Crystal structure of a secretory signalling glycoprotein from sheep at 2.0 Å resolution

Abstract

A 40 kDa glycoprotein from dry secretion of sheep is implicated as a signaling factor and is named as SPS-40. This protein is secreted only during the early phase of involution when the drastic tissue remodeling occurs in the mammary gland. SPS-40 was purified from sheep dry secretions and crystallized using hanging drop vapour diffusion method. The crystals belong to orthorhombic space group P2 12 12 1 with cell dimensions, a = 62.7 Å, b = 66.4 Å, c = 107.5 Å. The protein was also cloned for the determination of its complete amino acid sequence. The three-dimensional structure of SPS-40 was determined by X-ray crystallographic method at 2.0 Å resolution. The structure revealed the presence of an N-linked glycan chain at Asn39. The protein adopts a conformation with a classical (β/α) 8-barrel fold of triosephosphate isomerase (TIM) (residues 1–237 and 310–360) with an insertion of a small (α + β) domain (residues 240–307) similar to that observed in chitinases. However, the Leu substitution for Glu in the consensus catalytic sequence in SPS-40 causes a loss of chitinase activity. Furthermore, the sugar-binding groove in SPS-40 is distorted considerably from the standard chitin-binding site in chitinase enzymes and hence the binding of chitin-like oligosaccharides is considerably hampered. Three surface loops, His188–His197, Phe202–Arg212 and Phe244–Pro260 have exceptionally high values of B-factors (average = 70.5 Å 2), indicating the presence of a less defined region.