Western immunoblots were performed as described above. RESULTS Monocysteine Derivatives of BtuB and TonB Are Functional. insights into possible mechanisms of substrate transport and energy coupling. Both proteins span the outer membrane by means of a 22-stranded -barrel with short loops around the periplasmic face and longer loops around the extracellular face. The barrel is usually comprised of the residues from the C-terminal end of the polypeptide chain. The N-terminal 150C200 amino acids, which include the most conserved sequences of this family of transporters, form a novel structure that fits into the barrel and is ARPC1B held in place by at least 40 hydrogen bonds with residues in the barrel walls. Although this domain name was originally referred to as a plug or cork, we prefer the term hatch, reflecting its active role in the transport process, its probable retention in the barrel during substrate uptake, and its direct participation in substrate and TonB recognition (8). The structure of the ligand-bound form of FhuA confirmed previous suggestions that conformational changes occur on substrate binding (9C12), but these changes were surprisingly subtle. The most dramatic change on substrate binding occurs around the periplasmic Anle138b face of the hatch domain name, Anle138b far from the ligand-binding site. This change involves the unfolding of helix-1 and the movement of the N-terminal segment of the protein preceding helix-1 by as much as 17 ? relative to the rest of the protein (6, 7). This N-terminal segment has been implicated in active transport and was called Tonbox-1, here the TonB-box. The TonB-box consists of 7 amino acid residues (Asp6-Thr-Leu-Val-Val-Thr-Ala12 in BtuB), which are highly conserved in the TonB-dependent transporters. In all proteins that were examined, some mutations affecting this region eliminated TonB-dependent transport but not TonB-independent activities or substrate binding (2, 13). Surprisingly, extensive changes in the amino acid sequence of this region in BtuB had little or no effect on CN-Cbl uptake rates (14). The most deleterious changes for CN-Cbl uptake introduced glycine or proline substitutions at positions 8 and 10. These results suggested that this contribution of the TonB-box for transport activity was not strongly dependent on residue-specific interactions, but on polypeptide chain configuration, suggesting that other portions of BtuB also might interact with TonB. The TonB-uncoupled transport phenotype of these mutants was weakly suppressed in an allele-specific manner by mutations that changed glutamine-160 near a highly conserved a part of TonB (15, 16). These results suggested the Anle138b presence of a functional conversation between TonB and the TonB-box of the transporters. The physical conversation between TonB and two TonB-dependent transporters, FepA and FhuA, has been demonstrated by formaldehyde crosslinking (17C19). A mutation in the TonB-box of FepA, I14P, which rendered it inactive for transport, prevented crosslinking to TonB (19). This approach identified regions of TonB that were necessary for crosslinking with FepA but could not determine whether these residues were directly involved in complex formation. In the present study, site-directed disulfide crosslinking was used to study the potential interaction of the TonB-box of BtuB and the portion of TonB that was implicated by the genetic suppression analysis. For this purpose, single cysteine residues were introduced at successive positions in the TonB-box of BtuB and were found to form disulfide bonds with single cysteine residues in TonB at and near residue 160. The fact that different pairs of cysteine residues formed disulfide bonds with different efficiencies provides evidence for the potential structure and orientation of these interacting surfaces. Finally, changes in the pattern of disulfide bonding in the presence of TonB-uncoupling mutations indicated the relevance of the contact surfaces deduced from the cross-linking behavior. This approach demonstrates an conversation between specific residues of TonB and a TonB-dependent transporter. MATERIALS AND METHODS Bacterial Strains. The K12 strain used for plasmid construction and maintenance was JM109. Other strains used in this study were derived from strain MC4100 (20). Strain RK5016 carries the additional mutations plasmids and chloramphenicol (40 g/ml) for the plasmids. Construction of the Plasmids Made up of and gene in pBR322 was previously described (14). Plasmids made up of the and/or the genes were constructed by cloning these genes from the chromosome of strain JM109 using PCR with primers external to each coding sequence that add unique restriction sites. These genes were cloned with their own promoters, as a 988-bp as a 1,321-bp mutant plasmids were transferred by transformation into the strain RK5043, and the mutants were transformed into the strain RK5016 for phenotypic analysis. All and mutant plasmids were introduced in.