Crystallization and preliminary X-ray analysis of coagulation factor IX-binding protein from habu snake venom at pH 6.5 and 4.6

Abstract

Coagulation factor IX-binding protein isolated from Trimeresurus¯avoviridis (IX-bp) is a C-type lectin-like protein. It is an anticoagulant protein consisting of homologous subunits A and B. The subunits both contain a Ca 2+ -binding site with differing af®nity (K d values of 14 and 130 mM at pH 7.5). These binding characteristics are pH-dependent; under acidic conditions, the af®nity of the low-af®nity site was reduced considerably. In order to identify which site has high af®nity and also to investigate the Ca 2+ -releasing mechanism, IX-bp was crystallized at pH 6.5 and 4.6. The crystals at pH 6.5 and 4.6 diffracted to 1.72 and 2.29 A Ê resolution, respectively; the former crystals belong to the monoclinic space group P2 1 , with unit-cell parameters a = 60.7, b = 63.5, c = 66.9 A Ê , = 117.0 , while the latter belong to the monoclinic space group C2, with a = 134.1, b = 37.8, c = 55.8 A Ê , = 110.4 .

pH-Dependent Structural Changes at Ca2+-binding Sites of Coagulation Factor IX-binding Protein

“…Crystallization and data collection were done as described. 20 Structure determination and refinement Initial models of habu IX-bp at pH 6.5 and apo habu IX-bp were obtained by the molecular replacement method, with the program AMoRe 21 in the CCP4 program package, 22 using the coordinates from the previously solved crystal structure of habu IX-bp at pH 7.8 (PDB code 1bj3) 2 as a search model. The resultant models were first subjected to rigid-body refinement, followed by cycles of simulated annealing, energy minimization and individual B-factor refinement using CNS, 23 with visual inspection and manual modification of the models using QUANTA2000 (Accelrys) and XtalView.…”

Venomics and Cellular Toxicity of Thai Pit Vipers (Trimeresurus macrops and T. hageni)

“…C-type lectin protein is a non-enzymatic, calcium dependent protein that binds sugar residues [36]. Many snake venom c-type lectin-like proteins target coagulation factors [37]. The Southeast Asian T. albolabris c-type lectin protein binds directly to the von Willebrand factor receptor, which has key functions in both the hemostatic and thrombotic pathways, leading to platelet agglutination [38].…”