Glycine-Rich Transmembrane Helix 10 in the Staphylococcal Tetracycline Transporter TetA(K) Lines a Solvent-Accessible Channel
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- 作者：Karl A. Hassan ; Katie L. Robinson ; Alison N. Smith ; Joanne H. Gibson ; Ronald A. Skurray ; Melissa H. Brown
- 刊名：Biochemistry
- 出版年：2006
- 出版时间：December 26, 2006
- 年：2006
- 卷：45
- 期：51
- 页码：15661 - 15669
- 全文大小：350K
- 年卷期：v.45,no.51(December 26, 2006)
- ISSN：1520-4995
文摘
The staphylococcal TetA(K) tetracycline exporter is classified within the major facilitatorsuperfamily of transport proteins and contains 14 
hars/alpha.gif" BORDER=0>-helical transmembrane segments (TMS). Using cysteine-scanning mutagenesis, 27 amino acid residues across and flanking putative TMS 10 of the TetA(K)transporter were individually replaced with cysteine. The level of solvent accessibility to each of thetargeted amino acid positions was determined as a measure of fluorescein maleimide reactivity anddemonstrated that TMS 10 of TetA(K) has a cytoplasmic boundary at G313 and is likely to extend fromat least V298 on the periplasmic side. TMS 10 was found to be amphiphilic containing at least partiallysolvent accessible amino acid residues along the length of one helical face, suggesting that this helix mayline a solvent-exposed channel. Functional analyses of these cysteine mutants demonstrated a significantrole for a number of amino acid residues, including a predominance of glycine residues which werefurther analyzed by alanine substitution. These residues are postulated to allow interhelical interactionsbetween TMS 10 and distal parts of TetA(K) that are likely to be required for the tetracycline transportmechanism in TetA(K) and may be a general feature required by bacterial tetracycline transporters foractivity.
hars/alpha.gif" BORDER=0>-helical transmembrane segments (TMS). Using cysteine-scanning mutagenesis, 27 amino acid residues across and flanking putative TMS 10 of the TetA(K)transporter were individually replaced with cysteine. The level of solvent accessibility to each of thetargeted amino acid positions was determined as a measure of fluorescein maleimide reactivity anddemonstrated that TMS 10 of TetA(K) has a cytoplasmic boundary at G313 and is likely to extend fromat least V298 on the periplasmic side. TMS 10 was found to be amphiphilic containing at least partiallysolvent accessible amino acid residues along the length of one helical face, suggesting that this helix mayline a solvent-exposed channel. Functional analyses of these cysteine mutants demonstrated a significantrole for a number of amino acid residues, including a predominance of glycine residues which werefurther analyzed by alanine substitution. These residues are postulated to allow interhelical interactionsbetween TMS 10 and distal parts of TetA(K) that are likely to be required for the tetracycline transportmechanism in TetA(K) and may be a general feature required by bacterial tetracycline transporters foractivity.