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Crystal Structure of Human Angiogenin Reveals the Structural Basis for Its Functional Divergence from Ribonuclease K

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Crystal Structure of Human Angiogenin Reveals the Structural Basis for Its Functional Divergence from Ribonuclease K Proc. Natl. Acad. Sci. USA Vol. 91, pp. 2915-2919, April 1994 Biochemistry Crystal structure of human angiogenin reveals the structural basis for its functional divergence from ribonuclease K. RAvI ACHARYA*t, ROBERT SHAPIROO§, SIMON C. ALLEN*, JAMES F. RIORDAN*, AND BERT L. VALLEEO *School of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom; and tCenter for Biochemical and Biophysical Sciences and Medicine and §Department of Pathology, Harvard Medical School, 250 Longwood Avenue, Boston, MA 02115 Contributed by Bert L. Vallee, December 29, 1993 ABSTRACT Angiogenin, a potent inducer of neovascular- differs significantly from that of RNase in precisely those ization, is the only angiogenic molecule known to exhibit regions considered to be important for the characteristic ribonucleolytic activity. Its overall structure, as determined at enzymatic and biological activities of Ang. 2.4 A, is similarto that of pancreatic ribonuclease A, but it differs markedly in several distinct areas, particularly the ribonucleolytic active center and the putative receptor binding METHODS site, both ofwhich are critically involved in biological function. Recombinant human [Met-']Ang crystallizes in the orthorhom- Most sri ly, the site that is spatially analogous to that for bic system, space group C2221, with the unit cell dimensions a pyrimidine binding in ribonuclease A differs siificantly in = 83.4 A, b = 120.6 A, and c = 37.7 A (one molecule per conformation and is "obstructed" by glutamine-117. Move- asymmetric unit, 63% solvent) (18). Diffraction data (AD-lab ment of this and adjacent residues may be required for data set) were collected from native crystals (2.5 A) by using a substrate binding to anginin and, hence, constitute a key Siemens area detector mounted on a rotating-anode x-ray part of its menism of action. source operating at 45 kV and 80 mA. The data were processed with the XDS package (19). A second data set (SRS) was col- Human angiogenin (Ang), a single-chain polypeptide (Mr lected (2.4 A) on station PX 7.2 ofthe Science and Engineering 14,124) present in tumor cell conditioned medium and normal Research Council Synchrotron Radiation Source (Daresbury, serum (1, 2), is a potent inducer of neovascularization (1). It U.K.). The source was operated at an energy of 2 GeV and binds specifically to endothelial cells in culture (3) and elicits current ranging from 210 mA to 150 mA, with wavelength 1.488 second-messenger responses (4). It also binds heparin (38), A. The film data were processed with the mosco program (D. can serve as a substratum for endothelial cell adhesion (5), Stuart, University ofOxford) and the Daresbury CCP4 program and is translocated to the nucleus (39). Among angiogenic suite. The two data sets were scaled by using the program molecules, Ang is unique in that it is aribonucleolytic enzyme 3DSCALE (20). The structure was determined with the MERLOT (6) with an amino acid sequence 33% identical to that of (21) package ofmolecularreplacement programs and the search bovine pancreatic ribonuclease (RNase) A (7). Moreover, was based on the RNase Aprotein model (5RSA-PDB) (22) with although Ang has the same general catalytic properties as Ang sequence changes incorporated into the model. The angles RNase A-it cleaves preferentially on the 3' side of pyrim- from both rotation and translation function search gave a idines and follows a transphosphorylation/hydrolysis mech- consistent set of translation vectors on the Harker sections. anism-its activity differs markedly both in magnitude and in Cycles of positional and simulated annealing refinement using specificity (6, 8). the xPLOR package (23) and model building using FRODO (24) Efforts to delineate the structural basis for the character- gave the current model (using all data, 6609 reflections, istic enzymatic and biological activities of Ang have been 8.0-2.4 A) which has an R factor of 0.22 (rms deviation of guided in large part by the vast wealth ofexisting information bond lengths from ideality, 0.015 A; bond angles, 3.510) (R on RNase A, much of it derived from x-ray crystallography = 1I IFO - FC 1/z1 F0 1). The structure contains 54 water (9, 10). Residues in Ang corresponding to those in RNase