Roles of acidic residues in the hydrophilic loop regions of metal-tetracycline/H + antiporter Tet(K) of Staphylococcus aureus

Abstract

Three transmembrane glutamic acid residues play essential roles in the metal-tetracycline/H + antiporter Tet(K) of Staphylococcus aureus [Fujihira et al., FEBS Lett. 391 (1996) 243–246]. In the putative hydrophilic loop region of the Tet(K) and Tet(L) proteins, six acidic residues are conserved. Asp 74, Asp 200, Asp 318 and Glu 381 are located on the putative cytoplasmic side, and Asp 39 and Glu 345 on the putative periplasmic side. These residues were replaced by a neutral amino acid residue or a charge-conserved one. In contrast to the transmembrane glutamic acid residues, the replacement of the two glutamic acid residues (Glu 345 and Glu 381) did not affect the tetracycline resistance level. Out of the other four aspartic acid residues, the only essential residue is Asp 318, any replacement of which resulted in complete loss of the tetracycline resistance and transport activity. Asp 318 is located in cytoplasmic loop 10–11 in the putative 14-transmembrane-segment topology of Tet(K). In the case of the tetracycline exporters of Gram-negative bacteria, the only essential acidic residue in the cytoplasmic loop region is located in loop 2–3 [Yamaguchi et al., Biochemistry 31 (1992) 8344–8348]. It may be a general role for tetracycline efflux proteins that three transmembrane and one cytoplasmic acidic residues are mandatory for the tetracycline transport function.