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. The solvent-inaccessible residues (<20 A2 of exposed between residues 65 and 72 that is missing in Ang and whose surface) are indicatd by stars. Residues involved in lattice contacts A least- in the crystal for Ang are represented by plus signs. The secondary- position is occupied by the side chain of Leu-69. structure elements in human Ang bsed on _P M D (6] are H1 squares superposition of the Ang and RNase A structures (residues 3-14), H2 (21-33), B1 (41-47), H3 (4-58), B2 (62-65), B3 reveals the extent oftheir tertiary structural differences. The (69-73), B4 (76-84), B5 (93-101), B6 (103-108), B7 (111-116), and 31o overall rms deviation based on 116 equivalent Co atoms is (117-121) (Fig. 1). 2.26 A. The regions that deviate most significantly between the two structures involve residues 1-4, 17-23 (loop 1), 36-38 The active site of RNase is generally considered to consist (loop 2), 58-75 (loops 4 and 5 and strands B2 and B3), 85-93 of numerous subsites (see refs. 9 and 27): a P1 site, at which (loop 7), 109-111 (loop 9), and 117-123. phosphodiester bond cleavage occurs; a B1 site, for binding _ kycledyc Active Site. Site-directed mutagenesis of the pyrimidine whose ribose donates its 3' oxygen to the His-13, His-114, and Lys-40 has established that the ribonu- scissile bond; a B2 site, which preferentially binds a purmne cleolytic activity of Ang is crucial for angiogenesis (11, 12). ring on the opposite side of the scissile bond; and additional However, it is remarkable that this activity is several orders binding sites for peripheral bases and phosphates. The com- of nagtde less than that of RNase A toward commonly ponents of these various sites include the catalytic residues used substrates (6, 8). Although Ang shares certain aspects of His-12, Lys-l, and His-119 as well as Gln-li at P1; Thr-45 substrate specificity with RNase, there are also differences and Ser-123 at B1; and Asn-67, Gln-69, Asn-71, and Glu-111 such as a greater preference for C vs. U in the N position of at B2 (9, 28, 29). Based on the amino acid sequence of Aog, NpN' dinucleotide substrates and a lesser preference for A the P1 and B1 sites and part of the B2 site appear to be vs. G in the N' position (8). Clearly, these functional simi- conserved, with His-12, Lys41, His-119, Gln-li, Thr45, larities and differences are a reflection of the three- Ser-123, and Glu-111 maintained in Ang as His-13, Lys-40, dimensional structures of the two proteins. His-114, Gln-12, Thr-44, Ser-118, and Glu-108, respectively.

(.

NH10/ > N N

FIG. 1. The polypeptide fold for Ang (Left) and RNase A (Right) (22), drawn with the program MOLSCRIPT (25). Downloaded by guest on September 23, 2021 Biochemistry: Acharya et aL Proc. Natl. Acad. Sci. USA 91 (1994) 2917

Despite the striking sequence conservation between the either an alternative positioning of the pyrimidine ring (lim- active sites of Ang and RNase, the crystal structure of Ang ited by the constraints imposed by the catalytic histidines and reveals extensive architectural differences (Table 2). Thus, lysine, which are oriented essentially as in RNase), or some although the catalytic center (P1) appears to be fully intact conformational rearrangement of this site in Ang. Evidence (His-13, Lys-40, His-114, and Gln-12 occupy positions almost to support the latter view is presented in the accompanying indistinguishable from those of their RNase counterparts), paper (31). Blocked substrate binding sites have also been both the B1 and B2 sites differ considerably. In RNase, the B1 described for other enzymes [e.g., chymosin (32) and rat site is an open pocket with Thr45 on one side near the carboxypeptidase A2 (33)]. However, a more complex set of entrance and Ser-123 at the other end. In contrast, there is no side-chain and backbone movements would be required in such pocket in Ang and the side chain of Gln-117 occupies Ang to generate