Abstract
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.
Highlights
The amino acid/polyamine/organocation (APC)2 superfamily comprises about 250 members that occur in all phyla from prokaryotes to higher eukaryotes
One APC subfamily is established by L-amino acid transporters (LATs), which correspond to the light subunits of eukaryotic heteromeric amino acid transporters [2, 3]
If substrates are amino acids, three main features can be used for specific selection and binding: (i) the negatively charged ␣-carboxyl group, (ii) the positively charged ␣-amino group, and (iii) the electrostatic, hydrophobic, or spatial properties of the side chain [22,23,24]. ␣-Carboxyl and ␣amino groups of L-amino acids possess similar structural and chemical characteristics; their side chains differ in shape, size, and electrostatic properties
Summary
The amino acid/polyamine/organocation (APC) superfamily comprises about 250 members that occur in all phyla from prokaryotes to higher eukaryotes These membrane proteins function as solute/cation symporters or solute/solute antiporters [1]. The identified first prokaryotic member of the LAT family, SteT from Bacillus subtilis, is a serine/threonine antiporter, which shows high sequence identity (ϳ30%) to the light subunits of eukaryotic heteromeric amino acid transporters. ␣-Carboxyl and ␣amino groups of L-amino acids possess similar structural and chemical characteristics (except for proline); their side chains differ in shape, size, and electrostatic properties. Combinations of these features are assumed to establish different interactions within the side chain binding pocket, which determines the substrate specificity of the transporter. The height of a force peak measures the strength of an interaction with piconewton accuracy, and the pulling distance, at which the force peak occurs, allows the interaction within the membrane protein structure to be located [38]
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