FIG. 2.

Comparative modeling of CT441 Tsp against Protein Data Bank accession number 1FC7/P1D Ctp and verification of the active site of CT441 with mutagenesis. (A) Residues 194 to 527 of the CT441 precursor protein were aligned with the 336 residues of the C-terminal processing protease of photosystem II (P1D) (Protein Data Bank accession number 1FC7) using the MODELLER comparative modeling package (21). A model representing the PDZ domain and the catalytic unit of CT441 was obtained by satisfaction of spatial restraints. The Ser455 and Lys481 residues of the active site are shown as sticks. (B) Mutation of serine 455 or lysine 481 ablated p65 cleavage activity of CT441. wt CT441 or point mutations of S455A or K481T were expressed in 293T cells as N-terminal HA-tagged proteins by transient transfection. Protein expression and p65 cleavage were determined by immunoblotting (IB) analysis. The expression of Erk2 was used as a loading control.