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| Home > Publications database > Identifizierung von Bindungsdeterminanten von Tat-Vorläuferproteinen an den TatBC-Rezeptorkomplex während der Tat-abhängigen Proteintranslokation in $\textit{Escherichia coli}$ |
| Home > Publications database > Identifizierung von Bindungsdeterminanten von Tat-Vorläuferproteinen an den TatBC-Rezeptorkomplex während der Tat-abhängigen Proteintranslokation in $\textit{Escherichia coli}$ |
| Book/Dissertation / PhD Thesis | FZJ-2017-07951 |
2018
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
Jülich
ISBN: 978-3-95806-290-0
Please use a persistent id in citations: http://hdl.handle.net/2128/17389 urn:nbn:de:0001-2018032821
Abstract: The Tat translocase has the remarkable ability to transport fully folded proteins across the cytoplasmic membrane of bacteria. In contrast to the well-studied Sec system, many aspects of the Tat-dependent translocation mechanism are still unexplored. The present work was focused on the binding step of Tat precursor proteins to the TatBC receptor complex, which is a crucial part of the translocation process. A productive interaction between the Tat substrate and the TatBC receptor complex is required for a successful translocation across the membrane. Most studies of the interactions between Tat substrates and the TatBC receptor complex focused primarily on the Tat consensus motif in the n-region of the Tat signal peptide containing two highly conserved twin arginine residues, which are indisputably the most important binding determinants of Tat precursor proteins to the TatBC substrate receptor. However, it remained unclear whether other regions of the Tat signal peptide and/or the early mature protein region also contribute to the overall binding of the Tat substrate to TatBC. Previous studies of this working group revealed that, besides an intact twin arginine motif, a minimal uninterrupted hydrophobic (h-) region in the signal peptide is required for effective Tat-dependent export in $\textit{Escherichia coli}$. These results suggested that a certain minimum length of the h-region is required to establish a productive interaction between the Tat substrate and the TatBC receptor complex. In the first part of the present work, the direct participation of the hydrophobic core of the signal peptide of the reporter protein TorA-30aa-MalE in the binding process to the TatBC receptor complex was investigated using a genetic approach. Using an exportdefective reporter variant TorA[KQ]-30aa-MalE, in which the twin arginine residues had been substituted by a lysine-glutamine pair (KQ), as starting point for the mutagenesis study, suppressing mutations in the h region of the TorA[KQ] signal peptide were identified, which compensated to a certain degree for the loss of the important binding contact at the twin arginine position and significantly restored the Tat-dependent export. Combinations of these mutations strongly increased the export demonstrating that the h-region-located mutations synergistically act together in promoting export of the otherwise transport-incompetent TorA[KQ]-30aa-MalE precursor protein. Interestingly, all of these mutations increased the overall hydrophobicity of the h-region. Furthermore, a positive correlation between the hydrophobicity and the translocation efficiency of the respective TorA[KQ]-30aa-MalE reporter variant could be observed. These results indicated that the newly introduced hydrophobic amino acid residues in the h-region enhanced the substrate-receptor-interactions within the hydrophobic TatBC binding cavity at the stage of advanced binding, thereby sufficiently increasing the overall binding affinity required for productive binding of the otherwise export-defective reporter variant. To directly analyze whether the h-region is in fact directly involved in productive TatBC receptor binding, site-specific $\textit{in vitro}$ crosslinking experiments were performed. It was shown that – in contrast to the negative control harboring an unaltered h-region - the signal peptides of the mutated TorA[KQ]-30aa-MalE variants had been threaded deep into the TatBC binding cavity, even in the absence of an intact twin arginine motif. These results confirmed that the mutations in the h-region exert their effect by directly affecting the binding step of the TorA signal peptide to the TatBC receptor complex upon insertion into the TatBC binding cavity and prior to the translocation of the Tat substrate across the membrane. Taken together, the results of the first part of the present work show that, besides the Tat consensus motif, the h-region of Tat signal peptides is another important binding determinant of Tat precursor proteins to the TatBC receptor complex. In the second part of this work, the direct contribution of the the early mature protein region of Tat substrates to the overall binding affinity to the TatBC receptor complex was investigated. Suppressing mutations in the early mature protein region of TorA[KQ]-30aa-MalE (in the 30 amino acids following the signal peptide, i.e. the 30aa-region) could be successfully identified which restored the export of the otherwise transport-incompetent reporter protein by either increasing the overall hydrophobicity or by increasing the positive net charge of the early mature protein part. While the newly introduced hydrophobic amino acids in the N-terminal part of the 30aa-region - analogous to the h-region located mutations - most likely enhanced the hydrophobic interactions within the TatBC binding cavity, the additional positively charged residues in the C-terminal half of the 30aa-region presumably increased the binding affinity of the precursor protein to the cytoplasmically localized domains of the TatBC receptor complex. In both cases, the early mature protein part interacts specifically with the Tat translocase strongly suggesting that this region of the precursor protein is also directly involved in the binding process to the TatBC receptor complex. Taken together, the present work provides direct evidence that the highly conserved twin arginine residues in the Tat consensus motif of Tat signal peptides are not strictly required for Tat-dependent protein export across the membrane, if compensatory mutations in the h-region of the signal peptide or the early mature protein region were present which enhance binding of the Tat substrate to the TatBC receptor complex at a non-twin arginine position. The results clearly demonstrate that, besides the Tat consensus motif, the h-region of Tat signal peptides and the early mature protein region are other important binding determinants which play a pivotal role in the binding process of Tat precursor proteins to the TatBC receptor complex.
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