Structure of a Kunitz-Type Chymotrypsin from Winged Bean Seeds at 2.95 Å Resolution

Thc crystal structure of an α-chymotrypsin inhibitor (P6₁22; a = 61.4, c = 210.9 Å) isolated from winged bean (Psophocarpus. tetragonolobus) seeds has been determined at 2.95 Å resolution by the molecular-replacement method using the 2.6 Å coordinates of Erythrina trypsin inhibitor (ETI) as the starting model (57% sequence homology). This protease inhibitor, WCI, belongs to the Kunitz (STI) family and is a single polypeptide chain with 183 amino-acid residues having a molecular weight of 20 244 Da. Structure refinement with RESTRAIN and X-PLOR has led to a crystallographic R factor of 19.1% for 3469 observed reflections (I > 2σ) in the resolution range 8–2.95 Å. A total of 56 water molecules have been incorporated in the refined model containing 181 amino-acid residues. In the refined structure the deviations of bond lengths and bond angles from ideal values are 0.015 Å and 2.2°, respectively. The inhibitor molecule is spherical and consists of 12 antiparallel β-strands with connecting loops arranged in a characteristic folding (a six-stranded β-barrel and a six-stranded lid on one hollow end of the barrel) common to other homologous serine protease inhibitors in the Kunitz (STI) family as well as to some non-homologous proteins like interleukin-lα and interleukin-lβ. In the structure the conformation of the protruding reactive-site loop is stabilized through hydrogen bonds mainly formed by the side chain of Asnl4, which intrudes inside the cavity of the reactive-site loop, with the side-chain and main-chain atoms of some residues in the loop region. A pseudo threefold axis exists parallel to the barrel axis of the structure. Each of the three subdomains comprises of four β-strands with connecting loops.