Abstract
To evaluate the potential of using heme-containing lipocalin nitrophorin 1 (NP1) as a template for protein engineering, we have replaced the native axial heme-coordinating histidine residue with glycine, alanine, and cysteine. We report here the characterization of the cysteine mutant H60C_NP1 by spectroscopic and crystallographic methods. The UV/vis, resonance Raman, and magnetic circular dichroism spectra suggest weak thiolate coordination of the ferric heme in the H60C_NP1 mutant. Reduction to the ferrous state resulted in loss of cysteine coordination, while addition of exogenous imidazole ligands gave coordination changes that varied with the ligand. Depending on the substitution of the imidazole, we could distinguish three heme coordination states: five-coordinate monoimidazole, six-coordinate bisimidazole, and six-coordinate imidazole/thiolate. Ligand binding affinities were measured and found to be generally 2–3 orders of magnitude lower for the H60C mutant relative to NP1. Two crystal structures of the H60C_NP1 in complex with imidazole and histamine were solved to 1.7- and 1.96-Å resolution, respectively. Both structures show that the H60C mutation is well tolerated by the protein scaffold and suggest that heme–thiolate coordination in H60C_NP1 requires some movement of the heme within its binding cavity. This adjustment may be responsible for the ease with which the engineered heme–thiolate coordination can be displaced by exogenous ligands.
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Abbreviations
- BuImd:
-
N-(n-Butyl)-imidazole
- H60C_NP1:
-
Nitrophorin 1 mutant in which the axial His-60 ligand is replaced by Cys-60
- MCD:
-
Magnetic circular dichroism
- NP1:
-
Nitrophorin 1
- NP4:
-
Nitrophorin 4
- PBS:
-
Phosphate-buffered saline
- RMSD:
-
Root mean square deviation
- RR:
-
Resonance Raman
- Tris:
-
Tris(hydroxymethyl)aminomethane
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Acknowledgments
The plasmid for the NP1 gene was provided by F.A. Walker, who we thank for a number of helpful discussions. We thank C.D. Stout for advice and help during X-ray data acquisition and structure refinement. We also thank David Ginsberg for assistance during protein expression and purification. This work was supported by National Institute of Health Grants GM41049 (to D.B.G.) and GM26730 (to J.H.D.).
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Vetter, S.W., Terentis, A.C., Osborne, R.L. et al. Replacement of the axial histidine heme ligand with cysteine in nitrophorin 1: spectroscopic and crystallographic characterization. J Biol Inorg Chem 14, 179–191 (2009). https://doi.org/10.1007/s00775-008-0436-x
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DOI: https://doi.org/10.1007/s00775-008-0436-x