The less conserved metal-binding site in human CRISP1 remains sensitive to zinc ions to permit protein oligomerization

Abstract

Cysteine-rich secretory proteins (CRISPs) are a subgroup of the CRISP, antigen 5 and PR-1 (CAP) superfamily that is characterized by the presence of a conserved CAP domain. Two conserved histidines in the CAP domain are proposed to function as a Zn²⁺-binding site with unknown function. Human CRISP1 is, however, one of the few family members that lack one of these characteristic histidine residues. The Zn²⁺-dependent oligomerization properties of human CRISP1 were investigated using a maltose-binding protein (MBP)-tagging approach in combination with low expression levels in XL-1 Blue bacteria. Moderate yields of soluble recombinant MBP-tagged human CRISP1 (MBP-CRISP1) and the MBP-tagged CAP domain of CRISP1 (MBP-CRISP1^ΔC) were obtained. Zn²⁺ specifically induced oligomerization of both MBP-CRISP1 and MBP-CRISP1^ΔC in vitro. The conserved His142 in the CAP domain was essential for this Zn²⁺ dependent oligomerization process, confirming a role of the CAP metal-binding site in the interaction with Zn²⁺. Furthermore, MBP-CRISP1 and MBP-CRISP1^ΔC oligomers dissociated into monomers upon Zn²⁺ removal by EDTA. Condensation of proteins is characteristic for maturing sperm in the epididymis and this process was previously found to be Zn²⁺-dependent. The Zn²⁺-induced oligomerization of human recombinant CRISP1 may shed novel insights into the formation of functional protein complexes involved in mammalian fertilization.