Fig. 8
From: Molecular basis of egg coat cross-linking sheds light on ZP1-associated female infertility
Role of a conserved ZP1-N1 His pair in Zn2+ binding and homodimerisation. a Surface representation of the structure of the deglycosylated cZP1-N1 homodimer in the crystal soaked with Zn(OAc)2, highlighting the position of the Zn2+ ion bound to H71(A) and H91(A) (black sphere) relative to the protein subunits, two proximal disulphide bonds and N-acetylglucosamine (NAG) residues attached to N65. The A subunit of the homodimer is coloured gold according to Fig. 5, whereas subunit B is shown in lilac. The inset shows a detailed view of the region around the Zn2+, coloured by electrostatic potential using a gradient from red (−4 kT e−1) to blue (+ 4 kT e−1) through white (0 kT e−1). b Details of the asymmetric interface of the cZP1-N1 homodimer, as observed in the native crystal of the deglycosylated protein. Selected amino acids are shown as sticks, with hydrogen bonds indicated by dashed lines. c Surface representation of the symmetric interface of glycosylated cZP1-N1 (chain A, top left panel; chain B, bottom left panel), showing the position of H71 and H91 relative to I68. Notably, the H71/H91 pocket of the A′ subunit is more accessible than that of B′, due to apparent flexibility of the relatively small symmetric interface. The right panels, which are rotated by 30 degrees over the y-axis compared with the left ones, show zoomed views of the symmetric interfaces around I68(A) and I68(B), with the surface of the A′ and B′ molecules coloured by electrostatic potential as in the inset of panel a. d Details of the asymmetric interface of deglycosylated cZP1-N1, as observed in the Zn2+-soaked crystal. The green mesh is an anomalous difference map calculated at λ = 1.2825 Å and contoured at 6 σ. e 100-ns MD simulation snapshot of the Zn2+-binding site shown in panel a. H71 and H91, together with four water molecules, coordinate the zinc ion during the whole simulation time (mean Zn-His N distance 2.22 ± 0.06 Å; mean Zn–H2O O distance 2.10 ± 0.05 Å). f Anti-5His immunoblot analysis of wild-type (WT) and His mutant hZP1-N1 constructs secreted by HEK293T cells (100 μL medium). g, h Coomassie-stained SDS-PAGE analysis of IMAC (panel g) and SEC (panel h) fractions from the purification of the same constructs shown in panel f. The SEC profiles corresponding to panel h are reported in Supplementary Fig. 6
