| 1. |
- Di Yu, Xiao, et al.
(författare)
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Large Is Fast, Small Is Tight : Determinants of Speed and Affinity in Subunit Capture by a Periplasmic Chaperone
- 2012
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 417:4, s. 294-308
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Tidskriftsartikel (refereegranskat)abstract
- The chaperone/usher pathway assembles surface virulence organelles of Gram-negative bacteria, consisting of fibers of linearly polymerized protein subunits. Fiber subunits are connected through 'donor strand complementation': each subunit completes the immunoglobulin (Ig)-like fold of the neighboring subunit by donating the seventh beta-strand in trans. Whereas the folding of Ig domains is a fast first-order process, folding of Ig modules into the fiber conformation is a slow second-order process. Periplasmic chaperones separate this process in two parts by forming transient complexes with subunits. Interactions between chaperones and subunits are also based on the principle of donor strand complementation. In this study, we have performed mutagenesis of the binding motifs of the Caf1M chaperone and Caf1 capsular subunit from Yersinia pestis and analyzed the effect of the mutations on the structure, stability, and kinetics of Caf1M-Caf1 and Caf1-Caf1 interactions. The results suggest that a large hydrophobic effect combined with extensive main-chain hydrogen bonding enables Caf1M to rapidly bind an early folding intermediate of Caf1 and direct its partial folding. The switch from the Caf1M-Caf1 contact to the less hydrophobic, but considerably tighter and less dynamic Caf1-Caf1 contact occurs via the zip-out-zip-in donor strand exchange pathway with pocket 5 acting as the initiation site. Based on these findings, Caf1M was engineered to bind Caf1 faster, tighter, or both faster and tighter. To our knowledge, this is the first successful attempt to rationally design an assembly chaperone with improved chaperone function.
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| 2. |
- Kumar Gahlot, Dharmender, Senior Research Engineer, 1985-, et al.
(författare)
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Diversity in genetic regulation of bacterial fimbriae assembled by the chaperone usher pathway
- 2023
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Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 24:1
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Forskningsöversikt (refereegranskat)abstract
- Bacteria express different types of hair-like proteinaceous appendages on their cell surface known as pili or fimbriae. These filamentous structures are primarily involved in the adherence of bacteria to both abiotic and biotic surfaces for biofilm formation and/or virulence of non-pathogenic and pathogenic bacteria. In pathogenic bacteria, especially Gram-negative bacteria, fimbriae play a key role in bacteria–host interactions which are critical for bacterial invasion and infection. Fimbriae assembled by the Chaperone Usher pathway (CUP) are widespread within the Enterobacteriaceae, and their expression is tightly regulated by specific environmental stimuli. Genes essential for expression of CUP fimbriae are organised in small blocks/clusters, which are often located in proximity to other virulence genes on a pathogenicity island. Since these surface appendages play a crucial role in bacterial virulence, they have potential to be harnessed in vaccine development. This review covers the regulation of expression of CUP-assembled fimbriae in Gram-negative bacteria and uses selected examples to demonstrate both dedicated and global regulatory mechanisms.
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| 3. |
- Kumar Gahlot, Dharmender, 1985-, et al.
(författare)
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Optimised Heterologous Expression and Functional Analysis ofthe Yersinia pestis F1-Capsular Antigen Regulator Caf1R
- 2021
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Ingår i: International Journal of Molecular Sciences. - Switzerland : MDPI. - 1661-6596 .- 1422-0067. ; 22:18
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Tidskriftsartikel (refereegranskat)abstract
- The bacterial pathogen, Yersinia pestis, has caused three historic pandemics and continuesto cause small outbreaks worldwide. During infection, Y. pestis assembles a capsule-like protectivecoat of thin fibres of Caf1 subunits. This F1 capsular antigen has attracted much attention due to itsclinical value in plague diagnostics and anti-plague vaccine development. Expression of F1 is tightlyregulated by a transcriptional activator, Caf1R, of the AraC/XylS family, proteins notoriously prone toaggregation. Here, we have optimised the recombinant expression of soluble Caf1R. Expression fromthe native and synthetic codon-optimised caf1R cloned in three different expression plasmids wasexamined in a library of E. coli host strains. The functionality of His-tagged Caf1R was demonstratedin vivo, but insolubility was a problem with overproduction. High levels of soluble MBP-Caf1R wereproduced from codon optimised caf1R. Transcriptional-lacZ reporter fusions defined the PM promoterand Caf1R binding site responsible for transcription of the cafMA1 operon. Use of the identifiedCaf1R binding caf DNA sequence in an electrophoretic mobility shift assay (EMSA) confirmed correctfolding and functionality of the Caf1R DNA-binding domain in recombinant MBP-Caf1R. Availabilityof functional recombinant Caf1R will be a valuable tool to elucidate control of expression of F1 andCaf1R-regulated pathophysiology of Y. pestis.
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| 4. |
- Yu, Xiaodi, et al.
(författare)
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Allosteric Mechanism Controls Traffic in the Chaperone/Usher Pathway
- 2012
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Ingår i: Structure. - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 20, s. 1861-1871
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Tidskriftsartikel (refereegranskat)abstract
- Many virulence organelles of Gram-negative bacterial pathogens are assembled via the chaperone/usher pathway. The chaperone transports organelle subunits across the periplasm to the outer membrane usher, where they are released and incorporated into growing fibers. Here, we elucidate the mechanism of the usher-targeting step in assembly of the Yersinia pestis F1 capsule at the atomic level. The usher interacts almost exclusively with the chaperone in the chaperone:subunit complex. In free chaperone, a pair of conserved proline residues at the beginning of the subunit-binding loop form a "proline lock" that occludes the usher-binding surface and blocks usher binding. Binding of the subunit to the chaperone rotates the proline lock away from the usher-binding surface, allowing the chaperone-subunit complex to bind to the usher. We show that the proline lock exists in other chaperone/usher systems and represents a general allosteric mechanism for selective targeting of chaperone:subunit complexes to the usher and for release and recycling of the free chaperone.
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| 5. |
- Zavialov, Anton V, et al.
(författare)
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Resolving the energy paradox of chaperone/usher-mediated fibre assembly
- 2005
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 389:Pt 3, s. 685-694
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Tidskriftsartikel (refereegranskat)abstract
- Periplasmic chaperone/usher machineries are used for assembly of filamentous adhesion organelles of Gram-negative pathogens in a process that has been suggested to be driven by folding energy. Structures of mutant chaperone–subunit complexes revealed a final folding transition (condensation of the subunit hydrophobic core) on the release of organelle subunit from the chaperone–subunit pre-assembly complex and incorporation into the final fibre structure. However, in view of the large interface between chaperone and subunit in the pre-assembly complex and the reported stability of this complex, it is difficult to understand how final folding could release sufficient energy to drive assembly. In the present paper, we show the X-ray structure for a native chaperone–fibre complex that, together with thermodynamic data, shows that the final folding step is indeed an essential component of the assembly process. We show that completion of the hydrophobic core and incorporation into the fibre results in an exceptionally stable module, whereas the chaperone–subunit pre-assembly complex is greatly destabilized by the high-energy conformation of the bound subunit. This difference in stabilities creates a free energy potential that drives fibre formation.
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