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Title: Zn<sup>2+</sup>-dependent autocatalytic activity of the Bordetella pertussis CyaA-hemolysin
Authors: Veerada Raksanoh
Lalida Shank
Panchika Prangkio
Mattayaus Yentongchai
Somsri Sakdee
Chompounoot Imtong
Chanan Angsuthanasombat
Chiang Mai University
Mahidol University
Prince of Songkla University
Biophysics Institute for Research and Development (BIRD)
Keywords: Biochemistry, Genetics and Molecular Biology
Issue Date: 15-Apr-2017
Citation: Biochemical and Biophysical Research Communications. Vol.485, No.4 (2017), 720-724
Abstract: © 2017 Elsevier Inc. Proteolytic degradation of the ∼100-kDa isolated RTX (Repeat-in-ToXin) subdomain (CyaA-RTX) of the Bordetella pertussis CyaA-hemolysin (CyaA-Hly) was evidently detected upon solely-prolonged incubation. Here, a truncated CyaA-Hly fragment (CyaA-HP/BI) containing hydrophobic and acylation regions connected with the first RTX block (BI1015–1088) was constructed as a putative precursor for investigating its potential autocatalysis. The 70-kDa His-tagged CyaA-HP/BI fragment which was over-expressed in Escherichia coli as insoluble aggregate was entirely solubilized with 4 M urea. After re-naturation in a Ni2+-NTA affinity column, the purified-refolded CyaA-HP/BI fragment in HEPES buffer (pH 7.4) supplemented with 2 mM CaCl2was completely degraded upon incubation at 37 °C for 3 h. Addition of 1,10-phenanthroline‒an inhibitor of Zn2+-dependent metalloproteases markedly reduced the extent of degradation for CyaA-HP/BI and CyaA-RTX, but the degradative effect was clearly enhanced by addition of 100 mM ZnCl2. Structural analysis of a plausible CyaA-HP/BI model revealed a potential Zn2+-binding His-Asp cluster located between the acylation region and RTX-BI1015–1088. Moreover, Arg997‒one of the identified cleavage sites of the CyaA-RTX fragment was located in close proximity to the Zn2+-binding catalytic site. Overall results demonstrated for the first time that the observed proteolysis of CyaA-HP/BI and CyaA-RTX fragments is conceivably due to their Zn2+-dependent autocatalytic activity.
ISSN: 10902104
Appears in Collections:Scopus 2016-2017

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