Understanding the Structural Requirements for Hemolytic Protein Function

We study the hemolytic activity of a hemolysin protein (HpmA) secreted by the gram negative bacterium Proteus mirabilis. HpmA is the A component of a type Vb, or two-partner, secretion pathway (TPS pathway). To cross the outer membrane, unfolded periplasmic HpmA is recognized, exported, and folded by its membrane bound cognate B component. Once secreted, HpmA displays cytotoxic activity against mammalian cells, including red blood cells. HpmA is comprised of 1577 amino acids and contains at least two functional domains - a TPS domain that targets the protein to the correct B component and a hemolytic domain. Less than 20% of the HpmA structure is known, and none of the hemolytic domain structure is known. To address this problem, we used quantitative hemolytic activity assays to determine the contributions to protein function made by different segments of the hemolytic domain. Our data show a reduction but not elimination of function as the protein is progressively truncated. We have developed two models that could explain these results - an Assembly model and an Association model that differ in the stoichiometry of HpmA needed to lyse a red blood cell. The applicability of these models to different HpmA truncations and the implications for HpmA biological structure and function are discussed.