Dynamics of a bacterial multidrug ABC transporter in the inward- and outward-facing conformations.
The study of membrane proteins remains a challenging task, and approaches to unravel their dynamics are scarce. Here, we applied hydrogen/deuterium exchange (HDX) coupled to mass spectrometry to probe the motions of a bacterial multidrug ATP-binding cassette (ABC) transporter, BmrA, in the inward-facing (resting state) and outward-facing (ATP-bound) conformations. Trypsin digestion and global or local HDX support the transition between inward- and outward-facing conformations during the catalytic cycle of BmrA. However, in the resting state, peptides from the two intracellular domains, especially ICD2, show a much faster HDX than in the closed state. This shows that these two subdomains are very flexible in this conformation. Additionally, molecular dynamics simulations suggest a large fluctuation of the Cα positions from ICD2 residues in the inward-facing conformation of a related transporter, MsbA. These results highlight the unexpected flexibility of ABC exporters in the resting state and underline the power of HDX coupled to mass spectrometry to explore conformational changes and dynamics of large membrane proteins.
Institut de Biologie Structurale,  Université Joseph Fourier Grenoble 1, 38027 Grenoble Cedex 1,  France.
Shahid
S
Mehmood
Carmen
C
Domene
Eric
E
Forest
Jean-Michel
JM
Jault
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D003902:Detergents; D041961:Deuterium Exchange Measurement; D004926:Escherichia coli / Q000235:genetics; D013058:Mass Spectrometry; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis / Q000502:physiology; D010449:Peptide Mapping; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D014357:Trypsin / Q000378:metabolism / Q000494:pharmacology
D000251:Adenosine Triphosphatases; D001426:Bacterial Proteins; D003902:Detergents; D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D014357:Trypsin; C075041:Bmr protein, Bacillus subtilis