1KRJ

Engineering Calcium-binding site into Cytochrome c Peroxidase (CcP)

1KRJ の概要

• エントリーDOI: 10.2210/pdb1krj/pdb
• 分子名称: Cytochrome c Peroxidase, POTASSIUM ION, PROTOPORPHYRIN IX CONTAINING FE, ... (4 entities in total)
• 機能のキーワード: cation-binding loop, calcium selectivity, trp191 cationic-radical, open/closed conformer, bidentate bond, pentagonal bipyrimidal geometry, lignin peroxidase, oxidoreductase
• 由来する生物種: Saccharomyces cerevisiae (baker's yeast)
• 細胞内の位置: Mitochondrion matrix: P00431
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 34316.86

構造登録者

Bonagura, C.A.,Bhaskar, B.,Sundaramoorthy, M.,Poulos, T.L. (登録日: 2002-01-09, 公開日: 2002-01-23, 最終更新日: 2024-04-03)

主引用文献

Bonagura, C.A.,Bhaskar, B.,Sundaramoorthy, M.,Poulos, T.L.
Conversion of an engineered potassium-binding site into a calcium-selective site in cytochrome c peroxidase.
J.Biol.Chem., 274:37827-37833, 1999

PubMed Abstract: We have previously shown that the K(+) site found in ascorbate peroxidase can be successfully engineered into the closely homologous peroxidase, cytochrome c peroxidase (CCP) (Bonagura, C. A. , Sundaramoorthy, M., Pappa, H. S., Patterson, W. R., and Poulos, T. L. (1996) Biochemistry 35, 6107-6115; Bonagura, C. A., Sundaramoorthy, M., Bhaskar, B., and Poulos, T. L. (1999) Biochemistry 38, 5538-5545). All other peroxidases bind Ca(2+) rather than K(+). Using the K(+)-binding CCP mutant (CCPK2) as a template protein, together with observations from structural modeling, mutants were designed that should bind Ca(2+) selectively. The crystal structure of the first generation mutant, CCPCA1, showed that a smaller cation, perhaps Na(+), is bound instead of Ca(2+). This is probably because the full eight-ligand coordination sphere did not form owing to a local disordering of one of the essential cation ligands. Based on these observations, a second mutant, CCPCA2, was designed. The crystal structure showed Ca(2+) binding in the CCPCA2 mutant and a well ordered cation-binding loop with the full complement of eight protein to cation ligands. Because cation binding to the engineered loop results in diminished CCP activity and destabilization of the essential Trp(191) radical as measured by EPR spectroscopy, these measurements can be used as sensitive methods for determining cation-binding selectivity. Both activity and EPR titration studies show that CCPCA2 binds Ca(2+) more effectively than K(+), demonstrating that an iterative protein engineering-based approach is important in switching protein cation selectivity.

PubMed: 10608846
DOI: 10.1074/jbc.274.53.37827

実験手法

X-RAY DIFFRACTION (2 Å)