The role of helix VIII in the lactose permease ofEscherichia coli: I. Cys‐scanning mutagenesis

Abstract

Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in transmembrane domain VI11 and flanking hydrophilic loops (from Gln 256 to Lys 289) was replaced individually with Cys. Of the 34 single-Cys mutants, 26 accumulate lactose to >70% of the steady state observed with C-less permease, and an additional 7 mutants (Gly 262 4 Cys, Gly 268 + Cys, Asn 272 + Cys, Pro 280 + Cys, Asn 284 + Cys, Gly 287 + Cys, and Gly 288 + Cys) exhibit lower but significant levels of accumulation (30-50% of C-less). As a-helical conformation and demonstrate that, although only a single residue in this region of the permease is essential for activity (Glu 269), one face of the helix plays an important role in the transport mechanism. More direct evidence for the latter conclusion is provided in the companion paper (Frillingos S , Kaback HR, 1997, Protein Sci 6:438-443) by using site-directed sulfhydryl modification of the Cys-replacement mutants in situ. Keywords: active transport; bioenergetics; Cys modification; Cys replacementThe lactose permease of Escherichin coli, which catalyzes the coupled stoichiometric translocation of P-galactosides and H+ as a monomer, is an integral membrane protein with 12-transmembrane domains that are most probably in a-helical conformation (reviewed in . Site-directed mutagenesis of wild-type permease and Cys-scanning mutagenesis of a functional mutant devoid of Cys residues (C-less permease) have provided important insights into the structure and mechanism of the permease. Although as few as four amino acid residues [Glu 269 (helix VIII), Arg 302 (helix IX), His 322 (helix X), and Glu 325 (helix X)] appear to be critically involved in the symport mechanism, the activity of various active Cys-replacement mutants is altered by alkylation, and these mutants appear in clusters, suggesting that helical interfaces within the permease are important for turnover. Moreover, site-directed fluorescence spectroscopy (Jung et al

The role of helix VIII in the lactose permease of Escherichia coli: II. Site‐directed sulfhydryl modification

“…In this respect, it is noteworthy that these positions are on one face of helix VIII and distributed over two turns of the helix, which is approximately twice the length of a disaccharide. Moreover, the permease seems to behave in a similar manner with galactose as substrate (Frillingos et al, 1997). Therefore, the findings are more consistent with a "pathway" than a fixed binding site.…”

“…Single-Cys mutants (Frillingos et al, 1997) were transferred into plasmid pKR35/ C-less lacy-CXB by restriction fragment replacement. Mutations were verified by sequencing the length of the inserted DNA fragment through the ligation junctions using the dideoxynucleotide termination method (Sanger et al, 1977) after alkaline denaturation (Hattori & Sakaki, 1986).…”

“…Active lactose transport E. coli TI 84 (Z-Y-) transformed with each plasmid was grown, induced, and harvested as described previously (Frillingos et al, 1997). Cells were pretreated with NEM (2 mM) at 25°C in the absence or presence of TDG (10 mM) for a given time, the reactions were stopped by addition of 20 mM DlT, and cells were washed extensively with 1 0 0 mM K P i (pH 7.5)/10 mM MgS04 to remove TDG and excess reagent.…”

“…Keywords: active transport; bioenergetics; Cys modification; Cys replacement; ligand binding In the companion paper (Frillingos et al, 1997), Cys-scanning mutagenesis was employed to examine the role of putative transmembrane helix VI11 in the lactose permease of Escherichia coli. Residues were found on one face of putative helix VI11 where Cys-replacement followed by NEM treatment inactivates the permease.…”

“…Finally, no significant alteration in NEM reactivity is observed for mutant Gly 262 + Cys, Glu 269 + Cys, Ala 273 + Cys, Met 276 + Cys, Phe 277 + Cys, or Ala 279 -+ Cys. The findings indicate that a portion of one face of helix VI11 (Val 264, Gly 268, and Asn 272), which is in close proximity to Cys 148 (helix V), interacts with substrate, whereas another position bordering these residues (Thr 265) is altered by a ligandinduced conformational change.Keywords: active transport; bioenergetics; Cys modification; Cys replacement; ligand binding In the companion paper (Frillingos et al, 1997), Cys-scanning mutagenesis was employed to examine the role of putative transmembrane helix VI11 in the lactose permease of Escherichia coli. Residues were found on one face of putative helix VI11 where Cys-replacement followed by NEM treatment inactivates the permease.…”