molecules. considered important for enzymatic activity have been mod- ified chemically or by mutagenesis and, conversely, regions RESULTS AND DISCUSSION of dissimilarity to RNase have been probed for their role in angiogenic activity. To date, 16 individual residues and four Overall Structure. The three-dimensional structure of hu- segments of primary structure of Ang have been examined, man Ang [determined at 2.4 A (Table 1)¶] features a kidney- mostly by mutation (remarkably, few of their counterparts shaped tertiary fold reminiscent of RNase A with approxi- have been mutated in RNase A). The results have indicated mate dimensions 38 A x 43 A x 34 A (Fig. 1). The central that the ribonucleolytic active site of Ang is necessary core of the molecule consists of P structure with a pair of (11-14), but not sufficient, for angiogenic activity; minimally, antiparallel twisted (strands of residues 69-84 (B3-B4) and a second, distinct region ofAng, probably constituting a cell 93-108 (B5-B6) forming the main topology with residues or receptor binding site, is also required (15, 16). Ser-72 and Gly-99 at the apices. Two additional strands on Significant progress in defining structure-function rela- either side of these central strands (residues 41-47, B1; tionships in Ang has been made by the approaches described, 111-116, B7) complete the major sheet structure. Residues aided in part by a predicted structure based on its homology 62-65 (B2) form an additional short strand on one side of the to RNase A (17). However, these efforts have been limited by core scaffold. Helix 1 (H1, residues 3-14 at the N terminus) the absence of direct knowledge concerning the three- is vicinal to a short 310-helix (residues 117-121 at the C dimensional structure of the protein. Here we report the crystal structure ofAng at 2.4 A. Most notably, the structure Abbreviation: Ang, angiogenin. tTo whom reprint requests should be addressed. IThe atomic coordinates have been deposited in the Protein Data The publication costs ofthis article were defrayed in part by page charge Bank, Chemistry Department, Brookhaven National Laboratory, payment. This article must therefore be hereby marked "advertisement" Upton, NY 11973 (entry code lANG). This information is embar- in accordance with 18 U.S.C. §1734 solely to indicate this fact. goed for 1 year (coordinates) from the date of publication. 2915 Downloaded by guest on September 23, 2021 2916 Biochemistry: Acharya et al. Proc. Nad. Acad Sci. USA 91 (1994) Table 1. Human Ang x-ray data collection and + * * * * Ang - - q d n s R Y T HF L T Q H Y D A - k p q 19 refinement statistics RNase A k e -- -t AA A K F E R Q H M D S s t s a 19 + * * * * * Data set Nm* Nit complete - G r d d R Y C E S I M R R R G L t s - p C K D I N 43 Rsy,* a S s - s N Y C N Q M M K S R N L t k d r CK P V N 44 2.4-A native (SRS) 5,742 3968 0.12 51 2.5-A native (AD-lab) 35,983 6664 0.07 92 T F I H g N K R S I K A I C e n k n g n p h r - - - 66 2.4-A native (AD-lab + SRS) 41,725 7229 0.11 88 T F V H e S L A D V Q A V C s - - - q k n v a c k n 67 *No. of measurements. *+ +* **** + + - e n - 1 r i S k S S F Q V T T C K L H g g S P W - 89 tNo. of independent reflections. t n C S S t M S I T D C R E T g sS kY p 93 *Rs~y = ZX£hI(h) - Ith)I/ZXj£Ihh), where IKh) is the ith measure- g q Y q Y ment of reflection h and 1(h) is the mean of the intensity. ++* * + + * * ** + * p p c q Y R A T A G F R N V VV A C E n - - g 1 P V 113 terminus) which is not present in RNase A. Helix 2 (H2, n - c a Y K TT Q A N K H I I V A C E g n p y v P V 118 * * + + residues 22-33) and helix 3 (H3, residues 49-58) are oriented HL D q S i f r r p 123 at =70° to the plane of the a-sheet on either side of the H F D a - s v 124 backbone structure. These helices are connected through the solvent-exposed ,3-strand B1. The remaining residues form FIG. 2. Sequence alignment of Ang and RNase A based on structural superpositions. Residues judged to be s lly equiva- the loop structures. A structure-based sequence alignment is lent (represented by uppercase letters) are those whose CO positiOnS given in Fig. 2. All insertions/deletions and virtually all superimpose within 1.2 A in the two strct.I= I nonconservative replacements occur in the loop regions or at by lowercase letters deviate more sig tly. Hyphensidicate that the N and C termini. RNase A has three disulfide bridges that there is no coresp ing equivalent residue inthe three-dimensional have structurally equivalent counterparts in Ang and a fourth strwture.
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