The 0.25‐nm X‐ray structure of the Bowman‐Birk‐type inhibitor from mung bean in ternary complex with porcine trypsin

Abstract

The structure of the Bowman-Birk-type inhibitor from mung bean Phuseolus uureus has been determined in ternary complex with porcine trypsin. The complex formed crystals of the trigonal space group P3,21 which diffracted to a resolution of 250 pm. Each of the two mung bean protease reactive sites is bound to trypsin according to the standard mechanism for serine proteinase inhibition. The binding loops thereby adopt the canonical conformation for the standard mechanism; however, the sub-van der Waals contact between the active-site serine O Y (195) and the P1 carbonyl carbon of both loops is significantly smaller (210 pm) than hitherto observed, with continuous electron density connecting the two atoms. The inhibitor is formed by two double-stranded antiparallel P-sheets, which are connected into a moderately twisted P-sheet by a network of hydrogen bonds involving main-chain atoms and two water molecules. All contacts with neighbors in the crystal lattice occur between trypsin molecules. This apparently gives rise to an unusual form of disorder where the complexes pack in two orientations Ta : MaMb : Tb and Tb : MbMa : Ta (Ta, Tb = trypsin, Ma = mung bean loop I, Mb = mung bean loop II), such that the asymmetric unit consists of the ternary complex in two orientations, each with half occupancy. This is nearly equivalent to an asymmetric unit which has one trypsin molecule with full occupancy and one mung bean inhibitor with half occupancy and a crystallographic twofold symmetry axis through its center. Because of the approximate twofold symmetry of the inhibitor itself, however, the electron density was interpretable for most of the inhibitor (17 residues at the termini were not resolved) and shows evidence of its double orientation.The Bowman-Birk-type inhibitors, isolated from leguminous seeds (Bowman, 1946), are quite small (60-90 residues) and are characterized (Birk et. al., 1963) by a high sulfur content and the ability to independently and simultaneously inhibit two serine proteinases, typically trypsin or chymotrypsin. The covalent structure, first determined for the soybean inhibitor (Odani and Jkenaka, 1973), showed the residue sequence to contain seven disulfide bridges and two tandem repeats, each with an active binding loop. Their physiological function is not known, although many potential roles have been suggested; anticarcinogenic effects have been observed. For reviews of Bowman-Birk inhibitors, see Ikenaka and Norioka (1986) or Birk (1985).The specificity of the Bowman-Birk inhibitors indicated that they inhibit via the standard mechanism (Laskowski and Kato, 1980), by which inhibitors with lysine or arginine at the P1 residue typically bind trypsin, and those with an aro- Read and James, 1986), show that these inhibitors mimic substrates, whereby the binding loop adopts the 'canonical conformation' at the reactive site and the adjoining residues. This conformation is common to all the standard inhibitors despite quite different folding motifs and differing stabilizing interactions between th...

The Three-dimensional Structure of Recombinant Leech-derived Tryptase Inhibitor in Complex with Trypsin

“…A total of 139,563 reflections (21,466 independent reflections, 98.6% complete to 1.9 Å) were measured with an overall R sym of 0.082. The structure was solved using AMoRe (28), with the trypsin component of the porcine trypsin-mung bean inhibitor complex (29) For expression in S. cerevisiae, the mutated rLDTI genes were isolated by XbaI/HindIII cleavage and ligated into yeast shuttle vector pVT102U/␣. The resulting expression vectors pRM 20.2.1, pRM 21.2.1, and pRM 22.2.1 were used to transform S. cerevisiae S-78 (35).…”

“…With the exception of the side chain of Tyr 217 , which swings out to accommodate the amino-terminal residues of LDTI ( Fig. 1; see below), no significant conformational changes are seen in the trypsin component compared with other porcine trypsin structures (29,(47)(48)(49). The LDTI moiety is well defined in the vicinity of the proteinase, but it is characterized by elevated temperature factors and disrupted density further away from trypsin.…”

Structural Basis of Inhibition Revealed by a 1:2 Complex of the Two-headed Tomato Inhibitor-II and Subtilisin Carlsberg

“…Structures for a multidomain PI bound to a proteinase have only been determined for the two-headed Bowman-Birk inhibitors from mung bean (39), soybean (40), and wheat germ (41) bound to trypsin. The Bowman-Birk inhibitors consist of two domains of roughly 60 amino acids that are related by a nearly perfect 2-fold rotational symmetry axis.…”

Unbound Form of Tomato Inhibitor-II Reveals Interdomain Flexibility and Conformational Variability in the Reactive Site Loops

“…The structure of the proteinase:inhibitor ternary complex suggests that proteinase binding restricts the orientation of the two inhibitory domains, because the proteinase molecule bound to Domain II can form interactions with residues in Domain I (26). In the only other structure of a proteinase inhibitor bound to multiple proteinase molecules, individual trypsin molecules bind to single domains of two-headed Bowman-Birk inhibitors and do not appear to affect the orientation of the two inhibitory domains (53)(54)(55). In contrast, the flexible orientation of the two domains of TI-II may allow the inhibitor to fine tune its interactions with different proteinase molecules bound to Domain II.…”