Crystal structures of two aminoglycoside kinases bound with a eukaryotic protein kinase inhibitor

Abstract

Antibiotic resistance is recognized as a growing healthcare problem. To address this issue, one strategy is to thwart the causal mechanism using an adjuvant in partner with the antibiotic. Aminoglycosides are a class of clinically important antibiotics used for the treatment of serious infections. Their usefulness has been compromised predominantly due to drug inactivation by aminoglycoside-modifying enzymes, such as aminoglycoside phosphotransferases or kinases. These kinases are structurally homologous to eukaryotic Ser/Thr and Tyr protein kinases and it has been shown that some can be inhibited by select protein kinase inhibitors. The aminoglycoside kinase, APH(3')-IIIa, can be inhibited by CKI-7, an ATP-competitive inhibitor for the casein kinase 1. We have determined that CKI-7 is also a moderate inhibitor for the atypical APH(9)-Ia. Here we present the crystal structures of CKI-7-bound APH(3')-IIIa and APH(9)-Ia, the first structures of a eukaryotic protein kinase inhibitor in complex with bacterial kinases. CKI-7 binds to the nucleotide-binding pocket of the enzymes and its binding alters the conformation of the nucleotide-binding loop, the segment homologous to the glycine-rich loop in eukaryotic protein kinases. Comparison of these structures with the CKI-7-bound casein kinase 1 reveals features in the binding pockets that are distinct in the bacterial kinases and could be exploited for the design of a bacterial kinase specific inhibitor. Our results provide evidence that an inhibitor for a subset of APHs can be developed in order to curtail resistance to aminoglycosides.

Figure 1. Crystal structures of CKI-7-bound kinases.

(A) APH(3′)-IIIa, (B) APH(9)-Ia, and (C) CK1 (PDB 2CSN). The enzymes are shown in cartoon representation and the inhibitors are drawn as sticks. (D) Chemical structure of CKI-7.

Figure 2. Nucleotide/inhibitor binding sites of APH(3′)-IIIa and APH(9)-Ia.

The CKI-7- and nucleotide-bound APH(3′)-IIIa are shown in cartoon representation in panels A and C respectively; The CKI-7- and nucleotide-bound APH(9)-Ia are shown in cartoon representation in panels B and D, respectively. (A) CKI-7 bound to APH(3′)-IIIa in magenta sticks and its simulated annealing F_o-F_c omit map, contoured at 2.5σ, are shown. Tyr42, which forms stacking interactions with the isoquinoline, and Ala93, which hydrogen bonds with the inhibitor, are shown in sticks. (B) CKI-7 bound to APH(9)-Ia in dark blue sticks and its simulated annealing F_o-F_c omit map, contoured at 2.5σ, are shown. Phe50 whose aromatic ring stacks with the isoquinoline, and Ile103, which forms hydrogen bond interactions with the inhibitor, are shown in sticks. (C,D) Comparison of the inhibitor- and nucleotide-bound APHs. The homologous ePK gly-rich loop is highlighted in green for nucleotide-bound APH(3′)-IIIa (C) and red for APH(9)-Ia (D). Tyr42 in APH(3′)-IIIa and Phe50 in APH(9)-Ia, that stacks the adenine ring of the nucleotide are shown in sticks and their carbon atoms are colored green for APH(3′)-IIIa (C) and red for APH(9)-Ia (D). The CKI-7-binding sites are delineated by surface representation in their respective enzymes. CKI-7 bound to APH(3′)-IIIa and APH(9)-Ia are colored as in panels A and B. CKI-7 binds to the same region and in an analogous manner as the nucleotide to each of the enzyme. The phosphates of the nucleotide are buried in the surface representing the homologous gly-rich loop in the presence of CKI-7.

Figure 3. Comparisons of CKI-7 binding to APH(3′)-IIIa, APH(9)-Ia and CK1.

The structures are superposed using the conserved residues among the enzymes. (A) Superposition of CKI-7-bound APH(3′)-IIIa and APH(9)-Ia. The conserved active site residues between APH(3′)-IIIa and APH(9)-Ia are colored dark grey and light grey respectively. The APH(3′)-IIIa-bound CKI-7 is shown as magenta sticks and the APH(9)-Ia-bound CKI-7 is shown as dark blue sticks. The rotation between the planes of the isoquinoline rings of CKI-7 bound to APH(3′)-IIIa and APH(9)-Ia is approximately 40°. (B) Superposition of CKI-7-bound APH(3′)-IIIa and CK1. The same degree of rotation as that observed between the isoquinolines of the APH(3′)-IIIa- and APH(9)-Ia-bound CKI-7 is observed here. (C) Superposition of CKI-7-bound APH(9)-Ia and CK1. The CKI-7 isoquinoline rings are coplanar. In panels (B) and (C), APH(3′)-IIIa and APH(9)-Ia are colored as in panel A, and CK1 is colored in cyan. The residues that interact with CKI-7 and those with dissimilar properties from ePK nucleotide-binding site are shown in sticks. Hydrogen bond interactions between CKI-7 and CK1 are illustrated as dash lines. Water molecule is represented by a red sphere.