Abstract 1296 Investigating the calcium binding mechanism of Schizophyllum commune Type Ia metacaspases using circular dichroism spectroscopy

Metacaspase proteins are proteases that have been shown to be involved in the regulation of programmed cell death pathways in fungi, plants, and protozoa. This project studies a Type I metacaspase from the fungus Schizophyllum commune (ScMCA-Ia). These metacaspases require calcium for activity. A well-characterized calcium binding site binds calcium with a Kd in the micromolar range, but ScMCA-Ia only reaches maximal activity once calcium concentration is in the millimolar range. This leads to a hypothesis that there are two calcium binding sites. Previous results support the formation of a high-affinity calcium binding site in the protein and this site is widely accepted in the literature. However, the particular residues involved in the second binding site and the structural changes in the metacaspase induced by calcium binding to both sites are still unclear. Previous work on ScMCA-Ia suggests D279, D295, D296, and D326 compose the high-affinity calcium binding site and the residues D405 and D411 are involved in forming the low-affinity calcium binding site. Wildtype and mutant versions of ScMCA-Ia are designed and made by site-directed mutagenesis and the proteins are purified using nickel affinity chromatography. To further study the residues involved in calcium binding, we used circular dichroism spectroscopy. Secondary structural elements of proteins have strong signals from their interactions with the circularly polarized light at unique wavelengths. We observe a change in signal at 222 nm as calcium concentration in the protein sample increases, indicating a conformational change due to calcium binding. WT ScMCA-Ia fits best to a two-site model with one Kd value in the micromolar range and a second in the millimolar range. This supports the current high- and low- affinity calcium binding site hypothesis. A low-affinity binding site mutant, D405A, fits a one-site model with a Kd value in the micromolar range, corresponding to only the high-affinity site being unmutated. Results also indicate that circular dichroism is a better method for this study than the previously used differential scanning fluorimetry as the spectroscopic approach avoids high temperature denaturation of the proteins and allows for more reliable determination of calcium Kd values for the less stable mutant metacaspases.