Molecular and Conformational Basis of a Specific and High-Affinity Interaction between AlbA and Albicidin Phytotoxin

The albA gene of Klebsiella oxytoca encodes a protein of 221 amino acids that binds the albicidin phytotoxin with a high affinity (dissociation constant = 6.4 × 10⁻⁸ M). For this study, circular dichroism (CD) spectrometry and an alanine scanning mutagenesis approach were used in combination to investigate the molecular and conformational mechanisms of this high-affinity protein-ligand interaction. CD analysis revealed that AlbA contains a high-affinity binding site, and binding of the albicidin ligand to AlbA in a low-ionic-strength environment induced significant conformational changes. The ligand-dependent conformational changes of AlbA were specific and rapid and reached a stable plateau within seconds after the addition of the antibiotic. However, such conformational changes were not detected when AlbA and albicidin were mixed in the high-ionic-strength buffer that is required for maximal binding activity. Based on the conceptual model of protein-ligand interaction, we propose that a threshold ion strength allows AlbA to complete its conformational rearrangement and resume its original stable structure for accommodation of the bound albicidin. Mutagenesis analysis showed that the replacement of Lys¹⁰⁶, Trp¹¹⁰, Tyr¹¹³, Leu¹¹⁴, Tyr¹²⁶, Pro¹³⁴, and Trp¹⁶² with alanine did not change the overall conformational structure of AlbA but decreased the albicidin binding activity about 30 to 60%. We conclude that these residues, together with the previously identified essential residue His¹²⁵, constitute a high-affinity binding pocket for the ligand albicidin. The results also suggest that hydrophobic and electrostatic potentials of these key amino acid residues may play important roles in the AlbA-albicidin interaction.