coli MetNI Methionine Transporter. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/x9ed-9n64.


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 Å 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.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:ABC transporter, methionine transporter, methionine-binding protein, MetNI, MetQ, L-methionine, transinhibition, outward-facing conformati
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Biochemistry and Molecular Biophysics
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Rees, Douglas C.
Thesis Committee:
  • Beauchamp, Jesse L. (chair)
  • Shan, Shu-ou
  • Clemons, William M.
  • Rees, Douglas C.
Defense Date:19 January 2018
Non-Caltech Author Email:ptnguyen.cit (AT)
Funding AgencyGrant Number
Vietnam International Educational DevelopmentUNSPECIFIED
Record Number:CaltechTHESIS:04302018-143201092
Persistent URL:
Related URLs:
URLURL TypeDescription adapted for Chapter 2 adapted for a part of Chapter 3
Nguyen, Phong Thanh0000-0002-6390-7350
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:10840
Deposited By: Phong Nguyen
Deposited On:11 May 2018 21:50
Last Modified:04 Oct 2019 00:20

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