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Phenylalanine 30 plays an important role in receptor binding of verotoxin-1.

Publication ,  Journal Article
Clark, C; Bast, D; Sharp, AM; St Hilaire, PM; Agha, R; Stein, PE; Toone, EJ; Read, RJ; Brunton, JL
Published in: Molecular microbiology
February 1996

The homopentameric B subunit of verotoxin 1 (VT1) binds to the glycosphingolipid receptor globotriaosylceramide (Gb3). We produced mutants with alanine substitutions for residues found near the cleft between adjacent subunits. Substitution of alanine for phenylalanine 30 (Phe-30) resulted in a fourfold reduction in B subunit binding affinity for Gb3 and a 10-fold reduction in receptor density in a solid-phase binding assay. The interaction of wild-type and mutant B subunits with Pk trisaccharide in solution was examined by titration microcalorimetry. The carbohydrate binding of the mutant was markedly impaired compared with that of the wild type and was too weak to allow calculation of a binding constant. These results demonstrate that the mutation significantly impaired the carbohydrate-binding function of the B subunit. To ensure that the mutation had not caused a significant change in structure, the mutant B subunit was crystallized and its structure was determined by X-ray diffraction. Difference Fourier analysis showed that its structure was identical to that of the wild type, except for the substitution of alanine for Phe-30. The mutation was also produced in the VT1 operon, and mutant holotoxin was purified to homogeneity. The cytotoxicity of the mutant holotoxin was reduced by a factor of 10(5) compared to that of the wild type in the Vero cell cytotoxicity assay. The results suggest that the aromatic ring of Phe-30 plays a major role in binding of the B subunit to the Galalpha1-4Galbeta1-4Glc trisaccharide portion of Gb3. Examination of the VT1 B crystal structure suggests two potential carbohydrate-binding sites which lie on either side of Phe-30.

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Published In

Molecular microbiology

DOI

EISSN

1365-2958

ISSN

0950-382X

Publication Date

February 1996

Volume

19

Issue

4

Start / End Page

891 / 899

Related Subject Headings

  • Trihexosylceramides
  • Shiga Toxin 1
  • Receptors, Cell Surface
  • Protein Conformation
  • Protein Binding
  • Phenylalanine
  • Mutation
  • Molecular Sequence Data
  • Microbiology
  • Glycolipids
 

Citation

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Clark, C., Bast, D., Sharp, A. M., St Hilaire, P. M., Agha, R., Stein, P. E., … Brunton, J. L. (1996). Phenylalanine 30 plays an important role in receptor binding of verotoxin-1. Molecular Microbiology, 19(4), 891–899. https://doi.org/10.1046/j.1365-2958.1996.427962.x
Clark, C., D. Bast, A. M. Sharp, P. M. St Hilaire, R. Agha, P. E. Stein, E. J. Toone, R. J. Read, and J. L. Brunton. “Phenylalanine 30 plays an important role in receptor binding of verotoxin-1.Molecular Microbiology 19, no. 4 (February 1996): 891–99. https://doi.org/10.1046/j.1365-2958.1996.427962.x.
Clark C, Bast D, Sharp AM, St Hilaire PM, Agha R, Stein PE, et al. Phenylalanine 30 plays an important role in receptor binding of verotoxin-1. Molecular microbiology. 1996 Feb;19(4):891–9.
Clark, C., et al. “Phenylalanine 30 plays an important role in receptor binding of verotoxin-1.Molecular Microbiology, vol. 19, no. 4, Feb. 1996, pp. 891–99. Epmc, doi:10.1046/j.1365-2958.1996.427962.x.
Clark C, Bast D, Sharp AM, St Hilaire PM, Agha R, Stein PE, Toone EJ, Read RJ, Brunton JL. Phenylalanine 30 plays an important role in receptor binding of verotoxin-1. Molecular microbiology. 1996 Feb;19(4):891–899.
Journal cover image

Published In

Molecular microbiology

DOI

EISSN

1365-2958

ISSN

0950-382X

Publication Date

February 1996

Volume

19

Issue

4

Start / End Page

891 / 899

Related Subject Headings

  • Trihexosylceramides
  • Shiga Toxin 1
  • Receptors, Cell Surface
  • Protein Conformation
  • Protein Binding
  • Phenylalanine
  • Mutation
  • Molecular Sequence Data
  • Microbiology
  • Glycolipids