Physiological evidence for an interaction between helices II and XI in the melibiose carrier of Escherichia coli.
The melibiose carrier from Escherichia coli is a cation-substrate cotransporter that catalyzes the accumulation of galactosides at the expense of H(+), Na(+), or Li(+) electrochemical gradients. Charged residues on transmembrane domains in the amino-terminal portion of this carrier play an important role in the recognition of cations, while the carboxyl portion of the protein seems to be important for sugar recognition. In the present study, we substituted Lys-377 on helix XI with Val. This mutant carrier, K377V, had reduced melibiose transport activity. We subsequently used this mutant for the isolation of functional second-site revertants. Revertant strains showed the additional substitutions of Val or Asn for Asp-59 (helix II), or Leu for Phe-20 (helix I). Isolation of revertant strains where both Lys-377 and Asp-59 are substituted with neutral residues suggested the possibility that a salt bridge exists between helix II and helix XI. To further test this idea, we constructed three additional site-directed mutants: Asp-59-->Lys (D59K), Lys-377-->Asp (K377D), and a double mutant, Asp-59-->Lys/Lys-377-->Asp (D59K/K377D), in which the position of these charges was exchanged. K377D accumulated melibiose only marginally while D59K could not accumulate. However, the D59K/K377D double mutant accumulated melibiose to a modest level although this activity was no longer stimulated by Na(+). We suggest that Asp-59 and Lys-377 interact via a salt bridge that brings helix II and helix XI close to one another in the three-dimensional structure of the carrier.
Franco
P J
PJ
Department of Cell Biology, Harvard Medical School, 240 Longwood Ave, Boston, MA 02115, USA.
Jena
A B
AB
Wilson
T H
TH
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D001692:Biological Transport; D002412:Cations; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D005697:Galactosides / Q000378:metabolism; D007700:Kinetics; D008553:Melibiose; D026901:Membrane Transport Proteins / Q000032:analysis / Q000235:genetics*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017433:Protein Structure, Secondary; D027981:Symporters
D001425:Bacterial Outer Membrane Proteins; D002412:Cations; D005697:Galactosides; D026901:Membrane Transport Proteins; D027981:Symporters; C021548:melibiose permease; D008553:Melibiose