Solubility engineering and crystallization of human apolipoprotein D

Abstract

Human apolipoprotein D (ApoD) is a physiologically important member of the lipocalin protein family that was discovered as a peripheral subunit of the high-density lipoprotein (HDL) but is also abundant in other body fluids and organs, including neuronal tissue. Although it has been possible to produce functional ApoD in the periplasm of Escherichia coli and to demonstrate its ligand-binding activity for progesterone and arachidonic acid, the recombinant protein suffers from a pronounced tendency to aggregate and to adsorb to vessel surfaces as well as chromatography matrices, thus hampering further structural investigation. Here, we describe a systematic mutagenesis study directed at presumably exposed hydrophobic side chains of the unglycosylated recombinant protein. As a result, one ApoD mutant with just three new amino acid substitutions-W99H, I118S, and L120S-was identified, which exhibits the following features: (1) improved yield upon periplasmic biosynthesis in E. coli, (2) elution as a monomeric protein from a gel permeation chromatography column, and (3) unchanged binding activity for its physiological ligands. In addition, the engineered ApoD was successfully crystallized (space group I4 with unit cell parameters a = 75.1 Å , b = 75.1 Å , c = 166.0 Å , a = b = g = 90°), thus demonstrating its conformationally homogeneous behavior and providing a basis for the future X-ray structural analysis of this functionally still puzzling protein.

Apolipoprotein D: An overview of its role in aging and age-related diseases

“…This was recently confirmed by the 1.8A structure of a hydrophilized form of recombinant ApoD. 13,14 These studies highlighted the presence of hydrophobic residues outside of the binding pocket, which may allow the molecule to interact with membranes or HDL, and favor ligand exchange, while the base of the calyx could provide a docking site for an unidentified cellular receptor. Both the crystallization studies and earlier affinity assays, point to arachidonic acid (AA) and progesterone (PG) as putative physiological ligands for ApoD.…”

Measuring and Increasing Protein Solubility

“…Homology modeling is a specific type of this strategy where sequence and structural alignments of a protein of interest and relatively more soluble homologous proteins are used to identify hydrophobic surface residues that could be substituted with hydrophilic residues 31, 32, 35, 52, 68, 92–94. For example, the murine version of leptin is more soluble than human leptin.…”

Human ApoD, an apolipoprotein up-regulated in neurodegenerative diseases, extends lifespan and increases stress resistance in Drosophila

“…It is up-regulated 500-fold at the site of the sciatic nerve crush injury in the rat (13,14). In vitro evidence indicates that it can carry membrane lipids, such as arachidonic acid and sterols (15)(16)(17)(18), and may be involved in the clearance and/or repair of damaged membranes, perhaps quenching harmful material released by neurons and glial cells in response to damage or recruiting lipids to expanding membranes. In the etiology of many of the disorders in which ApoD is elevated, oxidative stress is thought to play an important part (19,20).…”