Refined crystal structure (2.3 �) of a double-headed winged bean ?-chymotrypsin inhibitor and location of its second reactive site

Abstract

The crystal structure of a doubleheaded ␣-chymotrypsin inhibitor, WCI, from winged bean seeds has now been refined at 2.3 Å resolution to an R-factor of 18.7% for 9,897 reflections. The crystals belong to the hexagonal space group P6 1 22 with cell parameters a ‫؍‬ b ‫؍‬ 61.8 Å and c ‫؍‬ 212.8 Å .The final model has a good stereochemistry and a root mean square deviation of 0.011 Å and 1.14°from ideality for bond length and bond angles, respectively. A total of 109 ordered solvent molecules were localized in the structure. This improved structure at 2.3 Å led to an understanding of the mechanism of inhibition of the protein against ␣-chymotrypsin. An analysis of this higher resolution structure also helped us to predict the location of the second reactive site of the protein, about which no previous biochemical information was available. The inhibitor structure is spherical and has twelve antiparallel ␤-strands with connecting loops arranged in a characteristic ␤-trefoil fold common to other homologous serine protease inhibitors in the Kunitz (STI) family as well as to some non homologous functionally unrelated proteins. A wide variation in the surface loop regions is seen in the latter ones. Proteins 1999;35:321-331.1999 Wiley-Liss, Inc.

Functional Analysis of the Kunitz Trypsin Inhibitor Family in Poplar Reveals Biochemical Diversity and Multiplicity in Defense against Herbivores

“…We used secondary and tertiary structure predictions to predict reactive loop position, which provides a better estimate than sequence alignments with divergent plant KTIs. A similar approach has been used to predict the second reactive site of a winged-bean chymotrypsin inhibitor (Dattagupta et al, 1999). However, precise definition of the reactive loops and sites of poplar KTIs will require site-directed mutagenesis or structural determination.…”

The Plasticity of the β-Trefoil Fold Constitutes an Evolutionary Platform for Protease Inhibition

“…However, despite the abundance of data obtained from x-ray crystallography for free inhibitors and complexes with serine proteases (7)(8)(9)(12)(13)(14), very little is known on the specifics of multiple target recognition by these Janus-type proteins with only one structure in the Protein Data Bank of a Kunitz-STI inhibitor bound to two protease molecules (7).…”

“…It is commonly assumed that most members of this family have only a single reactive site loop, which for the archetypical soybean trypsin inhibitor (STI) is located between residues Ser-60 and Phe-66. However, several cases of inhibitors possessing two reactive sites, and thus binding two target molecules simultaneously, have been reported (7)(8)(9)(10). These have been dubbed "double-headed" or "Janus-type" inhibitors.…”

The Ternary Structure of the Double-headed Arrowhead Protease Inhibitor API-A Complexed with Two Trypsins Reveals a Novel Reactive Site Conformation

“…Most members have only one reactive site located in the region of residues 60 -70 (7,10,(12)(13)(14). However, a few members possess two reactive sites that simultaneously bind two protease molecules and are thus termed double-headed inhibitors (15)(16)(17)(18). All of these inhibitors are classified into family I3 of peptidase inhibitors (19).…”

“…Both reactive sites adopt a typical noncovalent "lock and key" inhibitory mechanism. The two reactive sites of API-A are located in loops 6 and 10, respectively, in contrast to the sole reactive site in loop 5 (residues 60 -67), which is usually found in typical Kunitztype inhibitors (8,11,13,18,42). The phenomenon of switching reactive site loops for double-headed inhibitors is not unique to API-A and has been proposed earlier for loop 3 of winged bean chymotrypsin inhibitor based on its crystal structure (18).…”