Crystal structure and ligand affinity of avidin in the complex with 4′-hydroxyazobenzene-2-carboxylic acid

• 4′-hydroxyazobenzene-2-carboxylic acid affinity to avidin was measured by MST.
• The crystal structure of the avidin-HABA complex was determined.
• The interaction of the hydrazone tautomer of HABA with avidin was confirmed.
• The interactions of HABA and biotin with avidin were compared.

Avidin is a protein found in egg white that binds numerous organic compounds with high affinity, especially biotin and its derivatives. Due to its extraordinary affinity for its ligands, avidin is extensively used in biotechnology. X-ray crystallography and fluorescence-based biophysical techniques were used to show that avidin binds the dye 4′-hydroxyazobenzene-2-carboxylic acid (HABA) with a lower affinity than biotin. The apparent dissociation constant determined for the avidin complex with HABA by microscale thermophoresis (MST) is 4.12 μM. The crystal structure of avidin–HABA complex was determined at a resolution of 2.2 Å (PDB entry 5chk). The crystals belong to a hexagonal system, in the space group P6422. In that structure, the hydrazone tautomer of HABA is bound at the bottom part of the central calyx near the polar residues. We show interactions of the dye with avidin and compare them with the previously reported avidin-biotin complex.

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