an active center comparable to that of this position. Moreover, although the orientation ofThr-44 is RNase. almost identical to that of its counterpart in RNase, Ser-118 The B2 site is also poorly conserved in the Ang structure. is relatively far from the position of Ser-123 in RNase. Of the four RNase residues which have been proposed to Interestingly, the Gln-117 side chain interacts with the same interact with the purine component of the substrate, only two groups-the main-chain NH and the side chain OH of one, Glu-iii, is unambiguously maintained in the primary Thr-44-that in RNase are involved in binding the pyrimidine structure of Ang, as Glu-108. Although its orientation is ring of substrates and inhibitors. These B1-site changes are a similar in the two proteins, Glu-108 plays only a minor role consequence of the markedly different orientations of the in Ang as shown by mutagenesis studies (14). No Ang residue polypeptide backbones of the two proteins starting at Asp- is structurally equivalent to Asn-67 or Asn-71 of RNase. In 116 (Ang)/Asp-121 (RNase). fact, if the substrate purine nucleotide adopts the same Numerous substrate analogue and inhibitor complexes conformation when bound to Ang as it does with RNase, the have been used to define the active site of RNase. We have only apparent candidate for an important component of the superimposed the Ang crystal structure on that of one of B2 site is Asn-68, whose side chain is positioned similarly to these (6RSA-PDB) (30), the complex ofRNase A with uridine that of Gln-69 in RNase. vanadate (Fig. 3 Lower), an inhibitor that occupies the PI and Putative Receptor Binding Site. In addition to the ribonu- B1 sites. The most prominent feature of the resultant super- cleolytic active site, a second site on Ang-minimally en- position (Fig. 3 Upper) is the incompatibility of the positions compassing residues in the segment 60-68 and Asn-109-is of the side chain of Ang Gln-117 and the uracil ring: indeed, also required for angiogenic activity (13, 15, 16). In contrast

Gln-117 passes directly through the uracil ring! Thus it would to the active-site mutants H13A (His-13 -+ Ala) and H114A seem that binding of RNA substrates to Ang must require (11), variants modified in this region do not inhibit the Table 2. Comparison of selected residues in the active-site regions of RNase A and Ang RNase A Ang Comments based on the three-dimensional structures residue Proposed role(s) in RNase (9, 27-30) residue of Ang and RNase Catalytic residues (the catalytic triad is positioned almost identically in the two structures) His-12 Removes 2'-OH proton; hydrogen His-13 His-13 ND1 hydrogen bonds to Thr-44 C= O in Ang, as with bonds to phosphate His-12 and Thr-45 in RNase Lys-41 Stabilizes intermediate; hydrogen bonds Lys-40 Lys-40 NZ hydrogen bonds to Ile-42 C=O in Ang; Lys-41 NZ to 2'-OH hydrogen bonds to OD1 of Asn-44 in RNase His-i19 Protonates 5' 0 leaving group His-114 RNase His-i19 NE2 hydrogen bonds directly with Asp-121 OD1; in Ang, H20-mediated Substrate-binding residues Gln-li Hydrogen bonds to phosphate Gln-12 Residues positioned almost identically Thr-45 Hydrogen bonds from NH and OG to Thr-44 Similar positioning in the two structures, but the interactions pyrimidine 02 and N3; hydrogen differ markedly: NH of Thr-44 in Ang hydrogen bonds to OE1 bond from C=O to ND1 of His-12 - of Gln-117 and OG of Thr-80, interactions absent from RNase Asn-67 Hydrogen bonds to purine N6/06 No equivalent residue in Ang Gln-69 Hydrogen bonds to purine N6/06 or N1 Asn-68 Ca atoms are not equivalent, but orientations of the side chains are similar Asn-71 Hydrogen bonds to purine N6/06 or N1 - No equivalent residue in Ang Glu-iii Hydrogen bonds to purine N1 Glu-108 Positions correspond well in the two structures Ser-123 Hydrogen bonds to uracil 04 Ser-118 Ang Ser-118 OG is 7 A from position of RNase Ser-123 and makes a hydrogen bond to Asp-116 that is absent from RNase; C=O of