Probabilistic study on subunit mismatch arrangement in staphylococcal γ-hemolysin heteroheptameric transmembrane pore

Staphylococcal γ-hemolysin (Hlg), consists of two separate proteins, Hlg1 (LukF) of 34 kDa and Hlg2 of 32 kDa, which cooperatively lyse mammalian erythrocytes. Hlg is an illustrative molecule for the study of the assembly and membrane insertion of transmembrane proteins having the unique characteristic of being composed of two separate proteins. Our previous studies revealed that LukF and Hlg2 assemble alternately on a membrane to form ring-shaped heteroheptameric transmembrane pores, which form funnel-shaped structure with a subunit mismatch arrangement in which the distance between two of the adjacent subunits is significant larger than the others. In the present study, further analysis of the subunit mismatch arrangement in Hlg heteroheptameric pores was conducted by two-dimensional (2-D) image analysis. The distances between two adjacent subunits were numbered according to the size of the distance between adjacent subunits, and how each distance was arranged in the pore was investigated. As a result, the following results were clarified: (1) Not all distances between two adjacent subunits are even. This indicates that a mismatch arrangement exists at not only one site but also at other sites. (2) There is a high probability leading several patterns of mismatch arrangement. (3) These mismatch patterns tend to be polarized into a region in which the distance between subunits is great and a region in which such distance is small. The probabilistic analysis used in this study revealed that there is the intrinsic geometry with subunit arrangements in the heteroheptameric pore consists of two differential subunits.