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

Abstract

The double-headed arrowhead protease inhibitors API-A and -B from the tubers of Sagittaria sagittifolia (Linn) feature two distinct reactive sites, unlike other members of their family. Although the two inhibitors have been extensively characterized, the identities of the two P1 residues in both API-A and -B remain controversial. The crystal structure of a ternary complex at 2.48 Å resolution revealed that the two trypsins bind on opposite sides of API-A and are 34 Å apart. The overall fold of API-A belongs to the ␤-trefoil fold and resembles that of the soybean Kunitz-type trypsin inhibitors. The two P1 residues were unambiguously assigned as Leu 87 and Lys 145 , and their identities were further confirmed by site-directed mutagenesis. Reactive site 1, composed of residues P5 Met 83 to P5 Ala 92 , adopts a novel conformation with the Leu 87 completely embedded in the S1 pocket even though it is an unfavorable P1 residue for trypsin. Reactive site 2, consisting of residues P5 Cys 141 to P5 Glu 150 , binds trypsin in the classic mode by employing a two-disulfide-bonded loop. Analysis of the two binding interfaces sheds light on atomic details of the inhibitor specificity and also promises potential improvements in enzyme activity by engineering of the reactive sites.Protease inhibitors (PIs) 4 are ubiquitously distributed in all organisms, including plants, animals, and microorganisms (1). They play vital roles in regulating their corresponding proteases, which are involved in many biological processes such as protein digestion, cell signal transmission, inflammation, apoptosis, blood coagulation, and embryogenesis (2). The clinical applications of PIs are widespread, and there is great interest in developing more potent therapeutic PIs for treating human diseases related to cancer (3), pancreatitis (4), thrombosis (5), and AIDS (6). To this end, the soybean Kunitz-type serine proteases inhibitors have been extensively studied (1,(7)(8)(9)(10)(11). The inhibitors of this family generally contain 170 -200 residues and have two disulfide bonds. 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 (15-18). All of these inhibitors are classified into family I3 of peptidase inhibitors (19). Most members are further grouped into subfamily I3A. However, the double-headed arrowhead PIs API-A and -B are grouped in subfamily I3B because of their very low sequence similarity to other members (19). In contrast to other double-headed PIs such as the Bowman-Birk and ovomucoid inhibitors, which have two identical reactive sites that have evolved by domain shuffling and gene duplication (1, 20 -25), both API-A and -B have two distinct reactive sites.API-A and -B were first purified from the tubers of Sagittaria sagittifolia (Linn) in 1979 (26). Both consist of 179 residues with three disulfide bonds and can inhibit a variety of serine pr...

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

“…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.…”

“…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).…”

A Trypsin Inhibitor from Rambutan Seeds with Antitumor, Anti-HIV-1 Reverse Transcriptase, and Nitric Oxide-Inducing Properties

“…The Kunitz type trypsin inhibitors demonstrate a molecular weight of~20 kDa and two disulfide bonds [20,21,28]. Though most Kunitz type trypsin inhibitors have only one reactive site, a few that display two reactive sites are termed double-headed inhibitors, e.g., arrowhead protease inhibitors A and B whose tertiary structures have been published recently [29]. Among them, Kunitz type trypsin inhibitors have been extensively studied and multiple activities/functions have been disclosed, such as significant inhibition of proliferation and metastasis of tumors [20,30], and even utilization in assessing geographical variation and studying the evolutionary process [31].…”

Analysis and Ranking of Protein-Protein Docking Models Using Inter-Residue Contacts and Inter-Molecular Contact Maps

“…These involve the interaction of the trypsin active site with two distinct binding sites on the API-A protein, implicating respectively Lys145 (interface AC) and Leu87 (interface BC) as position P1 (before the scissile bond). Experimental evidence is available for the functionality of both of these binding modes [38,39]. The 3E8L complex is particularly interesting also because it was a target (T40) in CAPRI, Round 18 (see below).…”

“…As an exemplary case, we will use in the following a complex between bovine trypsin and the inhibitor API-A from S. sagittifolia (PDB ID: 3E8L) [38]. In this complex, the double-headed arrowhead protease inhibitor API-A (chain C) simultaneously binds to two copies of the second protein, trypsin (chains A and B), forming two distinct association modes, corresponding to interfaces AC and BC.…”