Ang Ser-118 hydrogen bonds to a-NH of Arg-121 Other residues in the C-terminal region Asp-121 Helps to orient catalytic site by Asp-116 Side chains differ in orientations/interactions; hydrogen bond hydrogen bonding to NE2 of His-ii9 between OD2 of Ang Asp-116 and OG of Ser-118 and H20-mediated hydrogen bond to NE2 of His-114; RNase Asp-121 carboxylate makes hydrogen bonds directly to His-1i9 and NZ of Lys-66 Ala-122 Gln-117 Ang Gln-117, unlike RNase Ala-122, occupies the pyrimidine binding cleft, potentially hindering substrate binding; C=O interacts with NH of Phe-120, OE1 hydrogen bonds to NH of Thr-44, and NE2 hydrogen bonds to OG of Thr-44 Phe-120 Side chain occupies a position similar to that of Ser-123 in RNase; NH hydrogen bonds to Gln-117 C=O; C==O hydrogen bonds to a-NH of Arg-122 Arg-121 No structurally equivalent RNase residue; a-NH hydrogen bonds to C=O of Ser-118 Downloaded by guest on September 23, 2021 2918 Biochemistry: Acharya et al. Proc. Nad. Acad. Sci. USA 91 (1994)

FIG. 3. (Upper) Stereoview ofthe modeled complex ofhuman Ang with uridine vanadate (UV), based on superposition ofthe Ang structure and the complex ofRNase A with uridine vanadate (30). Amg residues are represented by thick lines and uridine vanadate by thin lines. (Lower) Corresponding stereo view of the complex of RNase A with uridine vanadate. biological activity of Ang. Hence, it has been implicated in generated that contrast with the decreases observed for cell-surface receptor binding. similar substitutions in RNase (35). It is apparent from the Two of the segments of primary structure in Ang that present findings that such mutations of Asp-116 in Ang may comprise this site (residues 59-68 and 108-110) are on disrupt its interaction with Ser-118, thereby reducing steric adjacent loops and are among those that differ most markedly constraints on Gln-117 so as to allow pyrimidine binding and from their RNase counterparts (Fig. 2): they contain numer- enhance activity. Similarly, mutation of Ser-118 fails to alter ous deletions and only one conserved residue. Although the specificity (14), which was surprising in view of the role of corresponding segments of RNase are also in loop regions, Ser-123 in the B1 site of RNase, but is consistent with the there is little similarity with respect to either backbone markedly different location of this residue in Ang. structure or side-chain positions, and the overall rms devia- Arg-33 of Ang is neither expected nor found to be a tion in the positions ofall atoms in these two segments is 4.33 component ofthe ribonucleolytic active site, yet its mutation A. This is consistent with the view that these surface regions to Ala decreases enzymatic activity by a factor of7 (13). This ofthe two proteins have evolved to bind different ligands-a effect may be explained by the hydrogen bonds between the purine moiety in RNA for RNase and a cell-surface receptor guanidino group of Arg-33 and the carbonyl oxygens ofboth for Ang. Indeed, it is striking that no RNase residue side Thr-11 and Tyr-14 on either side of the catalytic residue chains occupy positions analogous to those of the three Ang His-13. The complete loss of enzymatic activity that occurs residues thus far implicated in receptor binding (i.e., Asn-61, upon treatment of Ang with arginine reagents, which is not Arg-66, and Asn-109), just as no Ang residues are equivalent due to modification of Arg-33, has remained enigmatic. to Asn-67 and Asn-71 in the B2 purine binding site ofRNase. Mutation ofthe arginines at positions 31, 32, 66, and 70 does The side chains of both Arg-66 and Asn-109 in Aug are not change activity (13), consistent with their positions in the exposed to solvent (Fig. 2) and are potentially available for three-dimensional structure, and mutation ofArg-5, which is ligand interactions. The Asn-61 side chain, however, hydro- found at the edge ofthe active-site region, reduces activity by gen bonds to both