Functional analysis of enzymes involved in the co-translational modification of nascent chains
Final Report Abstract
Bacteria, unlike eukaryotes, initiate protein synthesis with a specialized initiator tRNA loaded with a formylated methionine. However, functional proteins do not retain the N-terminal formyl-group and often contain N-terminal residues other than methionine. The variability of the protein N-termini is the consequence of two consecutive co-translational maturation processes, deformylation and methionine excision, which are catalyzed by the essential processing enzymes peptide deformylase (PDF) and methionine aminopeptidase (MAP). The main focus of the project was (i) to understand how PDF and MAP action is spatially and temporally coordinated with the translating ribosome and (ii) to understand how their interaction with ribosome and nascent polypeptide is coordinated with the action of other ribosome bound maturation factors such as the chaperone trigger factor and the targeting factors SRP and SecA to ensure highly efficient synthesis of functional proteins. In the course of the project we show via co-sedimentation assays that E. coli PDF and MAP interact with ribosomes through ionic interactions. We further identified the binding regions on PDF and MAP and their docking sites at the ribosome with a combined approach of chemical crosslinking methods, mass spectrometry, mutational analysis, X-ray crystallography and in silico modeling. This led to the first model of ribosomes engaged with PDF or MAP and provided significant new insights how early processing enzymes are arranged on the ribosomal surface near the polypeptide exit tunnel to catalyze N-terminal modifications of nascent chains. Furthermore, we established an in vitro transcription-translation assay that allowed measuring co- translational processing of nascent polypeptides. Using enolase and barnase as in vitro model substrates, we showed that PDF and MAP proteolytically process nascent polypeptides as soon as the N-terminus emerges from the tunnel exit. The action of PDF and MAP can occur simultaneously with SRP-mediated targeting of ribosome-nascent chain complexes to facilitate efficient processing and targeting of nascent inner-membrane proteins to the membrane-embedded translocon. In contrast processing precedes co-translational folding attempts of the nascent chain and nascent chain interaction with the ribosome-bound chaperone Trigger Factor. The coordination of processing, co-translational targeting and chaperone-assisted folding is facilitated (i) by very rapid interaction kinetics of the enzymes and SRP with the ribosome and (ii) by a delayed recruitment of trigger factor which binds translating ribosomes only after a significant part of the protein (about 80 residues) is already exposed on the ribosomal surface. This project led to the first detailed understanding of the coordination of factors involved in nascent chain maturation during protein synthesis in bacteria. This furthermore motivated new projects in our laboratory including studying the N-terminal acetylation of proteins in eukaryotic cells, a highly ubiquitous modification of nascent chains catalyzed by at least five different acteyltransferases that also must act in concert with methionine aminopeptidases, chaperones and targeting factors in eukaryotic cells. It will be highly important to find out to what extent the principles of the coordination of factors engaged in protein maturation in bacteria also apply to the more complicated scenario of protein synthesis in eukaryotic organisms.
Publications
- A peptide deformylase-ribosome complex reveals mechanism of nascent chain processing. Nature 452, 108-111 (2008)
Bingel-Erlenmeyer, R. et al.
(See online at https://doi.org/10.1038/nature06683) - The ribosome as a platform for co-translational processing, folding and targeting of newly synthesized proteins. Nature structural & molecular biology 16, 589-597 (2009)
Kramer, G., Boehringer, D., Ban, N. & Bukau, B.
(See online at https://doi.org/10.1038/nsmb.1614) - Selective ribosome profiling reveals the cotranslational chaperone action of trigger factor in vivo. Cell 147, 1295-1308 (2011)
Oh, E. et al.
(See online at https://doi.org/10.1016/j.cell.2011.10.044) - Dynamic enzyme docking to the ribosome coordinates N-terminal processing with polypeptide folding. Nature structural & molecular biology 20 (2013)
Sandikci, A. et al.
(See online at https://doi.org/10.1038/nsmb.2615) - Co-translational mechanisms of protein maturation. Current opinion in structural biology 24, 24-33 (2014)
Gloge, F., Becker, A. H., Kramer, G. & Bukau, B.
(See online at https://doi.org/10.1016/j.sbi.2013.11.004)
