Artificial Avidin-Based Receptors for a Panel of Small Molecules

Abstract

Proteins with high specificity, affinity, and stability are needed for biomolecular recognition in a plethora of applications. Antibodies are powerful affinity tools, but they may also suffer from limitations such as low stability and high production costs. Avidin and streptavidin provide a promising scaffold for protein engineering, and due to their ultratight binding to D-biotin they are widely used in various biotechnological and biomedical applications. In this study, we demonstrate that the avidin scaffold is suitable for use as a novel receptor for several biologically active small molecules: Artificial, chicken avidin-based proteins, antidins, were generated using a directed evolution method for progesterone, hydrocortisone, testosterone, cholic acid, ketoprofen, and folic acid, all with micromolar to nanomolar affinity and significantly reduced biotin-binding affinity. We also describe the crystal structure of an antidin, sbAvd-2(I117Y), a steroid-binding avidin, which proves that the avidin scaffold can tolerate significant modifications without losing its characteristic tetrameric beta-barrel structure, helping us to further design avidin-based small molecule receptors.

Molecular features of steroid-binding antidins and their use for assaying serum progesterone

“…Dissimilar to the apo structure of sbAvd-2 (I117Y) [PDB:4U46] published previously [38], the L1,2 loop of the sbAvd-2 (I117Y)–progesterone structure is clearly stabilized in one major conformation, very likely needed to accommodate ligand binding: in the antidin-progesterone complex, Met14 and His16 of the L1,2 loop have clearly moved (in comparison to the apo structure) towards the ligand and are within hydrophobic/van der Waals interaction distance of the ligand (Fig 3). However, the L3,4 loop of the progesterone complex structure is in the open conformation similar to the apo structure of sbAvd-2 (I117Y) [PDB:4U46].…”

“…We reported recently [38] that the I117Y point mutation improves the thermostability of sbAvd antidins similarly to what was originally shown for chicken Avd [22]. This form was found to crystallize in the presence of progesterone and was thus selected for structural analysis.…”

“…The binding site of Avds has been engineered by both mutating single residues involved directly in biotin binding and by manipulating the loop regions of Avds important for ligand recognition and specificity [37,38]. The L3,4-loop in particular, with a vital role for biotin binding, has been an active target for mutagenesis [5,7–10].…”

“…engineered Avds recognizing the non-native ligands progesterone, testosterone, hydrocortisone, cholic acid and ketoprofen. Those antidins were created by engineering the L1,2-loop together with the L3,4-loop or L5,6-loop regions using a directed evolution method for mutagenesis and the phage display technique for selection [38]. In comparison to natural Avd, antidins showed reduced binding affinity for biotin and had increased affinity towards progesterone, as analyzed in vitro using calorimetry, fluorometry and microplate assays, and hence antidins have potential to be used as novel diagnostic reagents [38].…”

Structural characterization of core-bradavidin in complex with biotin

“…Apart from the naturally occurring Avds, a number of genetically engineered Avds [7] have been produced, too. These include the dual-chain Avd (dcAvd) [24] and single-chain Avd (scAvd) [25], respectively with two and four simultaneously modifiable ligand-binding sites, the monomeric streptavidin [26–28], the steroid-binding Avd (sbAvd) [29,30] and an extremely thermostable and protease resistant chimeric Avd [31]. …”