Structure of recombinantly expressed cockroach Lili-Mip protein in glycosylated and deglycosylated forms

• Published in: Biochimica et biophysica acta. General subjects Vol. 1866; no. 3; p. 130064
• Main Authors: KanagaVijayan, Dhanabalan, Subramanian, Rudra, Santhakumari, Partha Radhakrishnan, Chavas, Leonard M.G., Subramanian, Ramaswamy, Banerjee, Sanchari
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.03.2022
• Subjects: Amino Acid Sequence; Animals; Cockroaches - metabolism; Crystallography, X-Ray; Diploptera punctata; Fatty Acids, Monounsaturated - chemistry; Fatty Acids, Monounsaturated - metabolism; Female; Glycosylation; Insect Proteins - chemistry; Insect Proteins - genetics; Insect Proteins - metabolism; Lipocalin-like Milk proteins; Models, Molecular; Oleic acid (OLA); Oleic Acid - chemistry; Oleic Acid - metabolism; Palmitoleic acid (PAM); Protein Conformation; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Glycosylation; Oleic acid (OLA); Lipocalin-like Milk proteins; Diploptera punctata; Saccharomyces cerevisiae; Palmitoleic acid (PAM)
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2021.130064

The Pacific Beetle Cockroach is the only known viviparous cockroach. The pregnant females provide nutrition to the embryos by secreting milk proteins (Lili-Mips), which crystallize in vivo. The crystals that grow in the embryo are heterogeneous in their protein sequence. It is not apparent from the structure determined what role heterogeneity and glycosylation played in crystallization. Lili-Mips are very nutritious. Here, we report the cloning of synthesized Lili-Mip genes, their expression in Saccharomyces cerevisiae as secreted proteins, purification, crystallization, and the determination of a three-dimensional structure of one glycosylated and one deglycosylated form. A 2.35 Å structure of the glycosylated form is bound to palmitoleic acid and has several Zn atom mediated interactions. A 1.45 Å structure of the deglycosylated protein reveals a binding pocket that has both oleic and palmitoleic acid bound. Mass-spectrometry shows that oleic acid and palmitoleic acid are bound to the protein. Docking studies suggest that aliphatic chains of lengths 15, 16, and 18 carbons bind well in the pocket. The recombinantly expressed and secreted protein is glycosylated, has a bound fatty acid, is homogenous in its protein sequences, and readily forms crystals. The deglycosylated protein also crystallizes readily, suggesting that the high crystallizability of this protein is independent of glycosylation. Lili-Mips belong to the ubiquitous lipocalin family of proteins that bind to a large variety of ligands. While the residues lining the barrel are essential for the affinity of the ligand, our results show the role of side-chain orientations to ligand selectivity. [Display omitted] •The recombinantly expressed Lili-Mip has a bound fatty acid, and it is 31% glycosylated.•Both glycosylated and deglycosylated proteins crystallize readily and their structures were very similar.•While only PAM was bound to the glycosylated protein, density for OLA and PAM is observed in the deglycosylated Lili-Mip.