A Conserved Motif in Intracellular Loop 1 Stabilizes the Outward-Facing Conformation of TmrAB

Cinthia R. Millan, Martina Francis, Nitesh Kumar Khandelwal, Valery F. Thompson, Tarjani M. Thaker, Thomas M. Tomasiak

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

The ATP binding cassette (ABC) family of transporters moves small molecules (lipids, sugars, peptides, drugs, nutrients) across membranes in nearly all organisms. Transport activity requires conformational switching between inward-facing and outward-facing states driven by ATP-dependent dimerization of two nucleotide binding domains (NBDs). The mechanism that connects ATP binding and hydrolysis in the NBDs to conformational changes in a substrate binding site in the transmembrane domains (TMDs) is currently an outstanding question. Here we use sequence coevolution analyses together with biochemical characterization to investigate the role of a highly conserved region in intracellular loop 1 we define as the GRD motif in coordinating domain rearrangements in the heterodimeric peptide exporter from Thermus thermophilus, TmrAB. Mutations in the GRD motif alter ATPase activity as well as transport. Disulfide crosslinking, evolutionary trace, and evolutionary coupling analysis reveal that these effects are likely due to the destabilization of a network in which the GRD motif in TmrA bridges residues of the Q-loop, X-loop, and ABC motif in the NBDs to residues in the TmrAB peptide substrate binding site, thus providing an avenue for conformational coupling. We further find that disruption of this network in TmrA versus TmrB has different functional consequences, hinting at an intrinsic asymmetry in heterodimeric ABC transporters extending beyond that of the NBDs. These results support a mechanism in which the GRD motifs help coordinate a transition to an outward open conformation, and each half of the transporter likely plays a different role in the conformational cycle of TmrAB.

Original languageEnglish (US)
Article number166834
JournalJournal of Molecular Biology
Volume433
Issue number16
DOIs
StatePublished - Aug 6 2021
Externally publishedYes

Keywords

  • ABC transporter
  • ATPase
  • Conformational coupling
  • Exporter
  • Transport cycle

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Molecular Biology

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