TY - JOUR
T1 - A Conserved Motif in Intracellular Loop 1 Stabilizes the Outward-Facing Conformation of TmrAB
AU - Millan, Cinthia R.
AU - Francis, Martina
AU - Khandelwal, Nitesh Kumar
AU - Thompson, Valery F.
AU - Thaker, Tarjani M.
AU - Tomasiak, Thomas M.
N1 - Funding Information:
We thank members of the Tomasiak lab and Prof. Wolfgang Peti for helpful discussions and critical reading of this manuscript. Negative-stain images were collected at the Life Sciences North Imaging Facility at the University of Arizona supported by funding provided by the NIH (S10 OD011981). We also thank Olivier Lichtarge and Panos Katsonis for technical assistance with Evolutionary Trace methods, and Kelly Brock for assistance with Evolutionary Couplings analysis. This work was supported by grants from the National Institute of General Medicine Sciences to T.M. Tomasiak (R00 GM114245).
Publisher Copyright:
© 2021 Elsevier Ltd
PY - 2021/8/6
Y1 - 2021/8/6
N2 - 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.
AB - 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.
KW - ABC transporter
KW - ATPase
KW - Conformational coupling
KW - Exporter
KW - Transport cycle
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U2 - 10.1016/j.jmb.2021.166834
DO - 10.1016/j.jmb.2021.166834
M3 - Article
C2 - 33524413
AN - SCOPUS:85101001961
VL - 433
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 16
M1 - 166834
ER -