Fig. 5.

Protein–protein interactions at the ToxR^cyt/toxTpro and ToxR^cyt/ompUpro structures. (A) Ribbon and surface representations of the ToxR^cyt monomers D and C bound to the toxTpro40 DNA. Monomers D and C are colored in dark green and light green, respectively. Top, front, and rear views are shown, and protein–protein interface areas are indicated in purple for monomer D and in gold for monomer C. (B) Detail of the main molecular contacts between ToxR^cyt monomers D and C. The color code is the same as in the previous panel. (C) Interactions between the Y (red) and Z (orange) monomers of ToxR^cyt bound to the ompUpro19 DNA. (D) Interactions between W (purple) and X (blue) monomers of ToxR^cyt bound to the ompUpro19 DNA. (E) Surface representation of the interaction between Phe69 of monomer W (purple) and Lys102 of monomer X (blue) of ToxR^cyt bound to the ompUpro19 DNA. (F) Binding of ToxR to sites centered at −65 and −51 is not required for toxT activation. Plasmids harboring the wild-type (WT) toxT promoter fused to lacZ, mutant toxT promoters, or no toxT promoter (−) were assessed for toxT activation in the presence or absence of ToxR in V. cholerae strain O395 (ToxR+) or EK307 (ToxR−). Mutant −65/−51 contained 10 nucleotide transversions in the downstream region of the toxT promoter. Mutants −94 and −84 were reported previously to fail to bind ToxR and cannot be activated in V. cholerae (17). Samples were tested four times in duplicate. *P < 0.01 relative to the wild-type toxT promoter in the presence of ToxR as assessed by Student’s t test. Levels of ToxR were confirmed with an anti-ToxR antibody (6). (G) ToxR binding to a FAM-labeled fluorescent wild-type toxT promoter probe from −72 to −36 was measured in the absence or presence of unlabeled (cold) competitor toxT promoter DNA added at a 25-fold excess. While the wild-type toxT promoter cold competitor DNA completely competed away ToxR binding to the FAM-labeled probe (lane 6), the toxT mutant probe with the −65 and −51 ToxR binding sites mutated (10 mutations in all) was unable to compete with the wild-type FAM-labeled toxT probe (lane 7). ToxR showed dose-dependent binding to the FAM-labeled toxT probe, and a ToxR mutant, ToxR-R84A, was unable to bind the toxT promoter (lane 8). R84 was shown previously to be critical for ToxR binding to the toxT promoter (5) and makes direct contact with DNA (Fig. 2B).