Magnetic circular dichroism studies of iron(ii) binding to human calprotectin. Issue 2 (21st October 2016)

• Record Type: Journal Article
• Authors: Baker, Tessa M.; Nakashige, Toshiki G.; Nolan, Elizabeth M.; Neidig, Michael L.
• Is Part Of: Chemical science. Volume 8:Issue 2(2017)
• Page Start: 1369
• Page End: 1377
• Publication Date: 2016-10-21
• Subjects: Chemistry -- Periodicals; 540.5
• DOI: 10.1039/c6sc03487j
• Languages: English
• ISSNs: 2041-6520

Abstract

Abstract : Magnetic circular dichroism studies of Fe(ii ) binding to human calprotectin demonstrate the nature of Fe(ii ) coordination at two different Fe(ii )-binding sites and provide insight into how Ca(ii ) modulates Fe(ii ) coordination. Abstract : Calprotectin (CP) is an abundant metal-chelating protein involved in host defense, and the ability of human CP to bind Fe(ii ) in a calcium-dependent manner was recently discovered. In the present study, near-infrared magnetic circular dichroism spectroscopy is employed to investigate the nature of Fe(ii ) coordination at the two transition-metal-binding sites of CP that are a His3 Asp motif (site 1) and a His6 motif (site 2). Upon the addition of sub-stoichiometric Fe(ii ), a six-coordinate (6C) Fe(ii ) center associated with site 2 is preferentially formed in the presence of excess Ca(ii ). This site exhibits an exceptionally large ligand field (10 D q = 11 045 cm −1 ) for a non-heme Fe(ii ) protein. Analysis of CP variants lacking residues of the His6 motif supports that CP coordinates Fe(ii ) at site 2 by employing six His ligands. In the presence of greater than one equiv. of Fe(ii ) or upon mutation of the His6 motif, the metal ion also binds at site 1 of CP to form a five-coordinate (5C) Fe(ii )–His3 Asp motif that was previously unidentified in this system. Notably, the introduction of His-to-Ala mutations at the His6 motif results in a mixture of 6C (site 2) and 5C (site 1) signals in the presence of sub-stoichiometric Fe(ii ). These results are consistent with a reduced Fe(ii )-binding affinity of site 2 as more weakly coordinating water-derived ligands complete the 6C site. In the absence of Ca(ii ), both sites 1 and 2 are occupied upon addition of sub-stoichiometric Fe(ii ), and a stronger ligand field is observed for the 5C site. These spectroscopic studies provide further evaluation of a unique non-heme Fe(ii )–His6 site for metalloproteins and support the notion that Ca(ii ) ions influence the Fe(ii )-binding properties of CP.