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

Human angiogenin (Ang), an unusual member of the pancreatic RNase superfami ly, is a potent inducer of angiogenesis in who. Its ribonucleolytic activit y is weak (10(4) to 10(6)-fold lower than that of bovine RNase A), but none theless seems to be essential for biological function. Ang has been implica ted 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 differ ent forms (Pyr1 at 1.8 Angstrom and Met-1 at 2.0 Angstrom resolution) and f or two active-site variants, K40Q and H13A, at 2.0 Angstrom 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 angio genin's RNase activity include: (i) the obstruction of the pyrimidine-bindi ng site by Gln117; (ii) the existence of a hydrogen bond between Thr44 and Thr80 that further suppresses the effectiveness of the pyrimidine site; (ii i) the absence of a counterpart for the His119-Asp121 hydrogen bond that po tentiates 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 p urine-binding residues of RNase A. Analysis of the native structures has re vealed details of the cell-binding region and nuclear localisation signal o f Ang that are critical for angiogenicity. The cell-binding site differs dr amatically from the corresponding regions of RNase A and two other homologu es, eosinophil-derived neurotoxin and onconase, all of which lack angiogeni c: activity. Determination of the structures of the catalytically inactive variants K40Q and H13A has now allowed a rigorous assessment of the relatio nship between the ribonucleolytic and biological activities of Ang. No sign ificant change outside the enzymatic active site was observed in K40Q, esta blishing that the loss of angiogenic activity for this derivative is direct ly attributable to disruption of the catalytic apparatus. The H13A structur e shows some changes beyond the ribonucleolytic site, but sites involved in cell-binding and nuclear translocation are essentially unaffected by the a mino acid replacement. (C) 1999 Academic Press.