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

Jie Sheng,J Bernd Helms,Nick K Olrichs,Dora V Kaloyanova,Bart M Gadella

doi:10.1038/s41598-021-84926-y

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-CRISP1ΔC) were obtained. Zn2+ specifically induced oligomerization of both MBP-CRISP1 and MBP-CRISP1ΔC 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-CRISP1ΔC 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.

Highlights

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
Different bacterial expression systems were employed with different induction conditions and different tags (His, glutathione S-transferase (GST), maltose-binding protein (MBP)) to improve the solubility, expression levels and purification of human Cysteine-rich secretory protein 1 (CRISP1)
Successful overexpression of MBP-CRISP1/CRISP1ΔC was achieved in the cloning strain XL-1 Blue

Summary

Introduction

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. The Zn2+-induced oligomerization of human recombinant CRISP1 may shed novel insights into the formation of functional protein complexes involved in mammalian fertilization. Spermatozoa pass through the epididymis where an extensive remodeling of the sperm plasma membrane takes place, including the acquisition of epididymal proteins[4,5]. Some of these proteins contribute to the acquisition of sperm’s ability to bind and penetrate through the extracellular structures surrounding the oocyte, while others are involved in preventing the occurrence of premature capacitation, known as decapacitation factors[5,6]. CRISP1 is present in the cumulus cell layer surrounding the ovulated oocyte and capable of modulating sperm motility and orientation before fertilisation[14]

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