Molecular basis for substrate-dependent transmembrane signaling in an outer-membrane transporter.
Transmembrane signaling events that propagate through receptors and transporters have critical roles in cellular function and regulation. In the Escherichia coli vitamin B(12) transporter, BtuB, substrate binding to the extracellular surface of the protein triggers the unfolding of an energy coupling motif at the periplasmic surface. Here, the molecular interactions mediating this substrate-dependent transmembrane signaling event were investigated in a novel way by combining a two mutant cycle analysis with site-directed spin labeling (SDSL). SDSL was used to monitor the unfolding and conformational equilibrium of the energy-coupling motif, and a thermodynamic two-mutant cycle analysis was used to estimate pair-wise interaction free energies for a pair of charged residues (D316 and R14) within the protein interior. The data indicate that D316 and R14 are critical to this structural transition. Substrate binding is shown to reduce the interaction free energy between these residues, thereby triggering the unfolding of the energy coupling motif of this membrane transporter. The result indicates that SDSL when used in combination with a mutant cycle analysis provides an approach to examine the molecular interactions mediating signaling events in membrane proteins.
Lukasik
Stephen M
SM
Chemistry and Biophysics Programs, Department of Chemistry, University of Virginia, Charlottesville, VA 22904-4319, USA.
Ho
K W David
KW
Cafiso
David S
DS
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D017510:Protein Folding; D015398:Signal Transduction; D013113:Spin Labels; D013379:Substrate Specificity; D014805:Vitamin B 12 / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; C044592:BtuB protein, E coli; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D013113:Spin Labels; D014805:Vitamin B 12