Ribosome protection by antibiotic resistance ATP-binding cassette protein Weixin Sua,b,1, Veerendra Kumarc,1, Yichen Dingd,1, Rya Eroa,b,2, Aida Serraa, Benjamin Sian Teck Leea, Andrew See Weng Wongb, Jian Shie, Siu Kwan Szea, Liang Yanga,d,2, and Yong-Gui Gaoa,b,c,2 aSchool of Biological Sciences, Nanyang Technological University, Singapore 637551; bInstitute of Structural Biology, Nanyang Technological University, Singapore 639798; cInstitute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore 138673; dSingapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University, Singapore 637551; and eCentre for BioImaging Sciences, National University of Singapore, Singapore 117557 Edited by Peter B. Moore, Yale University, New Haven, CT, and approved April 11, 2018 (received for review February 23, 2018) The ribosome is one of the richest targets for antibiotics. Unfortu- peptide bond formation that underlies translation arrest (10, 11). nately, antibiotic resistance is an urgent issue in clinical practice. However, certain oligopeptides are believed to lead to drug re- Several ATP-binding cassette family proteins confer resistance to sistance by “flushing out” the macrolides while passing through ribosome-targeting antibiotics through a yet unknown mechanism. the NPET (13, 14). Therefore, the fate of the macrolide-bound Among them, MsrE has been implicated in macrolide resistance. Here, ribosome is determined by the dynamic interactions among the we report the cryo-EM structure of ATP form MsrE bound to the bound drug, the PTC, and the sequence specificity of the emerging ribosome. Unlike previously characterized ribosomal protection pro- oligopeptide chain (5, 15). It should be noted, however, that teins,MsrEisshowntobindtoribosomal exit site. Our structure macrolides could induce ribosomal arrest by allosterically altering reveals that the domain linker forms a unique needle-like arrangement the PTC even without forming significant contacts with the nascent with two crossed helices connected by an extended loop projecting chain, demonstrating the existence of a functional link between the into the peptidyl-transferase center and the nascent peptide exit NPET and the PTC (16). tunnel, where numerous antibiotics bind. In combination with bio- A wide range of mechanisms mediate antibiotic resistance, one chemical assays, our structure provides insight into how MsrE binding of the greatest threats to public health and food security world- leads to conformational changes, which results in the release of the wide. In the case of macrolides, various bacterial species are in- drug. This mechanism appears to be universal for the ABC-F type trinsically insensitive due to chromosomal mutations in ribosomal ribosome protection proteins. genes causing reduced drug-binding efficiency (17, 18). The most common acquired resistance mechanism is the posttranscriptional ribosome protection | antibiotic resistance | ABC-F | MsrE | methylation of the 23S rRNA by methyltransferases (e.g., Erm protein synthesis family), which also results in decreased drug-binding efficiency (1, ore than one-half of the antibiotics in clinical use target Significance Mbacterial ribosome and protein synthesis, particularly the elongation step (1). The peptidyl-transferase center (PTC) and ARE ABC-F genes have been found in numerous pathogen genomes the adjacent nascent peptide exit tunnel (NPET) in the ribosomal and multi-drug resistance conferring plasmids. Further transmission large subunit are the key players in protein elongation, with func- will challenge the clinical use of many antibiotics. The development tions in catalyzing the peptide bond formation and the emergence of of improved ribosome-targeting therapeutics relies on the elucida- the nascent chain, respectively. PTC-targeting antibiotics, such as tion of the resistance mechanisms. Characterization of MsrE protein chloramphenicol, group A streptogramins, lincosamides, and pleu- bound to the bacterial ribosome is first of its kind for ARE ABC-F members. Together with biochemical data, it sheds light on the ri- romutilins, inhibit protein synthesis by interfering with the correct BIOCHEMISTRY positioning of the tRNA substrates (1, 2). In contrast, macrolides bosome protection mechanism by domain linker-mediated confor- and group B streptogramins bind to a site within the NPET adjacent mational change and displacement leading to drug release, to the PTC and immediately before the constriction point at which suggesting a mechanism shared by other ARE ABC-F proteins. These ribosomal proteins (r-proteins) L4 and L22 narrow the tunnel width proteins present an intriguing example of structure-function re- to approximately 10 Å (3, 4). lationship and a medically relevant target of study as they collec- The primary mechanism of macrolide action is believed to be the tively mediate resistance to the majority of antibiotic classes context-specific inhibition of peptide bond formation rather than targeting the peptidyl-transferase center region. the indiscriminate obstruction of nascent chain passage through the NPET. Namely, ribosome-profiling analyses have revealed that Author contributions: L.Y. and Y.