Bordetella pertussis CyaA-RTX subdomain requires calcium ions for structural stability against proteolytic degradation.
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.
Pojanapotha
Pichaya
P
Laboratory of Molecular Biophysics and Structural Biochemistry, Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand.
Thamwiriyasati
Niramon
N
Powthongchin
Busaba
B
Katzenmeier
Gerd
G
Angsuthanasombat
Chanan
C
D037361:Adenylate Cyclase Toxin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001886:Bordetella pertussis / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D037361:Adenylate Cyclase Toxin; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D002118:Calcium