Binding of Ca2+ and Zn2+ to factor IX/X-binding protein from venom of Agkistrodon halys Pallas: stabilization of the structure during GdnHCl-induced and thermally induced denaturation.
Coagulation factor IX/coagulation factor X binding protein from the venom of Agkistrodon halys Pallas (AHP IX/X-bp) is a unique coagulation factor IX/coagulation factor X binding protein (IX/X-bp). Among all IX/X-bps identified, only AHP IX/X-bp is a Ca(2+)- and Zn(2+)-binding protein. The binding properties of Ca(2+) and Zn(2+) ions binding to apo-AHP IX/X-bp and their effects on the stability of the protein have been investigated by isothermal titration calorimetry, fluorescence spectroscopy, and differential scanning calorimetry. The results show that AHP IX/X-bp has two metal binding sites, one specific for Ca(2+) with lower affinity for Zn(2+) and one specific for Zn(2+) with lower affinity for Ca(2+). The bindings of Ca(2+) and Zn(2+) in the two sites are entropy- and enthalpy-driven. The binding affinity of AHP IX/X-bp for Zn(2+) is 1 order of magnitude higher than for Ca(2+) for either high-affinity binding or low-affinity binding, which accounts for the existence of one Zn(2+) in the purified AHP IX/X-bp. Guanidine hydrochloride (GdnHCl)-induced and thermally induced denaturations of Ca(2+)-Ca(2+)-AHP IX/X-bp, Zn(2+)-Zn(2+)-AHP IX/X-bp, and Ca(2+)-Zn(2+)-AHP IX/X-bp are all a two-state processes with no detectable intermediate state(s), indicating the Ca(2+)/Zn(2+)-induced tight packing of the protein. Ca(2+) and Zn(2+) increase the structural stability of AHP IX/X-bp against GdnHCl or thermal denaturation to a similar extent. Although Ca(2+) and Zn(2+) have no obvious effect on the secondary structure of AHP IX/X-bp, they induce different rearrangements in local conformation. The Zn(2+)-stabilized specific conformation of AHP IX/X-bp may be helpful to its recognition of the structure of coagulation factor IX. This work suggests that in vitro, Ca(2+) plays a structural rather than an active role in the anticoagulation of AHP IX/X-bp, whereas Zn(2+) plays both structural and active roles in the anticoagulation. In blood, Ca(2+) binds to AHP IX/X-bp and stabilizes its structure, whereas Zn(2+) cannot bind to AHP IX/X-bp owing to the low Zn(2+) concentration. AHP IX/X-bp prolongs the clotting time in vivo through its binding only with coagulation factor X/activated coagulation factor X.
Wu
Hao
H
Department of Chemistry, University of Science and Technology of China, Hefei, People's Republic of China.
Xu
Xiaolong
X
Shen
Dengke
D
Peng
Lili
L
Song
Jiajia
J
Zhang
Yan
Y
D017836:Agkistrodon; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D003435:Crotalid Venoms / Q000737:chemistry*; D005164:Factor IX / Q000737:chemistry*; D005170:Factor X / Q000737:chemistry*; D019791:Guanidine / Q000494:pharmacology; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects; D013329:Structure-Activity Relationship; D013696:Temperature; D015032:Zinc / Q000737:chemistry*
C031442:Agkistrodon venoms; D003435:Crotalid Venoms; D005164:Factor IX; D005170:Factor X; D015032:Zinc; D019791:Guanidine; D002118:Calcium