Abstract
Messenger-RNA-directed protein synthesis is accomplished by the ribosome1,2,3. In eubacteria, this complex process is initiated by a specialized transfer RNA charged with formylmethionine (tRNAfMet)4,5,6. The amino-terminal formylated methionine of all bacterial nascent polypeptides blocks the reactive amino group to prevent unfavourable side-reactions and to enhance the efficiency of translation initiation7,8. The first enzymatic factor that processes nascent chains is peptide deformylase (PDF)5,9,10,11; it removes this formyl group as polypeptides emerge from the ribosomal tunnel12,13 and before the newly synthesized proteins can adopt their native fold, which may bury the N terminus. Next, the N-terminal methionine is excised by methionine aminopeptidase14. Bacterial PDFs are metalloproteases sharing a conserved N-terminal catalytic domain. All Gram-negative bacteria, including Escherichia coli, possess class-1 PDFs characterized by a carboxy-terminal α-helical extension15. Studies focusing on PDF as a target for antibacterial drugs14,16 have not revealed the mechanism of its co-translational mode of action despite indications in early work that it co-purifies with ribosomes17. Here we provide biochemical evidence that E. coli PDF interacts directly with the ribosome via its C-terminal extension. Crystallographic analysis of the complex between the ribosome-interacting helix of PDF and the ribosome at 3.7 Å resolution reveals that the enzyme orients its active site towards the ribosomal tunnel exit for efficient co-translational processing of emerging nascent chains. Furthermore, we have found that the interaction of PDF with the ribosome enhances cell viability. These results provide the structural basis for understanding the coupling between protein synthesis and enzymatic processing of nascent chains, and offer insights into the interplay of PDF with the ribosome-associated chaperone trigger factor.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Ramakrishnan, V. Ribosome structure and the mechanism of translation. Cell 108, 557–572 (2002)
Moore, P. B. & Steitz, T. A. The ribosome revealed. Trends Biochem. Sci. 30, 281–283 (2005)
Liljas, A. Deepening ribosomal insights. ACS Chem. Biol. 1, 567–569 (2006)
Marcker, K. & Sanger, F. N-formyl-methionyl-s-RNA. J. Mol. Biol. 8, 835–840 (1964)
Adams, J. M. & Capecchi, M. R. N-formylmethionyl-sRNA as the initiator of protein synthesis. Proc. Natl Acad. Sci. USA 55, 147–155 (1966)
Laursen, B. S., Sorensen, H. P., Mortensen, K. K. & Sperling-Petersen, H. U. Initiation of protein synthesis in bacteria. Microbiol. Mol. Biol. Rev. 69, 101–123 (2005)
Eisenstadt, J. & Lengyel, P. Formylmethionyl-tRNA dependence of amino acid incorporation in extracts of trimethoprim-treated Escherichia coli. Science 154, 524–527 (1966)
Harvey, R. J. Growth and initiation of protein synthesis in Escherichia coli in the presence of trimethoprim. J. Bacteriol. 114, 309–322 (1973)
Fry, K. T. & Lamborg, M. R. Amidohydrolase activity of Escherichia coli extracts with formylated amino acids and dipeptides as substrates. J. Mol. Biol. 28, 423–433 (1967)
Adams, J. M. On the release of the formyl group from nascent protein. J. Mol. Biol. 33, 571–589 (1968)
Pine, M. J. Kinetics of maturation of the amino termini of the cell proteins of Escherichia coli. Biochim. Biophys. Acta 174, 359–372 (1969)
Housman, D., Gillespie, D. & Lodish, H. F. Removal of formyl-methionine residue from nascent bacteriophage f2 protein. J. Mol. Biol. 65, 163–166 (1972)
Ball, L. A. & Kaesberg, P. Cleavage of the N-terminal formylmethionine residue from a bacteriophage coat protein in vitro. J. Mol. Biol. 79, 531–537 (1973)
Giglione, C., Boularot, A. & Meinnel, T. Protein N-terminal methionine excision. Cell. Mol. Life Sci. 61, 1455–1474 (2004)
Guilloteau, J. P. et al. The crystal structures of four peptide deformylases bound to the antibiotic actinonin reveal two distinct types: a platform for the structure-based design of antibacterial agents. J. Mol. Biol. 320, 951–962 (2002)
Giglione, C., Pierre, M. & Meinnel, T. Peptide deformylase as a target for new generation, broad spectrum antimicrobial agents. Mol. Microbiol. 36, 1197–1205 (2000)
Takeda, M. & Webster, R. E. Protein chain initiation and deformylation in B. subtilis homogenates. Proc. Natl Acad. Sci. USA 60, 1487–1494 (1968)
Meinnel, T. et al. The C-terminal domain of peptide deformylase is disordered and dispensable for activity. FEBS Lett. 385, 91–95 (1996)
Schuwirth, B. S. et al. Structures of the bacterial ribosome at 3.5 Å resolution. Science 310, 827–834 (2005)
Becker, A. et al. Iron center, substrate recognition and mechanism of peptide deformylase. Nature Struct. Biol. 5, 1053–1058 (1998)
