Crystal structure of the bovine α-chymotrypsin:kunitz inhibitor complex. An example of multiple protein:protein recognition sites

Capasso, Clemente; Rizzi, Menico; Menegatti, Enea; Ascenzi, Paolo; Bolognesi, M.

doi:10.1002/(sici)1099-1352(199701/02)10:1<26::aid-jmr351>3.0.co;2-n

Cited by 44 publications

(47 citation statements)

References 55 publications

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“…The bovine pancreatic trypsin inhibitor belongs to the Kunitz-type inhibitors and is a potent inhibitor of serine proteases. The crystal structures of BPTI with bovine trypsin and with bovine chymotrypsin have been determined [47][48][49]. Analysis of these complexes have shown that there are 10-13 residues on the inhibitor forming less than 4 Å contacts with 20-25 residues of the enzyme.…”

Section: Page 7 Of 22mentioning

confidence: 83%

A bifunctional sea anemone peptide with Kunitz type protease and potassium channel inhibiting properties

Peigneur¹,

Billen²,

Derua³

et al. 2011

Biochemical Pharmacology

103

101

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This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Page 1 of 22A c c e p t e d M a n u s c r i p t Abstract Sea anemone venom is a known source of interesting bioactive compounds, including peptide toxins which are invaluable tools for studying structure and function of voltage-gated potassium channels. APEKTx1 is a novel peptide isolated from the sea anemone Anthopleura elegantissima, containing 63 amino acids cross-linked by 3 disulfide bridges. Sequence alignment reveals that APEKTx1 is a new member of the type 2 sea anemone peptides targeting voltage-gated potassium channels (K V 's), which also include the kalicludines from Anemonia sulcata. Similar to the kalicludines, APEKTx1 shares structural homology with both the basic pancreatic trypsin inhibitor (BPTI), a very potent Kunitz-type protease inhibitor, and dendrotoxins which are powerful blockers of voltage-gated potassium channels. In this study, APEKTx1 has been subjected to a screening on a wide range of 23 ion channels expressed in Xenopus leavis oocytes: 13 cloned voltage-gated potassium channels (K V 1.1-K V 1.6, K V 1.1 triple mutant, K V 2.1, K V 3.1, K V 4.2, K V 4.3, hERG, the insect channel Shaker IR), 2 cloned hyperpolarization-activated cyclic nucleotidesensitive cation non-selective channels (HCN1 and HCN2) and 8 cloned voltage-gated sodium channels (Na V 1.2-Na V 1.8 and the insect channel DmNa V 1). Our data shows that APEKTx1 selectively blocks K V 1.1 channels in a very potent manner with an IC 50 value of 0.9 nM. Furthermore, we compared the trypsin inhibitory activity of this toxin with BPTI. APEKTx1 inhibits trypsin with a dissociation constant of 124 nM. In conclusion, this study demonstrates that APEKTx1 has the unique feature to combine the dual functionality of a potent and selective blocker of K V 1.1 channels with that of a competitive inhibitor of trypsin.Keywords Anthopleura elegantissima · K V channel inhibitor · sea anemone toxin · protease inhibitor ·

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Section: Page 7 Of 22mentioning

confidence: 83%

A bifunctional sea anemone peptide with Kunitz type protease and potassium channel inhibiting properties

Peigneur¹,

Billen²,

Derua³

et al. 2011

Biochemical Pharmacology

103

101

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“…Previously, the cDNA corresponding to AceKI was amplified from adult A. ceylanicum RNA by using a PCR-based approach (48). The translated cDNA suggested that AceKI represents a novel member of the Kunitz family of serine protease inhibitors, which are characterized by six cysteine residues that form three intramolecular disulfide bonds (8,43,44,49). Based on alignment of the AceKI amino acid sequence with the sequences of previously identified members of the Kunitz family, we hypothesized that Met 26 was the likely P1 inhibitory reactive site of the protein.…”

Section: Discussionmentioning

confidence: 99%

“…Although this interaction is reversible, the enzyme-inhibitor complex is sufficiently stable that considerable inhibition can be detected at nearly equimolar concentrations. Based on an alignment of AceKI with other members of the Kunitz-type family, we identified Met 26 as the likely P1 inhibitory reactive site amino acid (8,43,49,66). Therefore, in order to more clearly elucidate the role of Met 26 in defining the spectrum of inhibition of AceKI, a series of site-directed mutants were produced that had various substitutions incorporated at the P1 site (23,33,41,46,51,65).…”

