Structural and biophysical characterization of the tandem substrate-binding domains of the ABC importer GlnPQ

Evelyn Plötz, Geesina Schuurman-Wolters, Niels Zijlstra, Amarins Jager, Douglas Griffith, Albert Guskov, Giorgos Gouridis, Berend Poolman*, Thorben Cordes*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

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Abstract

The ATP-binding cassette transporter GlnPQ is an essential uptake system that transports glutamine, glutamic acid and asparagine in Gram-positive bacteria. It features two extra-cytoplasmic substrate-binding domains (SBDs) that are linked in tandem to the transmembrane domain of the transporter. The two SBDs differ in their ligand specificities, binding affinities and their distance to the transmembrane domain. Here, we elucidate the effects of the tandem arrangement of the domains on the biochemical, biophysical and structural properties of the protein. For this, we determined the crystal structure of the ligand-free tandem SBD1-2 protein from Lactococcus lactis in the absence of the transporter and compared the tandem to the isolated SBDs. We also used isothermal titration calorimetry to determine the ligand-binding affinity of the SBDs and single-molecule Förster resonance energy transfer (smFRET) to relate ligand binding to conformational changes in each of the domains of the tandem. We show that substrate binding and conformational changes are not notably affected by the presence of the adjoining domain in the wild-type protein, and changes only occur when the linker between the domains is shortened. In a proof-of-concept experiment, we combine smFRET with protein-induced fluorescence enhancement (PIFE–FRET) and show that a decrease in SBD linker length is observed as a linear increase in donor-brightness for SBD2 while we can still monitor the conformational states (open/closed) of SBD1. These results demonstrate the feasibility of PIFE–FRET to monitor protein–protein interactions and conformational states simultaneously.
Original languageEnglish
Article number200406
Number of pages16
JournalOpen Biology
Volume11
Issue number4
DOIs
Publication statusPublished - 7-Apr-2021

Keywords

  • substrate-binding protein
  • single-molecule spectroscopy
  • ABC transporter
  • tandem substrate-binding domains
  • Forster resonance energy transfer
  • protein-induced fluorescence enhancement

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