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Exploring the “N-Terminal Anchor” Binding Interface of the T3SS Chaperone–Translocator Complexes from P. aeruginosa
[Image: see text] The type III secretion system is a large multiprotein complex that many Gram-negative bacteria use for infection. A crucial part of the complex is its translocon pore formed by two proteins: the major and minor translocators. The pore completes a proteinaceous channel from the bact...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical Society
2023
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10116596/ https://www.ncbi.nlm.nih.gov/pubmed/36996474 http://dx.doi.org/10.1021/acs.biochem.3c00002 |
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author | Frankling, Charlotte L. Kang, Angray S. Main, Ewan R. G. |
author_facet | Frankling, Charlotte L. Kang, Angray S. Main, Ewan R. G. |
author_sort | Frankling, Charlotte L. |
collection | PubMed |
description | [Image: see text] The type III secretion system is a large multiprotein complex that many Gram-negative bacteria use for infection. A crucial part of the complex is its translocon pore formed by two proteins: the major and minor translocators. The pore completes a proteinaceous channel from the bacterial cytosol through the host cell membrane and allows the direct injection of bacterial toxins. Effective pore formation is predicated by the translocator proteins binding to a small chaperone within the bacterial cytoplasm. Given the vital role of the chaperone–translocator interaction, we investigated the specificity of the “N-terminal anchor” binding interface present in both translocator–chaperone complexes from Pseudomonas aeruginosa. Isothermal calorimetry (ITC), alanine scanning, and the selection of a motif-based peptide library using ribosome display were used to characterize the major (PopB) and minor (PopD) translocator interactions with their chaperone PcrH. We show that 10 mer PopB(51–60) and PopD(47–56) peptides bind to PcrH with a K(D) of 148 ± 18 and 91 ± 9 μM, respectively. Moreover, mutation to alanine of each of the consensus residues (xxVxLxxPxx) of the PopB peptide severely affected or completely abrogated binding to PcrH. When the directed peptide library (X-X-hydrophobic-X-L-X-X-P-X-X) was panned against PcrH, there was no obvious convergence at the varied residues. The PopB/PopD wild-type (WT) sequences were also not prevalent. However, a consensus peptide was shown to bind to PcrH with micromolar affinity. Thus, selected sequences were binding with similar affinities to WT PopB/PopD peptides. These results demonstrate that only the conserved “xxLxxP” motif drives binding at this interface. |
format | Online Article Text |
id | pubmed-10116596 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | American Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-101165962023-04-21 Exploring the “N-Terminal Anchor” Binding Interface of the T3SS Chaperone–Translocator Complexes from P. aeruginosa Frankling, Charlotte L. Kang, Angray S. Main, Ewan R. G. Biochemistry [Image: see text] The type III secretion system is a large multiprotein complex that many Gram-negative bacteria use for infection. A crucial part of the complex is its translocon pore formed by two proteins: the major and minor translocators. The pore completes a proteinaceous channel from the bacterial cytosol through the host cell membrane and allows the direct injection of bacterial toxins. Effective pore formation is predicated by the translocator proteins binding to a small chaperone within the bacterial cytoplasm. Given the vital role of the chaperone–translocator interaction, we investigated the specificity of the “N-terminal anchor” binding interface present in both translocator–chaperone complexes from Pseudomonas aeruginosa. Isothermal calorimetry (ITC), alanine scanning, and the selection of a motif-based peptide library using ribosome display were used to characterize the major (PopB) and minor (PopD) translocator interactions with their chaperone PcrH. We show that 10 mer PopB(51–60) and PopD(47–56) peptides bind to PcrH with a K(D) of 148 ± 18 and 91 ± 9 μM, respectively. Moreover, mutation to alanine of each of the consensus residues (xxVxLxxPxx) of the PopB peptide severely affected or completely abrogated binding to PcrH. When the directed peptide library (X-X-hydrophobic-X-L-X-X-P-X-X) was panned against PcrH, there was no obvious convergence at the varied residues. The PopB/PopD wild-type (WT) sequences were also not prevalent. However, a consensus peptide was shown to bind to PcrH with micromolar affinity. Thus, selected sequences were binding with similar affinities to WT PopB/PopD peptides. These results demonstrate that only the conserved “xxLxxP” motif drives binding at this interface. American Chemical Society 2023-03-30 /pmc/articles/PMC10116596/ /pubmed/36996474 http://dx.doi.org/10.1021/acs.biochem.3c00002 Text en © 2023 The Authors. Published by American Chemical Society https://creativecommons.org/licenses/by/4.0/Permits the broadest form of re-use including for commercial purposes, provided that author attribution and integrity are maintained (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Frankling, Charlotte L. Kang, Angray S. Main, Ewan R. G. Exploring the “N-Terminal Anchor” Binding Interface of the T3SS Chaperone–Translocator Complexes from P. aeruginosa |
title | Exploring the
“N-Terminal Anchor” Binding
Interface of the T3SS Chaperone–Translocator Complexes from P. aeruginosa |
title_full | Exploring the
“N-Terminal Anchor” Binding
Interface of the T3SS Chaperone–Translocator Complexes from P. aeruginosa |
title_fullStr | Exploring the
“N-Terminal Anchor” Binding
Interface of the T3SS Chaperone–Translocator Complexes from P. aeruginosa |
title_full_unstemmed | Exploring the
“N-Terminal Anchor” Binding
Interface of the T3SS Chaperone–Translocator Complexes from P. aeruginosa |
title_short | Exploring the
“N-Terminal Anchor” Binding
Interface of the T3SS Chaperone–Translocator Complexes from P. aeruginosa |
title_sort | exploring the
“n-terminal anchor” binding
interface of the t3ss chaperone–translocator complexes from p. aeruginosa |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10116596/ https://www.ncbi.nlm.nih.gov/pubmed/36996474 http://dx.doi.org/10.1021/acs.biochem.3c00002 |
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