Allosteric Inhibition of Aminoglycoside Phosphotransferase by a Designed Ankyrin Repeat Protein

• Published in: Structure (London) Vol. 13; no. 8; pp. 1131 - 1141
• Main Authors: Kohl, Andreas, Amstutz, Patrick, Parizek, Petra, Binz, H. Kaspar, Briand, Christophe, Capitani, Guido, Forrer, Patrik, Plückthun, Andreas, Grütter, Markus G.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.08.2005
• Subjects: Allosteric Regulation - physiology; Amino Acid Sequence; Ankyrin Repeat - physiology; Drug Resistance - physiology; Enterococcus - enzymology; Kanamycin Kinase - antagonists & inhibitors; Kanamycin Kinase - chemistry; Kanamycin Kinase - metabolism; Molecular Sequence Data; Protein Engineering; Protein Structure, Secondary; Protein Structure, Tertiary; Sequence Alignment; Staphylococcus - enzymology; Structural Homology, Protein
• ISSN: 0969-2126; 1878-4186
• DOI: 10.1016/j.str.2005.04.020

Aminoglycoside phosphotransferase (3′)-IIIa (APH) is a bacterial kinase that confers antibiotic resistance to many pathogenic bacteria and shares structural homology with eukaryotic protein kinases. We report here the crystal structure of APH, trapped in an inactive conformation by a tailor-made inhibitory ankyrin repeat (AR) protein, at 2.15 Å resolution. The inhibitor was selected from a combinatorial library of designed AR proteins. The AR protein binds the C-terminal lobe of APH and thereby stabilizes three α helices, which are necessary for substrate binding, in a significantly displaced conformation. BIAcore analysis and kinetic enzyme inhibition experiments are consistent with the proposed allosteric inhibition mechanism. In contrast to most small-molecule kinase inhibitors, the AR proteins are not restricted to active site binding, allowing for higher specificity. Inactive conformations of pharmaceutically relevant enzymes, as can be elucidated with the approach presented here, represent powerful starting points for rational drug design.