Hardesty, B. & Kramer, G. Folding of a nascent peptide on the ribosome. Prog. Nucleic Acid Res. Mol. Biol. 66, 41–66 (2001)
Ferbitz, L. et al. Trigger factor in complex with the ribosome forms a molecular cradle for nascent proteins. Nature 431, 590–596 (2004)
Agashe, V. R. et al. Function of trigger factor and DnaK in multidomain protein folding: increase in yield at the expense of folding speed. Cell 117, 199–209 (2004)
Kaiser, C. M. et al. Real-time observation of trigger factor function on translating ribosomes. Nature 444, 455–460 (2006)
Raue, U., Oellerer, S. & Rospert, S. Association of protein biogenesis factors at the yeast ribosomal tunnel exit is affected by the translational status and nascent polypeptide sequence. J. Biol. Chem. 282, 7809–7816 (2007)
Vetro, J. A. & Chang, Y. H. Yeast methionine aminopeptidase type 1 is ribosome-associated and requires its N-terminal zinc finger domain for normal function in vivo. J. Cell. Biochem. 85, 678–688 (2002)
Addlagatta, A. et al. Identification of an SH3-binding motif in a new class of methionine aminopeptidases from Mycobacterium tuberculosis suggests a mode of interaction with the ribosome. Biochemistry 44, 7166–7174 (2005)
Ragusa, S., Blanquet, S. & Meinnel, T. Control of peptide deformylase activity by metal cations. J. Mol. Biol. 280, 515–523 (1998)
Wegrzyn, R. D. & Deuerling, E. Molecular guardians for newborn proteins: ribosome-associated chaperones and their role in protein folding. Cell. Mol. Life Sci. 62, 2727–2738 (2005)
Cull, M. G. & Schatz, P. J. Biotinylation of proteins in vivo and in vitro using small peptide tags. Methods Enzymol. 326, 430–440 (2000)
Vila-Sanjurjo, A., Schuwirth, B. S., Hau, C. W. & Cate, J. H. Structural basis for the control of translation initiation during stress. Nature Struct. Mol. Biol. 11, 1054–1059 (2004)
Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Crystallogr. 26, 795–800 (1993)
Adams, P. D. et al. Recent developments in the PHENIX software for automated crystallographic structure determination. J. Synchrotron Radiat. 11, 53–55 (2004)
Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron-density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991)
Haldimann, A. & Wanner, B. L. Conditional-replication, integration, excision, and retrieval plasmid-host systems for gene structure-function studies of bacteria. J. Bacteriol. 183, 6384–6393 (2001)
Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. USA 97, 6640–6645 (2000)
Harlow, E. & Lane, D. P. Antibodies: A Laboratory Manual 313–315 (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1988)
Acknowledgements
Crystallographic data were collected at the beamline X06SA at the Swiss Light Source (SLS). We are grateful to C. Schulze-Briese, S. Gutmann, E. Pohl, S. Russo and T. Tomizaki for their outstanding support at the SLS. We thank B. Mikolasek for ribosome preparation, F. Parmeggiani and A. Plückthun at the University of Zurich for assistance with the surface plasmon resonance measurements and access to the Biacore 3000 instrument, R. Brunisholz at the Functional Genomics Center Zurich for mass-spectrometric analysis, D. Böhringer, J. Erzberger, S. Jenni and M. Müller for critically reading the manuscript and all members of the Ban laboratory for suggestions and discussions. This work was supported by the Swiss National Science Foundation (SNSF) and the National Center of Excellence in Research (NCCR) Structural Biology programme of the SNSF.
Author information
Authors and Affiliations
Corresponding author
Supplementary information
Supplementary Information
The file contains Supplementary Results, Supplementary Figures 1-6 with Legends, Supplementary Tables 1-2 and additional references. (PDF 3323 kb)
Rights and permissions
About this article
Cite this article
Bingel-Erlenmeyer, R., Kohler, R., Kramer, G. et al. A peptide deformylase–ribosome complex reveals mechanism of nascent chain processing. Nature 452, 108–111 (2008). https://doi.org/10.1038/nature06683
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nature06683
This article is cited by
-
Quality control of protein synthesis in the early elongation stage
Nature Communications (2023)
-
Kinetic control of nascent protein biogenesis by peptide deformylase
Scientific Reports (2021)
-
A Review: Molecular Chaperone-mediated Folding, Unfolding and Disaggregation of Expressed Recombinant Proteins
Cell Biochemistry and Biophysics (2021)
-
Structural basis of translation inhibition by cadazolid, a novel quinoxolidinone antibiotic
Scientific Reports (2019)
-
Multitasking of Hsp70 chaperone in the biogenesis of bacterial functional amyloids
Communications Biology (2018)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.