Sites of pH regulation of the urea channel of Helicobacter pylori.
Helicobacter pylori (Hp) and Streptococcus salivarius (Ss) require intrabacterial urease for acid resistance and express a urea channel, UreI. The presence of UreI was shown to increase urea permeability approximately 300-fold over that of a non-polar ureI deletion mutant. Expression of SsUreI in Xenopus oocytes increased urea uptake pH independently, whereas HpUreI shows an acidic pH dependence, half-maximal at pH 6.0. Mutagenesis of all histidines, aspartates, glutamates and the lysine in the periplasmic domain of HpUreI showed that His-123, His-131, Asp-129, Asp-140, Glu-138 and Lys-132 in the second periplasmic loop (PL2) and His-193 in the C-terminus (Ct) were important for activation of transport. With the exception of a lysine that was shown to substitute for His-193 in HpUreI, these charged amino acids are absent in SsUreI. A chimera in which PL1 of HpUreI was replaced by PL1 of SsUreI retained activity at acidic pH and gained partial activity at neutral pH. Exchange of PL2 inactivated transport, whereas exchange of Ct had no effect. Chimeras, in which either PL1 or PL2 of HpUreI replaced those of SsUreI, retained wild-type transport, but replacement of the Ct or both loops inactivated transport. PL1 appears to be important for restricting transport through HpUreI at neutral pH, whereas protonation of three histidines in PL2 and Ct and the presence of three dicarboxylic amino acids in PL2 appears to be necessary to activate HpUreI at acidic pH.
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University of California, Los Angeles, and Building 113, Room 324, VA Greater Los Angeles Health Care System, Los Angeles, CA 90073, USA.
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D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001692:Biological Transport; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D005260:Female; D016480:Helicobacter pylori / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D019897:Periplasm / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013291:Streptococcus / Q000235:genetics; D014508:Urea / Q000378:metabolism; D014981:Xenopus
D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; C114527:UreI protein, Helicobacter pylori; D014508:Urea