the main-chain oxygen of Ser-52 on helix only a factorof4. Arg-121, which was not mutated previously H3 and the side chain of Ser-74 on (-strand B3, and hence as it was considered to be too farfrom the active site, is in fact some conformational change would be required for this within this region (Fig. 3). Hence, the loss of enzymatic residue to interact with the receptor. Alternatively, its role in activity may be a consequence of modification of Arg-121. receptor binding may be indirect. Concluion. The three-dimensional structure of Ang ob- ImlcatIons Regarding Ang Mutants. The Ang crystal tained by x-ray diffraction is simultaneously familiar and structure suggests a molecular basis for hitherto unexplained unfamiliar. In terms ofcore topology and the arrangement of effects of mutagenesis on enzymatic activity. The most the catalytic histidines and lysine in its enzymatic active striking of these are the results of replacing Asp-116 by His, center, it looks similar to RNase A. Outside this core, new Ala, or Asn (34), where large increases in activity are motifs appear that are probably responsible for the charac- Downloaded by guest on September 23, 2021 Biochemistry: Acharya et al. Proc. NatL Acad. Sci. USA 91 (1994) 2919

teristic activities of Ang. Some of these differences were 3. Badet, J., Soncin, F., Guitton, J.-D., Lamare, O., Cartwright, anticipated based on earlier findings: thus, it was predicted T. & Barritault, D. (1989) Proc. Natd. Acad. Sci. USA 86, to bind cellular receptors 8427-8431. that the region of Ang thought 4. Bicknell, R. & Vallee, B. L. (1989) Proc. Natd. Acad. Sci. USA would not resemble its counterpart in RNase. X-ray crystal- 86, 1573-1577. lography now provides the detailed structure of this site and 5. Soncin, F. (1992) Proc. Natd. Acad. Sci. USA 89, 2232-2236. reveals interactions with additional areas of the molecule- 6. Shapiro, R., Riordan, J. F. & Vallee, B. L. (1986) Biochemistry helix H3 and strand B3-that had not been investigated 25, 3527-3532. previously for their functional importance. Other differences 7. Strydom, D. J., Fett, J. W., Lobb, R. R., Alderman, E. M., observed in the Ang structure vis-a-vis that of RNase were Bethune, J. L., Riordan, J. F. & Vallee, B. L. (1985) Biochem- most these being the complete istry 24, 5486-5494. not expected, the startling of 8. Harper, J. W. & Vallee, B. L. (1989) Biochemistry 28, 1875- reorientation of the C-terminal segment such that Gln-117 1884. obstructs the "normal" pyrimidine binding site; Asp-116 and 9. Richards, F. M. & Wyckoff, H. W. (1971) Enzymes 4,647-806. Ser-118 move 3-7 A from the positions of Asp-121 and 10. Blackburn, P. & Moore, S. (1982) Enzymes 15, 317-433. Ser-123 in RNase; and several new residues, including Phe- 11. Shapiro, R. & Vallee, B. L. (1989)Biochemistry 28, 7401-7408. 120 and Arg-121, are introduced into the active site. This 12. Shapiro, R., Fox, E. A. & Riordan, J. F. (1989) Biochemistry conformation, together with the absence of the 65-72 disul- 28, 1726-1732. fide loop (8), could well account for much of the decreased 13. Shapiro, R. & Vallee, B. L. (1992) Biochemistry 31, 12477- enzymatic potency of Ang toward typical RNase substrates. 12485. 14. Curran, T. P., Shapiro, R. & Riordan, J. F. (1993)Biochemistry The region around Lys-40 also plays a role in attenuating 32, 2307-2313. activity (36), but the structural basis for this is unclear from 15. Hallahan, T. W., Shapiro, R. & Vallee, B. L. (1991) Proc. Nat!. the present study. Acad. Sci. USA 88, 2222-2226. The mechanistic significance of the Ang C-terminal struc- 16. Hallahan, T. W., Shapiro, R., Strydom, D. J. & Vallee, B. L. ture remains to be investigated. One intriguing possibility is (1992) Biochemistry 31, 12477-12485. that it provides a means by which Ang can be activated at the 17. Palmer, K. A., Scheraga, H. A., Riordan, J. F. & Vallee, B. L. appropriate time and location in vivo to become a more potent (1986) Proc. Nat!. Acad. Sci. USA 83, 1965-1969. RNase. Thus, binding of Ang to a receptor, for example, 18. Acharya, K. R., Subramanian, V., Shapiro, R., Riordan, J. F. might reorganize this segment so as to move Gln-117 and & Vallee, B. L. (1992) J. Mol. Biol. 228, 1269-1270. 