Section: Vol 72 2004 Hookworm Kunitz Inhibitor 2215mentioning

confidence: 99%

Molecular Characterization ofAncylostoma ceylanicumKunitz-Type Serine Protease Inhibitor: Evidence for a Role in Hookworm-Associated Growth Delay

Chu¹,

Bungiro²,

Ibanez³

et al. 2004

Infect Immun

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Hookworm infection is a major cause of iron deficiency anemia and malnutrition in developing countries. The Ancylostoma ceylanicum Kunitz-type inhibitor (AceKI) is a 7.9-kDa broad-spectrum inhibitor of trypsin, chymotrypsin, and pancreatic elastase that has previously been isolated from adult hookworms. Site-directed mutagenesis of the predicted P1 inhibitory reactive site amino acid confirmed the role of Met 26 in mediating inhibition of the three target serine proteases. By using reverse transcription-PCR, it was demonstrated that the level of AceKI gene expression increased following activation of third-stage larvae with serum and that the highest level of expression was reached in the adult stage of the parasite. Immunohistochemistry studies performed with polyclonal immunoglobulin G raised against recombinant AceKI showed that the inhibitor localized to the subcuticle of the adult hookworm, suggesting that it has a potential in vivo role in neutralizing intestinal proteases at the surface of the parasite. Immunization with recombinant AceKI was shown to confer partial protection against hookworm-associated growth delay without a measurable effect on anemia. Taken together, the data suggest that AceKI plays a role in the pathogenesis of hookworm-associated malnutrition and growth delay, perhaps through inhibition of nutrient absorption in infected hosts.Hookworm infection remains a major global health problem, and over one billion people are reportedly infected in developing countries (9, 14). Hookworms, which are bloodfeeding intestinal nematodes, are a major cause of iron deficiency anemia and malnutrition (15,20,(59)(60)(61)64). While the anemia is presumably due to the cumulative effect of chronic intestinal blood loss, the molecular mechanisms underlying the pathogenesis of hookworm malnutrition remain unknown. Although it has been suggested that hookworm malnutrition and growth delay occur secondary to chronic iron deficiency, particularly in children, evidence from prior clinical studies suggests that hookworm infection is also associated with various degrees of intestinal malabsorption (18,35,54,57,62). It has been hypothesized that this hookworm malabsorption syndrome might occur secondary to mucosal inflammation triggered by the adult worm attached to the intestinal epithelium or might be a result of secretion of parasite inhibitors of host digestive enzymes (18).As part of a series of ongoing studies aimed at characterizing adult hookworm secretory proteins, a cDNA corresponding to the gene encoding a putative Kunitz-type serine protease inhibitor was previously identified from adult Ancylostoma ceylanicum RNA by using a PCR-based approach (48). The A. ceylanicum Kunitz-type inhibitor (AceKI) cDNA was expressed in Escherichia coli, and the recombinant protein was found to inhibit the pancreatic enzymes chymotrypsin, pancreatic elastase, and trypsin in vitro, with equilibrium inhibitory dissociation constant (K i ) values ranging from picomolar levels to low nanomolar levels. The native AceKI prote...

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“…The analysis of the crystal structures of bovine trypsin-BPTI [6] and bovine chymotrypsin-BPTI [7][8] complexes shows that there are 13 residues of BPTI forming < 4 Å contacts with the enzyme (Table (1)). Large part of All values were calculated using HBPLUS program [18] and structural data for respective trypsin-BPTI [6] and chymotrypsin-BPTI complexes [8]; in case of Arg15, values were calculated from data for APPI complexed with trypsin or chymotrypsin [8].…”

Section: Accommodation Of the P1 Residues In The S1 Pocket Of Trypsinmentioning

confidence: 99%

Structure-Function Relationships in Serine Protease-Bovine Pancreatic Trypsin Inhibitor Interaction

Krowarsch¹,

Zakrzewska²,

Smalås³

et al. 2005

PPL

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Crystal structure of the bovine α-chymotrypsin:kunitz inhibitor complex. An example of multiple protein:protein recognition sites

Cited by 44 publications

References 55 publications

A bifunctional sea anemone peptide with Kunitz type protease and potassium channel inhibiting properties

A bifunctional sea anemone peptide with Kunitz type protease and potassium channel inhibiting properties

Molecular Characterization ofAncylostoma ceylanicumKunitz-Type Serine Protease Inhibitor: Evidence for a Role in Hookworm-Associated Growth Delay

Structure-Function Relationships in Serine Protease-Bovine Pancreatic Trypsin Inhibitor Interaction

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