| Solution structure of the main alpha-amylase inhibitor from amaranth seeds. | |
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MedLine Citation:
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PMID: 11298757 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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The most abundant alpha-amylase inhibitor (AAI) present in the seeds of Amaranthus hypochondriacus, a variety of the Mexican crop plant amaranth, is the smallest polypeptide (32 residues) known to inhibit alpha-amylase activity of insect larvae while leaving that of mammals unaffected. In solution, 1H NMR reveals that AAI isolated from amaranth seeds adopts a major trans (70%) and minor cis (30%) conformation, resulting from slow cis-trans isomerization of the Val15-Pro16 peptide bond. Both solution structures have been determined using 2D 1H-NMR spectroscopy and XPLOR followed by restrained energy refinement in the consistent-valence force field. For the major isomer, a total of 563 distance restraints, including 55 medium-range and 173 long-range ones, were available from the NOESY spectra. This rather large number of constraints from a protein of such a small size results from a compact fold, imposed through three disulfide bridges arranged in a cysteine-knot motif. The structure of the minor cis isomer has also been determined using a smaller constraint set. It reveals a different backbone conformation in the Pro10-Pro20 segment, while preserving the overall global fold. The energy-refined ensemble of the major isomer, consisting of 20 low-energy conformers with an average backbone rmsd of 0.29 +/- 0.19 A and no violations larger than 0.4 A, represents a considerable improvement in precision over a previously reported and independently performed calculation on AAI obtained through solid-phase synthesis, which was determined with only half the number of medium-range and long-range restraints reported here, and featured the trans isomer only. The resulting differences in ensemble precision have been quantified locally and globally, indicating that, for regions of the backbone and a good fraction of the side chains, the conformation is better defined in the new solution structure. Structural comparison of the solution structure with the X-ray structure of the inhibitor when bound to its alpha-amylase target in Tenebrio molitor shows that the backbone conformation is only slightly adjusted on complexation, while that of the side chains involved in protein-protein contacts is similar to those present in solution. Therefore, the overall conformation of AAI appears to be predisposed to binding to its target alpha-amylase, confirming the view that it acts as a lid on top of the alpha-amylase active site. |
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Authors:
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J C Martins; M Enassar; R Willem; J M Wieruzeski; G Lippens; S J Wodak |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't |
Journal Detail:
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Title: European journal of biochemistry / FEBS Volume: 268 ISSN: 0014-2956 ISO Abbreviation: Eur. J. Biochem. Publication Date: 2001 Apr |
Date Detail:
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Created Date: 2001-04-12 Completed Date: 2001-05-31 Revised Date: 2008-11-21 |
Medline Journal Info:
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Nlm Unique ID: 0107600 Medline TA: Eur J Biochem Country: Germany |
Other Details:
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Languages: eng Pagination: 2379-89 Citation Subset: IM |
Affiliation:
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High Resolution NMR Centrum (HNMR), Vrije Universiteit Brussel, Belgium. jose@hnmr.vub.ac.be |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Amaranth Dye
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chemistry* Binding Sites Crystallography, X-Ray Disulfides Hydrogen Bonding Magnetic Resonance Spectroscopy Models, Molecular Peptides / pharmacology Proline / chemistry Protein Binding Protein Conformation Protein Folding Seeds / chemistry* Stereoisomerism Valine / chemistry alpha-Amylases / antagonists & inhibitors* |
| Chemical | |
Reg. No./Substance:
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0/Disulfides; 0/Peptides; 147-85-3/Proline; 7004-03-7/Valine; 915-67-3/Amaranth Dye; EC 3.2.1.1/alpha-Amylases |
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