Analysis of Tryptophan Residues in the Staphylococcal Multidrug Transporter QacA Reveals Long-Distance Functional Associations of Residues on Opposite Sides of the Membrane

Abstract

Tryptophan residues can possess a multitude of functions within a multidrug transport protein, e.g., mediating interactions with substrates or distal parts of the protein, or fulfilling a structural requirement, such as guiding the depth of membrane insertion. In this study, the nine tryptophan residues of the staphylococcal QacA multidrug efflux protein were individually mutated to alanine and phenylalanine, and the functional consequences of these changes were determined. Phenylalanine substitutions for each tryptophan residue were functionally tolerated. However, alanine modifications revealed an important functional role for three tryptophan residues, W58, W149, and W173, each of which is well conserved among QacA-related transport proteins in the major facilitator superfamily. The most functionally compromising mutation, an alanine substitution for W58, likely to be located at the extracellular interface of transmembrane segment 2, abolished all detectable QacA-mediated resistance and transport function. Second-site suppressor analyses identified several mutations that rescued the function of the W58A QacA mutant. Remarkably, all of these suppressor mutations were shown to be located in cytoplasmic loops between transmembrane helices 2 and 3 or 12 and 13, demonstrating novel functional associations between amino acid positions on opposite sides of the membrane and in distal Nand C-terminal regions of the QacA protein.

Efflux-Mediated Drug Resistance in Bacteria

“…[75] Site-directed mutagenesis studies with QacA produced interesting results, for example, a tryptophan-to-alanine change in the outside surface was compensated by changes in the inside loops of the protein. [76] An in-vitro study revealed that EmrAB of E. coli forms a dimer in contrast to the trimeric RND AcrB. [77]…”

Transcriptomic and biochemical analyses identify a family of chlorhexidine efflux proteins

“…Genes were amplified by PCR with Pfx supermix (Invitrogen) and cloned into the pTTQ18-RGSH6 vector as reported (7). Site-directed mutagenesis was conducted by using the QuikChange method as described (30), but using KOD hot start polymerase (Novagen). The ACIAD1978 gene was deleted from the A. baylyi ADP1 genome by allelic exchange as described (31).…”

The AlternativeTranslational Profile That Underlies the Immune-Evasive State of Persistence in Chlamydiaceae Exploits Differential Tryptophan Contents of the Protein Repertoire

“…This is not surprising per se because Trp residues in transmembrane proteins have special properties that can facilitate long-distance functional associations on opposite sides of the membrane. For example, in the staphylococcal multidrug transporter QacA, a number of Trp residues appear to be located at the periphery of transmembrane segments, where they guide the depth of the transmembrane insertion (47). Aromatic stacking interactions accommodated by Trp residues may also assist the stable binding of aromatic substrates.…”