19. Kabsch, W. J. (1988) J. Appl. Crystallogr. 21, 61-71. open the pyrimidine binding site. [As the accompanying 20. Stuart, D. I., Levine, M., Muirhead, H. & Stammers, D. K. paper shows (31), such a conformational rearrangement (1979) J. Mol. Biol. 134, 109-142. appears to be part of the in vitro catalytic pathway of Ang.] 21. Fitzgerald, P. M. (1988) J. App!. Crystallogr. 21, 274-278. An analogous mechanism has been proposed for regulation of 22. Wlodawer, A., Bott, R. & Sjolin, L. (1982) J. Biol. Chem. 257, cyclin-dependent kinase 2, which contains a unique helix- 1325-1332. loop segment that interferes with binding of substrates to the 23. Brdnger, A. T., Kuriyan, J. & Karplus, M. (1987) Science 235, native enzyme (37). 458-460. Finally, we note that the three-dimensional structure re- 24. Jones, T. A. (1985) Methods Enzymol. 115, 90-111. ported here was determined with unliganded Ang and, hence, 25. Kraulis, P. J. (1991) J. Appl. Crystallogr. 24, 946-950. struc- 26. Kabsch, W. J. & Sander, C. (1983) Biopolymers 22, 2577-2637. functional implications can only be inferred. Crystal 27. Pares, X., Nogues, M. V., de Llorens, R. & Cuchillo, C. M. tures of substrate and inhibitor complexes and of Ang mu- (1991) Essays Biochem. 26, 89-103. tants will be required to obtain more direct information. At 28. Wodak, S. Y., Liu, M. Y. & Wyckoff, H. W. (1977) J. Mol. the same time, the present Ang structure generates questions Biol. 116, 855-875. and allows predictions readily examined by mutagenic 29. Brflnger, A. T., Brooks, C. L. & Karplus, M. (1985) Proc. and/or kinetic studies (see ref. 31). Moreover, we believe Natd. Acad. Sci. USA 82, 8458-8462. that this structure will prove useful for the design of novel 30. Bornh, B., Chen, C., Egan, M., Miller, M., Wlodawer, A. & inhibitors of Ang. It has already provided insight into the Cohen, J. S. (1985) Biochemistry 24, 2058-2067. structural basis for the catalytic activity ofAng, which differs 31. Russo, N., Shapiro, R., Acharya, K. R., Riordan, J. F. & Studies with Vallee, B. L. (1994) Proc. Nat!. Acad. Sci. USA 91, 2920-2924. from that of the homologous protein RNase. 32. Gilliland, G. L., Winborne, E. L., Nachman, J. & Wlodawer, receptors or receptor analogues should extend this under- A. (1990) Proteins 8, 82-101. standing to the unique biological activity of Ang as an 33. Faming, Z., Kobe, B., Stewart, C. B., Rutter, W. J. & Gold- angiogenic protein. smith, E. J. (1991) J. Biol. Chem. 266, 24606-24612. 34. Harper, J. W. & Vallee, B. L. (1988) Proc. Nat!. Acad. Sci. We thank David Phillips and Tony Rees for support and encour- USA 85, 7139-7143. agement, Vasanta Subramanian and David Stuart for discussions, 35. DeMel, V. S. J., Martin, P. D., Doscher, M. S. & Edwards, and the staff at the Synchrotron Radiation Source, Daresbury, for B. F. P. (1992) J. Biol. Chem. 267, 247-256. facilities. This work was supported by the Medical Research Council 36. Harper, J. W., Fox, E. A., Shapiro, R. & Vallee, B. L. (1990) and the Leverhulme Trust, U.K., and the Endowment for Research Biochemistry 29, 7297-7302. in Human Biology, Inc., Boston. 37. De Bondt, H. L., Rosenblatt, J., Jancarik, J., Jones, H. D., Morgan, D. 0. & Kim, S.-H. (1992) Nature (London) 363, 1. Fett, J. W., Strydom, D. J., Lobb, R. R., Alderman, E. M., 595-602. Bethune, J. L., Riordan, J. F. & Vallee, B. L. (1985) Biochem- 38. Soncin, F., Shapiro, R. & Fett, J. W. (1994) J. Biol. Chem. 269, istry 24, 5480-5486. in press. 2. Shapiro, R., Strydom, D. J., Olson, K. A. & Vallee, B. L. 39. Moroianu, J. & Riordan, J. F. (1994) Proc. Nat!. Acad. Sci. (1987) Biochemistry 26, 5141-5146. USA 91, 1677-1681. Downloaded by guest on September 23, 2021