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Chaperone/usher machinery

a catalyst of virulence organelle assembly

Yu, Xiaodi (2012). Chaperone/usher machinery. Diss. (sammanfattning/summary) Uppsala : Sveriges lantbruksuniv., Acta Universitatis Agriculturae Sueciae, 1652-6880 ; 2012:41
ISBN 978-91-576-7677-1
[Doctoral thesis]

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Many virulence organelles of Gram-negative bacterial pathogens are assembled via the periplasmic chaperone/usher (CU) pathway. The assembly process is a complex task, involving secretion of organelle subunits via the two membranes and periplasm, subunit folding and assembly. In this thesis, the mechanism of the organelle subunit trafficking and assembly via the CU pathway was investigated at different steps, subunit capture by the chaperone (paper II), usher targeting (paper III), and transport through the usher (paper I), using the Caf1M/Caf1M CU system that assembles the Yersinia pestis F1 capsular antigen from Caf1 subunits.
In paper II, we performed mutagenesis of the binding motifs of the Caf1M chaperone and Caf1 subunit and analyzed the effect of the mutations on structure, stability, and kinetics of Caf1M-Caf1 and Caf1-Caf1 interactions. We show that a large hydrophobic effect combined with extensive main-chain hydrogen bonding enable Caf1M to rapidly capture/fold Caf1 subunit. The switch from the Caf1M-Caf1 contact to the 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 with improved chaperone function was engineered. In paper III, we elucidated the mechanism of the usher-targeting step at atomic resolution. We show that a pair of conserved proline residues in free chaperone forms a ‘proline lock’, which blocks the usher binding. Binding of subunit to the chaperone opens the proline lock and allows the chaperone-subunit complex to bind to the usher. We show that this proline lock exists in other CU systems and represents a general allosteric mechanism for selective targeting of chaperone:subunit complexes to the usher and for release and recycling of free chaperone. In paper I, a novel middle domain of Caf1A usher (UMD) was isolated and its crystal structure was determined. We show that UMD and Caf1 fibre subunit displayed significant structural similarity. UMD did not bind Caf1M-Caf1 complexes, but its presence was shown to be essential for Caf1-fibre secretion. The study suggests that UMD may play the role of a subunit-substituting protein (dummy subunit), plugging or priming secretion through the channel in the Caf1A usher.

Authors/Creators:Yu, Xiaodi
Title:Chaperone/usher machinery
Subtitle:a catalyst of virulence organelle assembly
Series Name/Journal:Acta Universitatis Agriculturae Sueciae
Year of publishing :2012
Number of Pages:49
i.Yu, X.*, Visweswaran, G.R.*, Duck, Z.*, Marupakula, S., MacIntyre, S., Knight, S.D. & Zavialov, A.V. (2009). Caf1A usher possesses a Caf1 subunit-like domain that is crucial for Caf1 fibre secretion. Biochem J 418(3):541-51
II.Yu, X.*, Fooks, L.J.*, Moslehi-Mohebi, E., Tischenko, V.M., Askarieh, G., Knight, S.D., Macintyre, S. & Zavialov, A.V. (2012). Large Is Fast, Small Is Tight: Determinants of Speed and Affinity in Subunit Capture by a Periplasmic Chaperone. J Mol Biol. 471(4), 294-308.
III.Yu, X.*, Dubnovitsky, A.P.*, Alex, F., MacIntyre, S., Knight, S.D. & Zavialov, A.V. Allosteric mechanism controls traffic in the chaperone/usher pathway (Submitted)
Place of Publication:Uppsala
Publisher:Dept. of Molecular Biology, Swedish University of Agricultural Sciences
ISBN for printed version:978-91-576-7677-1
Publication Type:Doctoral thesis
Full Text Status:Public
Agris subject categories.:X Agricola extesions > X30 Life sciences
X Agricola extesions > X60 Technology
Subjects:Obsolete subject words > NATURAL SCIENCES
Obsolete subject words > TECHNOLOGY > Bioengineering
Agrovoc terms:proteins, pathogens, biochemical pathways
Keywords:chaperone/usher pathway, protein-protein interaction, protein structure, allosteric regulation, assembly, folding, secretion
Permanent URL:
ID Code:8873
Department:(NL, NJ) > Dept. of Molecular Biology (until 131231)
External funders:FORMAS and Swedish Research Council
Deposited By: Dr Xiaodi Yu
Deposited On:15 May 2012 09:00
Metadata Last Modified:02 Dec 2014 10:50

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