Macrolide-Peptide Conjugates as Probes of the Path of Travel of the Nascent Peptides through the Ribosome

• Type of Material: Article
• Authors: Arren Z. Washington, Georgia Institute of Technology; Derek B. Benicewicz, Georgia Institute of Technology; Joshua C. Canzoneri, Georgia Institute of Technology; Crystal E. Fagan, Emory University; Sandra C. Mwakwari, Georgia Institute of Technology; Tatsuya Maehigashi, Emory University; Christine Dunham, Emory University; Adegboyega K. Oyelere, Georgia Institute of Technology
• Language: English
• Date: 2014-11-01
• Publisher: American Chemical Society
• Publication Version: Final Published Version
• Copyright Statement: © 2014 American Chemical Society
• Final Published Version (URL): http://dx.doi.org/10.1021/cb5003224
• Title of Journal or Parent Work: ACS Chemical Biology
• ISSN: 1554-8929
• Volume: 9
• Issue: 11
• Start Page: 2621
• End Page: 2631
• Grant/Funding Information: This work is based on research conducted at the Advanced Photon Source on the NE-CAT beamlines, which is supported by National Center for Research Resources (NIH) Award RR-15301, and at the SER-CAT beamline.; Use of the Advanced Photon Source, an Office of Science User Facility operated for the US Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the US DOE under Contract DE-AC02-06CH11357.; CEF support was provided by the Department of Defense through the National Defense Science and Engineering Graduate Fellowship Program (NDSEG) and NIH Training Grant T32 GM8367. C.M.D. is a Pew Scholar in the Biomedical Sciences.; This work was financially supported in part by NASA Astrobiology Institute [NNA09DA78A] (A.K.O.) and National Institutes of Health (NIH) Award R01GM093278 (to C.M.D.). J.C.C. and A.Z.W. are thankful recipients of the GAANN predoctoral fellowships from the Georgia Tech Center for Drug Design, Development, and Delivery and from the School of Chemistry and Biochemistry, respectively.; National Institutes of Health, United States
• Keywords: MESSENGER-RNA; Biochemistry & Molecular Biology; ANGSTROM RESOLUTION; TERMINAL ALKYNES; HISTONE DEACETYLASE INHIBITORS; ANTIBACTERIAL ACTIVITY; ESCHERICHIA-COLI RIBOSOME; STRUCTURAL INSIGHT; TRANSFERASE CENTER; Science & Technology; 70S RIBOSOME; EXIT TUNNEL; Life Sciences & Biomedicine
• Research Categories: Chemistry, Biochemistry

Abstract

Despite decades of research on the bacterial ribosome, the ribosomal exit tunnel is still poorly understood. Although it has been suggested that the exit tunnel is simply a convenient route of egress for the nascent chain, specific protein sequences serve to slow the rate of translation, suggesting some degree of interaction between the nascent peptide chain and the exit tunnel. To understand how the ribosome interacts with nascent peptide sequences, we synthesized and characterized a novel class of probe molecules. These peptide-macrolide (or "peptolide") conjugates were designed to present unique peptide sequences to the exit tunnel. Biochemical and X-ray structural analyses of the interactions between these probes and the ribosome reveal interesting insights about the exit tunnel. Using translation inhibition and RNA structure probing assays, we find the exit tunnel has a relaxed preference for the directionality (N → C or C → N orientation) of the nascent peptides. Moreover, the X-ray crystal structure of one peptolide derived from a positively charged, reverse Nuclear Localization Sequence peptide, bound to the 70S bacterial ribosome, reveals that the macrolide ring of the peptolide binds in the same position as other macrolides. However, the peptide tail folds over the macrolide ring, oriented toward the peptidyl transferase center and interacting in a novel manner with 23S rRNA residue C2442 and His69 of ribosomal protein L4. These data suggest that these peptolides are viable probes for interrogating nascent peptide-exit tunnel interaction.