Sequence changes in the Ton box region of BtuB affect its transport activities and interaction with TonB protein

Uptake of cobalamins by the transporter protein BtuB in the outer membrane of Escherichia coil requires the proton motive force and the transperiplasm ic protein TonB. The Ton box sequence near the amino terminus of BtuB is co nserved among all TonB-dependent transporters and is the only known site of mutations that confer a transport-defective phenotype which can be suppres sed by certain substitutions at residue 160 in TonB. The crystallographic s tructures of the TonB-dependent transporter FhuA revealed that the region n ear the Ton box, which itself was not resolved, is exposed to the periplasm ic space and undergoes an extensive shift in position upon binding of subst rate. Site-directed disulfide bonding in intact cells has been used to show that the Ton box of BtuB and residues around position 160 of TonB approach each other in a highly oriented and specific manner to form BtuB-TonB hete rodimers that are stimulated by the presence of transport substrate. Here, replacement of Ton box residues with proline or cysteine revealed that resi due side chain recognition is not important for function, although replacem ent with proline at four of the seven Ton box positions impaired cobalamin transport. The defect in cobalamin utilization resulting from the L8P subst itution was suppressed by cysteine substitutions in adjacent residues in Bt uB or in TonB. This suppression did not restore active transport of cobalam ins but may allow each transporter to function at most once. The uncoupled proline substitutions in BtuB markedly affected the pattern of disulfide bo nding to TonB, both increasing the extent of cross-linking and shifting the pairs of residues that can be joined. Cross-linking of BtuB and TonB in th e presence of the BtuB V10P substitution became independent of the presence of substrate, indicating an additional distortion of the exposure of the T on box in the periplasmic space. TonB action thus requires a specific orien tation for functional contact with the Ton box, and changes in the conforma tion of this region block transport by preventing substrate release and rep eated transport cycles.