Bordetella pertussis CyaA-RTX subdomain requires calcium ions for structural stability against proteolytic degradation

• Main Authors: Pichaya Pojanapotha, Niramon Thamwiriyasati, Busaba Powthongchin, Gerd Katzenmeier, Chanan Angsuthanasombat
• Other Authors: Mahidol University
• Format: Article
• Published: 2018
• Subjects: Biochemistry, Genetics and Molecular Biology
• Online Access: https://repository.li.mahidol.ac.th/handle/123456789/11593

Previously, the 126-kDa Bordetella pertussis CyaA pore-forming (CyaA-PF) domain expressed in Escherichia coli was shown to retain its hemolytic activity. Here, a 100-kDa RTX (Repeat-in-ToXin) subcloned fragment (CyaA-RTX) containing a number of putative calcium-binding repeats was further investigated. The recombinant CyaA-RTX protein, although expressed as a soluble form in a protease-deficient E. coli strain BL21(DE3)pLysS, was found to be highly sensitive to proteolytic degradation. Interestingly, the addition of calcium ions in a millimolar range into the CyaA-RTX preparation significantly prevented the degradation. Moreover, levels of proteolytic degradation were dependent on calcium concentrations, implying an important role for calcium-binding sites in the RTX subdomain for structural stability. Homology-based modeling of the repetitive blocks in the CyaA-RTX subdomain supports that this calcium-bound protein folds into a parallel β-roll structure with calcium ions acting as a structural stabilizing bridge. © 2010 Elsevier Inc. All rights reserved.