Refined crystal structures of native human angiogenin and two active site variants: Implications for the unique functional properties of an enzyme involved in neovascularisation during tumour growth

Demetres D. Leonidas, Robert Shapiro, Simon C. Allen, Gowtham V. Subbarao, Kasinadar Veluraja, K. Ravi Acharya

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Abstract

Human angiogenin (Ang), an unusual member of the pancreatic RNase superfamily, is a potent inducer of angiogenesis in vivo. Its ribonucleolytic activity is weak (104 to 106-fold lower than that of bovine RNase A), but nonetheless seems to be essential for biological function. Ang has been implicated in the establishment of a wide range of human tumours and has therefore emerged as an important target for the design of new anti-cancer compounds. We report high-resolution crystal structures for native Ang in two different forms (Pyr1 at 1.8 Å and Met-1 at 2.0 Å resolution) and for two active-site variants, K40Q and H13A, at 2.0 Å resolution. The native structures, together with earlier mutational and biochemical data, provide a basis for understanding the unique functional properties of this molecule. The major structural features that underlie the weakness of angiogenin's RNase activity include: (i) the obstruction of the pyrimidine-binding site by Gln117; (ii) the existence of a hydrogen bond between Thr44 and Thr80 that further suppresses the effectiveness of the pyrimidine site; (iii) the absence of a counterpart for the His119-Asp121 hydrogen bond that potentiates catalysis in RNase A (the corresponding aspartate in Ang, Asp116, has been recruited to stabilise the blockage of the pyrimidine site); and (iv) the absence of any precise structural counterparts for two important purine-binding residues of RNase A. Analysis of the native structures has revealed details of the cell-binding region and nuclear localisation signal of Ang that are critical for angiogenicity. The cell-binding site differs dramatically from the corresponding regions of RNase A and two other homologues, eosinophil-derived neurotoxin and onconase, all of which lack angiogenic activity. Determination of the structures of the catalytically inactive variants K40Q and H13A has now allowed a rigorous assessment of the relationship between the ribonucleolytic and biological activities of Ang. No significant change outside the enzymatic active site was observed in K40Q, establishing that the loss of angiogenic activity for this derivative is directly attributable to disruption of the catalytic apparatus. The H13A structure shows some changes beyond the ribonucleolytic site, but sites involved in cell-binding and nuclear translocation are essentially unaffected by the amino acid replacement.

Original languageEnglish
Pages (from-to)1209-1233
Number of pages25
JournalJournal of Molecular Biology
Volume285
Issue number3
DOIs
Publication statusPublished - 22 Jan 1999

Keywords

  • Angiogenesis
  • Angiogenin
  • Nuclear translocation
  • Ribonuclease superfamily
  • X-ray crystallography

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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