1krj

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spinqualitylabelsall models 1krj, resolution 2.00Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

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

Overview

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.

About this Structure

1KRJ is a Single protein structure of sequence from Saccharomyces cerevisiae with K and HEM as ligands. Active as Cytochrome-c peroxidase, with EC number 1.11.1.5 Full crystallographic information is available from OCA.

Reference

Conversion of an engineered potassium-binding site into a calcium-selective site in cytochrome c peroxidase., Bonagura CA, Bhaskar B, Sundaramoorthy M, Poulos TL, J Biol Chem. 1999 Dec 31;274(53):37827-33. PMID:10608846

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