NMR Solution Structure of a Chymotrypsin Inhibitor from the Taiwan Cobra Naja naja atra

Yi-Jan Lin,Peter Güntert,Teppei Ikeya,Long-Sen Chang

doi:10.3390/molecules18088906

Abstract

The Taiwan cobra (Naja naja atra) chymotrypsin inhibitor (NACI) consists of 57 amino acids and is related to other Kunitz-type inhibitors such as bovine pancreatic trypsin inhibitor (BPTI) and Bungarus fasciatus fraction IX (BF9), another chymotrypsin inhibitor. Here we present the solution structure of NACI. We determined the NMR structure of NACI with a root-mean-square deviation of 0.37 Å for the backbone atoms and 0.73 Å for the heavy atoms on the basis of 1,075 upper distance limits derived from NOE peaks measured in its NOESY spectra. To investigate the structural characteristics of NACI, we compared the three-dimensional structure of NACI with BPTI and BF9. The structure of the NACI protein comprises one 310-helix, one α-helix and one double-stranded antiparallel β-sheet, which is comparable with the secondary structures in BPTI and BF9. The RMSD value between the mean structures is 1.09 Å between NACI and BPTI and 1.27 Å between NACI and BF9. In addition to similar secondary and tertiary structure, NACI might possess similar types of protein conformational fluctuations as reported in BPTI, such as Cys14–Cys38 disulfide bond isomerization, based on line broadening of resonances from residues which are mainly confined to a region around the Cys14–Cys38 disulfide bond.

Highlights

The Naja naja atra chymotrypsin inhibitor (NACI) is a protease inhibitor present in the venom of the Taiwan cobra (Naja naja atra). This chymotrypsin inhibitor belongs to the class of non-neurotoxic snake Kunitz/bovine pancreatic trypsin inhibitor (BPTI) inhibitors [1], which are different from their snake Kunitz/BPTI neurotoxic homologues, such as dendrotoxins, calcicludine and the B chain of β-bungarotoxin which act as Ca2+
Several unusual chemical shifts were observed in NACI
Based on the three-dimensional structure of NACI, the observed unusual chemical shifts can be attributed to aromatic ring current effects

Summary

Introduction

The Naja naja atra chymotrypsin inhibitor (NACI) is a protease inhibitor present in the venom of the Taiwan cobra (Naja naja atra). Variable residues located in the weak contact loop provide different interactions with various proteases. These surrounding residues are important for inhibitor protease specificity as well [3,7]. In addition to the P1 residue, based on the amino acid sequence alignment of NACI with other Kunitz/BPTI protease inhibitors, some conserved residues, such as Gly, Tyr, Phe, Tyr, Gly, Asn, and Phe probably play important roles in contact with the protease [3,8]. Due to isomerization of the Cys14–Cys disulfide bond, two conformational isomers with different chirality were observed in BPTI in the NMR spectra [9,10]. It was reported that isomerization of the Cys side chain between χ1 rotamers is faster than the corresponding Cys isomerization [10]

Chemical Shift Assignment

Structure Determination and Solution Structure of NaCl

Sample Preparation

NMR Measurements

Structure Calculation

Conclusions