-G.G. initiated the project; Y.-G.G. directed the project; W.S., V.K., Y.D., R.E., A.S., B.S.T.L., and S.K.S. performed the experiments; W.S., V.K., R.E., translation of most genes proceeds past the first six to eight codons and Y.-G.G. analyzed the data, determined the structure, and built the model; A.S.W.W. and can be arrested at any point during the translation when the and J.S. helped with data collection; and W.S., V.K., R.E., and Y.-G.G. wrote the manu- ribosome encounters specific short-sequence motifs (5–7). The script with input from all members. problematic sequence motifs are confined to the nascent peptide The authors declare no conflict of interest. residues in the PTC, not the peptide segment in contact with the This article is a PNAS Direct Submission. macrolide further down the NPET (5). Therefore, it appears that This open access article is distributed under Creative Commons Attribution-NonCommercial- the general mode of macrolide action involves selective inhibition NoDerivatives License 4.0 (CC BY-NC-ND). of peptide bond formation between specific combinations of donor Data deposition: The atomic coordinates and cryo-EM map have been deposited in the and acceptor substrates. In some cases, the macrolide-induced and Protein Data Bank, www.wwpdb.org (PDB ID code 5ZLU) and Electron Microscopy Data leader peptide-mediated translational arrest is used to regulate the Bank (accession no. EMD-6934), respectively. expression of downstream macrolideresistancemethyltransferase 1W.S., V.K., and Y.D. contributed equally to this work. genes, such as ermB and ermC (8–12). 2To whom correspondence may be addressed. Email: [email protected], yangliang@ntu. Structural characterization of the erythromycin-ErmBL leader edu.sg, or [email protected]. peptide-ribosome complex reveals that the drug redirects the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. path of the peptide in the tunnel and leads to conformational 1073/pnas.1803313115/-/DCSupplemental. changes in PTC and tRNA substrates unable to participate in the Published online April 30, 2018. www.pnas.org/cgi/doi/10.1073/pnas.1803313115 PNAS | May 15, 2018 | vol. 115 | no. 20 | 5157–5162 Downloaded by guest on September 24, 2021 19). Ribosome protection mechanism, by which the drug is actively removed from the ribosome, has recently become of great interest. The ATP-binding cassette F (ABC-F) family proteins confer resistance to a number of clinically relevant antibiotics targeting the ribosome PTC/NPET region (20). These proteins are collectively referred to as antibiotic resistance (ARE) ABC-F proteins (21). Unlike other ARE ABC family members that are shown to actively pump drugs out of the cells, ARE ABC-F proteins lack the trans- membrane domain characteristic to transporters and are believed to confer antibiotic resistance via ribosomal protection mechanism by interacting with the ribosome and displacing the bound drug (17, 20, 22–24). ARE ABC-F proteins have been classified into three groups based on antibiotic resistance: (i) Msr homologs (macrolides and streptogramin B), (ii) Vga/Lsa/Sal homologs (lincosamides, pleuromutilins, and streptogramin A), and (iii) OptrA homologs (phenicols and oxazolidinones) (20) (SI Appendix,Fig.S1). Notably, these proteins compose two ATP-binding domains connected by a linker of various lengths, which appears to be crucial for the effi- ciency and specificity of antibiotic resistance (20). However, an understanding of the molecular mechanism of how ARE ABC-F proteins interact with ribosomes to mediate antibiotic resistance requires a high-resolution structure of the complex. Here we report the cryo-EM structure of the ARE ABC-F protein MsrE bound to the bacterial ribosome at 3.6-Å resolution. Note that the Msr homologs (divided into four classes: A, C, D, and E) have the longest domain linkers among the ARE ABC-F members (SI Appendix,Fig.S1). Furthermore, we have previously identified a Pseudomonas aeruginosa clinical isolate carrying the msrE gene obtained through horizontal gene transfer (NCBI ac- Fig. 1. Cryo-EM structure of the MsrE-ribosome complex. (A) Electron density cession ID: CP020704), which highlights the importance of the of the overall complex. Ribosome 50S and 30S subunits are shown in pale ARE ABC-F