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