3-DIMENSIONAL SOLUTION STRUCTURE OF CUCURBITA-MAXIMA TRYPSIN INHIBITOR-V DETERMINED BY NMR-SPECTROSCOPY

The solution structure of Cucurbita maxima trypsin inhibitor-V (CMTI-V ), which is also a specific inhibitor of the blood coagulation protein , factor XII(a), was determined by H-1 NMR spectroscopy in combination with a distance-geometry and simulated annealing algorithm. Sequence- specific resonance assignments were made for all the main-chain and mo st of the side-chain hydrogens. Stereospecific assignments were also m ade for some of the beta-, gamma-, delta-, and epsilon-hydrogens and v aline methyl hydrogens. The ring conformations of all six prolines in the inhibitor were determined on the basis of H-1-H-1 vicinal coupling constant patterns; most of the proline ring hydrogens were stereospec ifically assigned on the basis of vicinal coupling constant and intrar esidue nuclear Overhauser effect (NOE) patterns. Distance constraints were determined on the basis of NOEs between pairs of hydrogens. Dihed ral angle constraints were determined from estimates of scalar couplin g constants and intraresidue NOEs. On the basis of 727 interproton dis tance and 111 torsion angle constraints, which included backbone phi a ngles and side-chain chi(1), chi(2), chi(3), and chi(4) angles, 22 str uctures were calculated by a distance geometry algorithm and refined b y energy minimization and simulated annealing methods. Both main-chain and side-chain atoms are well-defined, except for a loop region, two terminal residues, and some side-chain atoms located on the molecular surface. The average root mean squared deviation in the position for e quivalent atoms between the 22 individual structures and the mean stru cture obtained by averaging their coordinates is 0.58 +/- 0.06 Angstro m for the main-chain atoms and 1.01 +/- 0.07 Angstrom for all the non- hydrogen atoms of residues 3-40 and 49-67. These structures were compa red to the X-ray crystallographic structure of another protein of the same inhibitor family-chymotrypsin inhibitor-2 from barley seeds [CI-2 ; McPhalen, C. A., & James, M. N. G. (1987) Biochemistry 26, 261-269]. The main-chain folding patterns are highly similar for the two protei ns, which possess 62% sequence differences. However, major differences are noted in the N- and C-terminal segments, which may be due to the presence of a disulfide bridge in CMTI-V, but not in CI-2.