Substrate Binding Tunes Conformational Flexibility and Kinetic Stability of an Amino Acid Antiporter

• Published in: The Journal of biological chemistry Vol. 284; no. 28; pp. 18651 - 18663
• Main Authors: Bippes, Christian A., Zeltina, Antra, Casagrande, Fabio, Ratera, Merce, Palacin, Manuel, Muller, Daniel J., Fotiadis, Dimitrios
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 10.07.2009; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acids - chemistry; Antiporters - chemistry; Antiporters - metabolism; Bacillus subtilis - metabolism; Bacterial Proteins - chemistry; Binding Sites; Biological Transport; Biophysics - methods; Cloning, Molecular; Ion Transport; Kinetics; Membrane Transport, Structure, Function, and Biogenesis; Molecular Conformation; Substrate Specificity; Thermodynamics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M109.004267

We used single molecule dynamic force spectroscopy to unfold individual serine/threonine antiporters SteT from Bacillus subtilis. The unfolding force patterns revealed interactions and energy barriers that stabilized structural segments of SteT. Substrate binding did not establish strong localized interactions but appeared to be facilitated by the formation of weak interactions with several structural segments. Upon substrate binding, all energy barriers of the antiporter changed thereby describing the transition from brittle mechanical properties of SteT in the unbound state to structurally flexible conformations in the substrate-bound state. The lifetime of the unbound state was much shorter than that of the substrate-bound state. This leads to the conclusion that the unbound state of SteT shows a reduced conformational flexibility to facilitate specific substrate binding and a reduced kinetic stability to enable rapid switching to the bound state. In contrast, the bound state of SteT showed an increased conformational flexibility and kinetic stability such as required to enable transport of substrate across the cell membrane. This result supports the working model of antiporters in which alternate substrate access from one to the other membrane surface occurs in the substrate-bound state.