Substrate-dependent unfolding of the energy coupling motif of a membrane transport protein determined by double electron-electron resonance.
BtuB is a TonB-dependent transport protein that binds and carries vitamin B(12) across the outer membrane of Gram negative bacteria such as Escherichia coli. Previous work has demonstrated that the Ton box, a highly conserved segment near the N-terminus of the protein, undergoes an order-to-disorder transition upon the binding of substrate. Here, we incorporate pairs of nitroxide spin labels into membrane reconstituted BtuB and utilize a four-pulse double electron-electron resonance (DEER) experiment to measure distances between the Ton box and the periplasmic surface of the transporter with and without substrate. During reconstitution, the labeled membrane protein was diluted with wild-type protein, which significantly reduced the intermolecular electron spin-spin relaxation rate and increased the DEER signal-to-noise ratio. In the absence of substrate, each spin pair gives rise to a single distribution of distances that is consistent with the crystal structure obtained for BtuB; however, distances that are much longer are found in the presence of substrate, and the data are consistent with the existence of an equilibrium between folded and unfolded states of the Ton box. From these distances, a model for the position of the Ton box was constructed, and it indicates that the N-terminal end of the Ton box extends approximately 20 to 30 A into the periplasm upon the addition of substrate. We propose that this substrate-induced extension provides the signal that initiates interactions between BtuB and the inner membrane protein TonB.
Xu
Qi
Q
Department of Chemistry and Biophysics Program, University of Virginia, Charlottesville, Virginia 22904-4319, USA.
Ellena
Jeffrey F
JF
Kim
Miyeon
M
Cafiso
David S
DS
D020816:Amino Acid Motifs; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008280:Magnetics; D008565:Membrane Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D019897:Periplasm / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D013113:Spin Labels; D014805:Vitamin B 12 / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; C044592:BtuB protein, E coli; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D013113:Spin Labels; C489181:tonB protein, E coli; D014805:Vitamin B 12