Structural Basis for Kinase-Mediated Macrolide Antibiotic Resistance Desiree H. Fong, David L. Burk, Jonathan Blanchet, Amy Y. Yan, Albert M. Berghuis  Structure  Volume 25, Issue 5, Pages 750-761.e5 (May 2017) DOI: 10.1016/j.str.2017.03.007 Copyright © 2017 Elsevier Ltd Terms and Conditions

Structure 2017 25, 750-761.e5DOI: (10.1016/j.str.2017.03.007) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Arrangement of MPH(2′)-I and MPH(2′)-II Secondary Structure Cartoon depictions of (A) apo MPH(2′)-I (see also Table 1), (B) apo MPH(2′)-II (see also Table 2), and (C) GMPPNP-bound APH(2″)-Ia (PDB: 5IQA). The N-terminal lobes are colored red (MPH(2′)-I: 1–95; MPH(2′)-II: 32–93), the linker segments green (MPH(2′)-I: 96–119; MPH(2′)-II: 94–119), and the C-terminal lobes blue (MPH(2′)-I: 120–301; MPH(2′)-II: 120–299). The core and helical subdomains of the C-terminal lobes are colored light blue (MPH(2′)-I: 120–150, 193–260; MPH(2′)-II: 120–150, 193–260) and dark blue (MPH(2′)-I: 151–192, 261–301; MPH(2′)-II: 151–192, 193–299), respectively. The α helices are labeled, and GMPPNP bound to APH(2″)-Ia is shown as sticks. Structure 2017 25, 750-761.e5DOI: (10.1016/j.str.2017.03.007) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Cofactor and Substrate Binding Sites of MPH(2′)-I and MPH(2′)-II A cartoon depiction of (A) MPH(2′)-I (see also Table 1) and (B) MPH(2′)-II (see also Table 2) in their apo forms superimposed on their ternary complexes with GDP and erythromycin. The MPH(2′)-I and MPH(2′)-II complexes are colored green and red, respectively, with the apo structures shown in gray. Cofactors and macrolides are shown as yellow and magenta sticks, respectively (see also Figure S2). The conformation of the linker loop in the apo structure is labeled I, and that in the ternary complexes is labeled II. Structure 2017 25, 750-761.e5DOI: (10.1016/j.str.2017.03.007) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Nucleotide Binding Sites of MPH(2′)-I and MPH(2′)-II A detailed view of the nucleotide binding site of (A) the MPH(2′)-I ternary complex with erythromycin and (B) the MPH(2′)-II ternary complex with erythromycin, with notable residues shown as sticks. Hydrogen bonds to the linker are shown as yellow dashed lines and the proposed general base (Asp200) is shown as magenta sticks. See also Figure S3. Structure 2017 25, 750-761.e5DOI: (10.1016/j.str.2017.03.007) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 The MPH(2′)-I Macrolide Binding Site (A) Cartoon showing the superimposed structures of the four MPH(2′)-I ternary complexes. For clarity, only the protein component of the erythromycin complex is shown. Macrolide substrates from the complexes are shown as green (erythromycin), magenta (oleandomycin), orange (clarithromycin), and blue (azithromycin) sticks. The cofactor from the erythromycin complex is shown as yellow sticks. The N-terminal lobe is colored red, the linker segment green, and the C-terminal lobe light blue (core region) and dark blue (helical region). (B) Zoomed-in view of the MPH(2′)-I macrolide binding site. The macrolides, cofactors, and protein are as described in (A). The linker segment in the apo and ternary complex is highlighted in light brown and green, respectively. The modification site on the macrolide is denoted with an asterisk. See also Figures S1 and S2. Structure 2017 25, 750-761.e5DOI: (10.1016/j.str.2017.03.007) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 The MPH(2′)-II Macrolide Binding Site (A) MPH(2′)-II macrolide binding site with bound 14- and 15-membered macrolides. Macrolides are shown as red (erythromycin), blue (azithromycin), and orange (clarithromycin) sticks. For clarity, only the cartoon for the protein in the erythromycin complex is shown. The N-terminal lobe is colored red, the linker segment green, and the C-terminal lobe light blue (core region) and dark blue (helical region). Notable binding site residues are shown as sticks and hydrogen bonds by yellow dashes. The hydroxyl group modified by the enzyme is indicated with an asterisk. The conformation of the linker segment in the apo structure is shown in light brown. (B) Macrolide binding site of the MPH(2′)-II-spiramycin ternary complex. Spiramycin is shown as white sticks. Other features are shown as described in (A). (C) Macrolide binding site of the MPH(2′)-II-josamycin ternary complex. Josamycin is shown as white sticks. Other features are shown as described in (A). (D) Superposition of the MPH(2′)-II-spiramycin and MPH(2′)-II-josamycin ternary complexes. The cofactor and substrates in the spiramycin and josamycin complexes are shown as blue and brown sticks, respectively. See also Figures S1, S2, and S4. Structure 2017 25, 750-761.e5DOI: (10.1016/j.str.2017.03.007) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 6 Macrolide Binding to MPH(2′)-I, MPH(2′)-II, and a Bacterial Ribosome The cartoon depicts the binding of erythromycin (14-membered ring) and spiramycin (16-membered ring) to MPH(2′)-I and MPH(2′)-II, respectively (see also Figure S1) Also shown are the macrolides bound to the 50S ribosomal subunit from Haloarcula marismortui (PDB: 1YI2 and 1KD1). The macrolides are shown as sticks and the surface of the enzyme and ribosome is shown in gray. The C9–C14 edge of the lactone ring is highlighted in the ribosomal structures by an orange arrow. In the MPH(2′)-I and MPH(2′)-II complexes, the site of enzymatic modification is indicated with an asterisk. Structure 2017 25, 750-761.e5DOI: (10.1016/j.str.2017.03.007) Copyright © 2017 Elsevier Ltd Terms and Conditions