Both polarity and aromatic ring in the side chain of tryptophan 246 are involved in binding activity of Vibrio vulnificus hemolysin to target cells.

Vibrio vulnificus secretes a hemolysin/cytolysin (VVH) that induces cytolysis against a variety of mammalian cells by forming pores on the cellular membrane. VVH is known to bind to the cellular membrane as a monomer, and then convert to a pore-forming oligomer. However, the structural basis for binding of this toxin to target cells remains unknown. We show here that the polarity and indole ring on the side chain of Trp 246 (W246) of VVH, which sits on a bottom loop, participates in binding to cellular membrane. To clarify the binding mechanisms of VVH, we generated a series of W246 point mutants that were substituted with Arg (W246R), Ala (W246A), or Tyr (W246Y), and tested their binding and cytotoxicity on Chinese hamster ovary (CHO) cells. At a final concentration of 1 μg/ml of VVH, wild type (Wt), W246A and W246Y could bind and induce cytotoxicity to CHO cells, whereas W246R could not. The cytotoxic activity of W246A was significantly lower than that of Wt. These findings indicate that both the polarity and indole ring on the side chain of W246 were involved in the binding of this toxin to the target cellular membrane. The indole ring plays a particularly important role in toxin binding.