Transmembrane glutamic acid residues play essential roles in the metal‐tetracycline/H⁺ antiporter of Staphylococcus aureus

Abstract

; however, it has no transmembrane aspartyl residue. On the other hand, Tet(K) has three glutamyl residues, Glu-30, Glu-152 and Glu-397, in the putative transmembrane regions. In the present work, tet(K) gene was expressed in Escherichia coli and the transport activity was measured in everted membrane vesicles. When these glutamyl residues were replaced with Gin, the tetracycline transport activity was almost completely lost, indicating the important roles of these residues in Tet(K). In the case of Glu-397, even the charge-conserved mutation to Asp caused complete loss of the activity. On the other hand, the mutation of Glu-30 and Glu-152 to Asp resulted in significant retention of transport activity. These results are similar to those on the mutation of the three transmemhrane aspartyl residues in Tet(B), indicating that the transmembrane glutamyl residues in Tet(K) play roles similar to those of the transmemhrane aspartyl residues in Tet(B).

Tet(L) and Tet(K) Tetracycline-Divalent Metal/H + Antiporters: Characterization of Multiple Catalytic Modes and a Mutagenesis Approach to Differences in Their Efflux Substrate and Coupling Ion Preferences

“…It has been suggested that the 14-TMS Tet(L) and Tet(K) proteins and the 12-TMS Tet(B) protein have a three-dimensionally similar "catalytic core" that includes three carboxylates in distinct TMS regions (11). Recent mutagenesis studies of Tet(L) supported the notion of such a common set of crucial acidic residues (27).…”

“…Complete resolution of these major questions will require high-resolution structural data in addition to more detailed studies of binding and transport kinetics and more extensive mutagenesis studies in combination with approaches to provide additional structural information. However, the kinetic work and mutational work in this and other recent studies of Tet(L) and Tet(K) (11,13,(25)(26)(27), taken together, do provide information that is relevant to these important issues.…”

“…1). It includes an essential glutamate residue (E152) in Tet(L) and Tet(K) (11,27) and the consensus sequence GX 8 GX 3 GPX 2 GG. This consensus sequence is associated with antiporters, as opposed to symporters or uniporters; thus, motif C was hypothesized by others to be involved in the specific translocation mechanism of antiporters (24,47).…”

Membrane topology of the metal-tetracycline/H+ antiporter TetA(K) from Staphylococcus aureus

“…Although TetA(K) had previously been predicted to possess 14 TMS (8,21), it has recently been proposed by Fujihira et al (6) to contain only 12 TMS, with the ␣-helices of TMS 7 and TMS 8 (as shown in the 14-TMS model in Fig. 1) located in the cytoplasm.…”

Importance of the GP Dipeptide of the Antiporter Motif and Other Membrane-Embedded Proline and Glycine Residues in Tetracycline Efflux Protein Tet(L)

“…E152Q and R110C mutations were earlier shown to result in complete loss of tetracycline-resistance (41). Finally, we tested the effect on tetracycline-resistance of Tet(L) mutations in membrane-embedded glycines and prolines of two additional regions that are in the C-terminal domain of Tet(L): (i) a distinct GP dipeptide in TMS X that is in a functionally important region of a B. subtilis multi-drug resistance protein (43); and (ii) a glycine-rich region of TMS XIII, another helix that contains an essential acidic residue in Tet(K) and Tet(L) (41,44). The residues studied are highlighted in Figure 1.…”