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 1 , Robert Shapiro 2,3 , Simon C. Allen 1 , Gowtham V. Subbarao 1 , Kasinadar Veluraja 1 and K. Ravi Acharya 1 * 1 Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK 2 Center for Biochemical and Biophysical Sciences and Medicine and 3 Department of Pathology Harvard Medical School Boston, MA 02115, USA Human angiogenin (Ang), an unusual member of the pancreatic RNase superfamily, is a potent inducer of angiogenesis in vivo. Its ribonucleoly- tic 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 Ê and Met-1 at 2.0 A Ê res- olution) and for two active-site variants, K40Q and H13A, at 2.0 A Ê resol- ution. The native structures, together with earlier mutational and biochemical data, provide a basis for understanding the unique func- tional 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 stabil- ise the blockage of the pyrimidine site); and (iv) the absence of any pre- cise 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 cor- responding 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 signi®- cant 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 unaf- fected by the amino acid replacement. # 1999 Academic Press Keywords: angiogenesis; angiogenin; X-ray crystallography; ribonuclease superfamily; nuclear translocation *Corresponding author This article is dedicated to the Lord Phillips of Ellesmore (David C. Phillips) on his 75th birthday. Present address: K. Veluraja, Department of Physics, Manonmaniam Sundaranar University, Tirunelveli 627 002, India. Abbreviations used: Ang, human angiogenin; RNase A, bovine pancreatic ribonuclease A; EDN, eosinophil derived neurotoxin; ONC, onconase; AngBP, angiogenin binding protein; RI, ribonuclease inhibitor; NLS, nuclear localisation signal; r.m.s., root-mean-square; Pyr, pyroglutamic acid. E-mail address of the corresponding author: K.R.Acharya@bath.ac.uk Article No. jmbi.1998.2378 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 285, 1209±1233 0022-2836/99/031209±25 $30.00/0 # 1999 Academic Press