Solution Structures of a Kunitz-type chymotrypsin inhibitor from Bungarus fasciatus
Chun-Hua Hsu1,2,3, Shih-Hsiung Wu1,3, Shyh-Horng Chiou1,3, Ning-Yuan Su2, Chinpan Chen2
1Institute of Biochemical Sciences, National Taiwan University, and 2Institute of Biomedical Sciences, 3Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, R.O.C.
BF9 is the fraction IX component isolated from the venom of Bungarus fasciatus and consists of 65 amino acid residues with three disulfide bridges. It is a chymotrypsin inhibitor and belongs to a member of the BPTI-like superfamily. The 3D NMR solution structures of BF9 were determined on the basis of 536 interproton, 33 tortional-angle and 30 hydrogen-bond restraints by simulated annealing and energy minimization calculations using the X-PLOR program. The final set of 10 NMR structures is exceptionally well defined with average pairwise RMS differences of 0.47 Å for the backbone atoms in the secondary structure regions and 0.86 Å in residues 3-58. Based on the 3D structure, the unusual chemical shifts observed for BF9 were found to be due to an aromatic ring current effect. The side chains of Phe23, Tyr24, Tyr25, Phe35 and Phe47 exhibit a large number of long-range NOEs and are the principal components of the hydrophobic core in BF9. Most of the residues with slowly exchanging amide protons were found to be located in the b-sheet and C-terminal a-helical regions. This indicates that the secondary structures in BF9 are stable and rigid. To gain insight into the structure-function among the proteins in the BPTI-like superfamily, we compared the 3D structure of BF9 with three BPTI-like proteins (BPTI, DTK and TAP) that possess distinct biological functions. We found that these proteins possess similar secondary elements, but the loop regions and b-turn are different from each other. Based on the functional site residues of each protein, the amino acid types, the flexibility and the rigidity in the loop regions and the b-turn are considered to be related to various biological functions in BPTI-like superfamily.