The host thwarts pathogenic invasion through a multimodal offensive, including the restriction of metal nutrient availability. This strategy of microbial growth inhibition involves the transition-metal-scavenging protein calprotectin. In 2008, calprotectin was shown to deplete Mn(II) from tissue abscesses and thereby limit bacterial growth in mice (Corbin, et al. Science). To understand the molecular underpinnings of this observation, we biochemically characterized the Mn(II) coordination sphere of murine calprotectin. This protein is a heterooligomer of murine S100A8 (10.3 kDa) and murine S100A9 (13.0 kDa), and amino acid sequence alignment with the human protein indicates that it contains two metal-binding sites at the S100A8/S100A9 interface. Similar to human calprotectin, site 1 is a His3Asp and site 2 is a His6 motif. A series of calprotectin variants with mutated metal-binding site residues were prepared to both ascertain the site of high-affinity Mn(II) binding and elucidate the coordinating residues. Size exclusion chromatography indicated that the His6 site is the high-affinity Mn(II) site, and that Mn(II) binds causes a change in oligomerization state. Competition titrations with murine calprotectin variants and a Mn(II)-responsive small-molecule fluorescent sensor revealed the importance of six His residues in high-affinity Mn(II) chelation and the importance of excess Ca(II) ions in this process. Low-temperature continuous wave EPR spectroscopic investigations supported a highly symmetric Mn(II) coordination sphere, and provided further support for the involvement of (A9)His105 and (A9)His107 in Mn(II) binding. In total, this work defined the primary coordination sphere of the Mn(II)-His6 motif in murine calprotectin. Moreover, the results indicated that, similar to human calprotectin, the murine orthologue encapsulates Mn(II) in a nearly-octahedral binding site in a Ca(II)-dependent manner. These properties allow murine calprotectin to sequester Mn(II) and limit bacterial growth, ultimately helping the host overcome pathogenic incursion.

Murine calprotectin sequesters Mn(II) at a hexahistidine site

256th National Meeting (2018)

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