Role of the charge pair aspartic acid-237-lysine-358 in the lactose permease of Escherichia coli

Abstract

Using a lactose permease mutant devoid of Cys residues (C-less permease), Asp237 and Lys358 were replaced with Cys or other amino acids to pursue the proposal that the two residues form a charge pair [King, S. C., Hansen, C. L., & Wilson, T.H. (1991) Biochim. Biophys. Acta 1062, 177-186]. Individual replacement of Asp237 with Cys, Ala, or Lys or replacement of Lys358 with Cys, Ala, or Asp virtually abolishes active lactose transport. However, simultaneous replacement of both residues with Cys and/or Ala yields permease with high activity. Therefore, neutral amino acid substitutions at either position are detrimental only because they leave the opposing charge unpaired. Strikingly, moreover, when Asp237 is interchanged with Lys358, high activity is observed. The results indicate strongly that Asp237 and Lys358 interact to form a salt bridge and that neither residue nor the salt bridge per se is important for activity. Immunoblots reveal low membrane levels of the active mutants lacking the putative salt bridge, suggesting a role for the salt bridge in either permease folding or stability and raising the possibility that the salt bridge may exist in a folding intermediate but not in the mature protein. Remarkably, however, a mutant with Cys in place of Asp237 is restored to full activity by carboxymethylation which recreates a negative charge at position 237. Pulse-chase analysis and heat-inactivation studies indicate that the stability of the double mutant with Cys at positions 237 and 358 is comparable to C-less. Therefore, the interaction between Asp237 and Lys358 is likely to be important for permease folding and is maintained in the mature protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Secondary solute transport in bacteria

“…However, some evidence has been presented that indicates that at least the interaction between Asp-237 and Lys-358 is also present in the mature protein. A single cysteine mutant with Cys at position 237, which is virtually inactive, is restored to full activity upon carboxymethylation with iodoacetic acid thereby compensating the positive charge (Lys) at position 358 [146]. Iodoacetamide which is uncharged does not restore the activity of the D237C mutant.…”

“…Iodoacetamide which is uncharged does not restore the activity of the D237C mutant. By allowing lipophilic and lipophobic sulfhydryl reagents to react with single cysteines at position 237 and 358, and analysis of the inactivation of the lactose transport protein, indications have obtained that the residues at these positions are within the membrane bilayer but close to the membrane/water interface at the outer surface of the membrane [146].…”

Analysis of Substrate-Binding Elements in OxlT, the Oxalate:Formate Antiporter of Oxalobacter formigenes

“…This hypothesis is consistent with the findings that among derivatives at these sites, only the R272K and K355R variants retain measurable function (11,17), although at relatively low levels (see below). Such work did not identify whether these null responses relate to OxlT biogenesis or OxlT function, and to address this issue we adopted a strategy resembling that used to identify a salt bridge in LacY (23,24). Thus, we studied OxlT after reconstitution into proteoliposomes, where the already-assembled molecule is accessible to a variety of informative probes (see 15,16,21,22).…”

A general method for determining helix packing in membrane proteinsin situ: Helices I and II are close to helix VII in the lactose permease ofEscherichia coli

“…13). Furthermore, the presence of two pairs of charged residues that interact functionally-Asp-237 (helix VII) with Lys-358 (helix XI) (14)(15)(16)(17)(18) and Asp-240 (helix VII) with Lys-319 (helix X) (15,17,19)-indicates that helix VII is close to helices X and XI. The proximity relationships have been confirmed by engineering divalent metal-binding sites (bis-or tris-His residues) within the permease (20)(21)(22) and by the demonstration that monoclonal antibody 4B11 binds to the last two cytoplasmic loops (7).…”