Structural characterization and low-resolution model of BJ-48, a thrombin-like enzyme from Bothrops jararacussu venom.
Thrombin-like enzymes (TLEs) are important components of snake venoms due to their involvement in coagulopathies occurring on envenoming. Structural characterization of this group of serine proteases is of utmost importance for better understanding their unique properties. However, the high carbohydrate content of some members of this group prevents successful crystallization for structural determination. Circumventing this difficulty, the structure of BJ-48, a highly glycosylated TLE from Bothrops jararacussu venom, was studied in solution. At pH 8.0, where the enzyme displays maximum activity, BJ-48 has a radius of gyration (Rg) of 37 A and a maximum dimension (D(max)) of 130 A as measured by small-angle X-ray scattering (SAXS) and a Stokes radius (SR) of 50 A according to dynamic light scattering (DLS) data. At the naturally more acidic pH (6.0) of the B. jararacussu venom BJ-48 behaves as a more compact particle as evidenced by SAXS (R(g)=27.9 A and D(max)=82 A) and DLS (SR=30 A) data. In addition, Kratky plot analysis indicates a rigid shape at pH 8.0 and a flexible shape at pH 6.0. On the other hand, the center of mass of intrinsic fluorescence was not changed while varying pH, possibly indicating the absence of fluorescent amino acids in the regions affected by pH variation. Circular dichroism experiments carried out with BJ-48 indicate a substantially random coiled secondary structure that is not affected by pH. Low-resolution model of BJ-48 presented a prolate elongated shape at pH 8.0 and a U-shape at 6.0. BJ-48 tertiary structure at pH 6.0 was maintained on heating up to 52 degrees C and was completely lost at 75 degrees C. The possible existence of two pH-induced folding states for BJ-48 and its importance for the biological role and stability of this enzyme was discussed.
Guedes
Herbert L M
HL
Laboratório de Bioquímica de Proteínas e Peptídeos, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, RJ, Brazil.
Silva
Floriano P
FP
Netto
Carlos Correa
CC
de Salles
Cristiane M C
CM
Alexandre
Giani
G
Oliveira
Cristiano L P
CL
Torriani
Iris
I
De Simone
Salvatore Giovanni
SG
D000818:Animals; D017837:Bothrops; D003435:Crotalid Venoms / Q000737:chemistry*; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D015394:Molecular Structure; D011487:Protein Conformation; D017510:Protein Folding; D013917:Thrombin / Q000737:chemistry
D003435:Crotalid Venoms; C030961:botrocetin; D013917:Thrombin