Cryo-EM structure of Mycobacterium tuberculosis 50S ribosomal subunit bound with clarithromycin reveals dynamic and specific interactions with macrolides

ABSTRACT

Tuberculosis (TB) is the leading infectious disease caused by Mycobacterium tuberculosis (Mtb). Clarithromycin (CTY), an analog of erythromycin (ERY), is more potent against multidrug-resistance (MDR) TB. ERY and CTY were previously reported to bind to the nascent polypeptide exit tunnel (NPET) near peptidyl transferase center (PTC), but the only available CTY structure in complex with D. radiodurans (Dra) ribosome could be misinterpreted due to resolution limitation. To date, the mechanism of specificity and efficacy of CTY for Mtb remains elusive since the Mtb ribosome-CTY complex structure is still unknown. Here, we employed new sample preparation methods and solved the Mtb ribosome-CTY complex structure at 3.3Å with cryo-EM technique, where the crucial gate site A2062 (E. coli numbering) is located at the CTY binding site within NPET. Two alternative conformations of A2062, a novel syn-conformation as well as a swayed conformation bound with water molecule at interface, may play a role in coordinating the binding of specific drug molecules. The previously overlooked C–H hydrogen bond (H-bond) and π interaction may collectively contribute to the enhanced binding affinity. Together, our structure data provide a structural basis for the dynamic binding as well as the specificity of CTY and explain of how a single methyl group in CTY improves its potency, which provides new evidence to reveal previously unclear mechanism of translational modulation for future drug design and anti-TB therapy. Furthermore, our sample preparation method may facilitate drug discovery based on the complexes with low water solubility drugs by cryo-EM technique.

KEYWORDS:

• Mycobacterium tuberculosis
• 50S ribosomal subunit
• clarithromycin
• gate site a2062
• dynamic interaction
• Cryo-EM

Acknowledgements

We are grateful to Prof. Jamie Cate for constructive suggestions and You Xu, Jingdong Chen and Alastair Murchie for valuable discussions. We thank Yao Cong for help with the initial data collection and Jianxun Qi and Jiawei Wang for help with the data processing and model building. We also thank Prof. Fei Lan, Jianxin Gu and Yuanyuan Ruan Xiaodan Zhang, Rui Sun and Gang Chen for help with the experiments. We thank the Electron Microscopy facility of the National Center for Protein Science of Shanghai (NCPSS) and the Center of Cryo-Electron Microscopy of Fudan University for the support of the initial cryo-EM data collection, also thank the image center at Institute of Biophysics of Chinese Academy of Sciences (IBP-CAS) for the support of the final full data set collection. The data processing and modeling were supported by the Medical Research Data Center of Fudan University.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the National Natural Science Foundation of China (Grant No. 31725007 to G.N., 11179012 to W.Z, 81772231 to Y.Z., 81673335 to J.L.), Key Discipline Construction Plan from Shanghai Municipal Health Commission (GWV-10.1-XK01 to W-H.Z.), Shanghai Hospital Development Center (SHDC2020CR1011B to W-H.Z), Shanghai Science and Technology Committee(20dz2260100 and 20dz2210400 to W-H.Z), Shanghai Municipal Commission of Science and Technology Research Project(19JC1411001 to Q.X.) and the National Key Basic Research and Development Plan (2011CB710800 to W.Z.)