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Human angiogenin (Ang), an unusual member of the pancreatic RNase superfamily,
is a potent inducer of angiogenesis in vivo. Its ribonucleolytic activity is
weak (10(4) to 10(6)-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 A and Met-1 at 2.0 A resolution) and for two active-site variants, K40Q
and H13A, at 2.0 A 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.
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