Human apolipoprotein D (ApoD) occurs in plasma associated with high density lipoprotein. Apart from the involvement in lipid metabolism, its binding activity for progesterone and arachidonic acid plays a role in cancer development and neurological diseases. The crystal structures of free ApoD and its complex with progesterone were determined at 1.8 Å resolution and reveal a lipocalin fold. The narrow, mainly uncharged pocket within the typical -barrel accommodates progesterone with its acetyl side chain oriented toward the bottom. The cavity adopts essentially the same shape in the absence of progesterone and allows complexation of arachidonic acid as another cognate ligand. Three of the four extended loops at the open end of the -barrel expose hydrophobic side chains, which is an unusual feature for lipocalins and probably effects association with the high density lipoprotein particle by mediating insertion into the lipid phase. This mechanism is in line with an unpaired Cys residue in the same surface region that can form a disulfide cross-link with apolipoprotein A-II.Human ApoD 2 (1, 2) is a glycoprotein of 169 amino acids, which was discovered in plasma (3) as an atypical lipoprotein. There, ApoD is peripherally associated with HDL via disulfide bond with ApoA-II (4), which itself forms an amphipathic ␣-helical belt that wraps around the lipid disc (5).ApoD was also isolated as a progesterone-binding protein abundant in mammary gross cystic disease fluid (6), and it was found as a monomeric protein in apocrine secretion, where it seems to bind odorants (7). ApoD mRNA is expressed in a variety of organs (8) as well as in certain human cancers. Indeed, ApoD has received attention as prognostic marker for various, often steroid-responsive tumors, including prostate cancer (9), breast carcinoma, and cutaneous malignant melanoma (10).In addition, ApoD is synthesized by astrocytes in the central nervous system (11), and there, it seems to be involved in arachidonic acid transport, metabolism, and signaling (12). Notably, ApoD plays a well documented pathophysiological role in several psychiatric disorders (12), especially in schizophrenia (13).ApoD has been prepared as a soluble recombinant protein via secretion into the periplasm of Escherichia coli, and its binding activity for progesterone and arachidonic acid, both with K D values around 1 M, was demonstrated (14). However, recombinant ApoD reveals a pronounced tendency to adsorb to surfaces and to form aggregates upon storage, which may be explained by hydrophobic surface properties that enable ApoD to interact with HDL and/or with lipid membranes in its physiological environment. Using systematic substitution of presumably exposed hydrophobic residues, we were able to construct a "solubilized" mutant of ApoD that retained native ligand binding function and appeared to be suitable for structural analysis (15).
EXPERIMENTAL PROCEDURESProtein Production and Crystallization-An engineered version of ApoD was produced in E. coli via periplasmic secretion essent...