Crystal structure of the Kunitz (STI)-type inhibitor from Delonix regia seeds

Krauchenco, Sandra; Pando, Silvana Cristina; Marangoni, Sérgio; Polikarpov, Igor

doi:10.1016/j.bbrc.2003.11.062

Cited by 47 publications

(29 citation statements)

References 32 publications

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“…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).…”

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confidence: 94%

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

Bao

Zhou

Jiang

et al. 2009

Journal of Biological Chemistry

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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...

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confidence: 94%

“…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)(16)(17)(18).…”

mentioning

confidence: 99%

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

Bao

Zhou

Jiang

et al. 2009

Journal of Biological Chemistry

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“…These bioactive macromolecules have been implicated in various physiological functions, such as regulation of proteolytic cascades and safe storage of proteins, as well as defense molecules against plant pests and pathogens [12]. Kunitz-type inhibitors are characterized by molecular masses around 20 kDa, a low cysteine content forming two disulphide bonds and a common structural fold composed of a ␤-trefoil formed by 12 antiparallel ␤-strands with long interconnecting loops presenting one or two reactive sites for serine proteinases [11,[13][14][15][16]. Although several common properties have been found in Kunitz inhibitors, some secondary activities have been reported in the literature.…”

Section: Introductionmentioning

confidence: 99%

Structural and mechanistic insights into a novel non-competitive Kunitz trypsin inhibitor from Adenanthera pavonina L. seeds with double activity toward serine- and cysteine-proteinases

Migliolo

Oliveira

Santos

et al. 2010

Journal of Molecular Graphics and Modelling

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“…This canonical conformation of the reactive loop characterizes a mechanism of competitive inhibition and is also found in the trypsin inhibitors from Erythrina caffra seeds and Psophocarpus tetragonolobus chymotrypsin inhibitor (Song & Suh, 1998;Krauchenco et al, 2003). Trypsin inhibitors, such as from Swartzia pickelii, can present also glutamine residue in the reactive site (Cavalcanti et al, 2002).…”

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confidence: 89%

Affinity Chromatography as a Key Tool to Purify Protein Protease Inhibitors from Plants

Santos¹,

Oliveira²,

Rabêlo³

et al. 2012

Affinity Chromatography

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Crystal structure of the Kunitz (STI)-type inhibitor from Delonix regia seeds

Cited by 47 publications

References 32 publications

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

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

Structural and mechanistic insights into a novel non-competitive Kunitz trypsin inhibitor from Adenanthera pavonina L. seeds with double activity toward serine- and cysteine-proteinases

Affinity Chromatography as a Key Tool to Purify Protein Protease Inhibitors from Plants

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