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

Article No. jmbi.1998.2378 available online at http://www.idealibrary.com on

J. Mol. Biol. (1999) 285, 1209±1233

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. Leonidas1, Robert Shapiro2,3, Simon C. Allen1, Gowtham V. Subbarao1, Kasinadar Veluraja1 and K. Ravi Acharya1* 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 3Department 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 ribonucleolytic activity is weak (104 to 106-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 Ê and Met-1 at 2.0 A Ê resnative Ang in two different forms (Pyr1 at 1.8 A Ê resololution) and for two active-site variants, K40Q and H13A, at 2.0 A ution. 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, eosinophilderived 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 unaffected by the amino acid replacement. # 1999 Academic Press

*Corresponding author

Keywords: angiogenesis; angiogenin; X-ray crystallography; ribonuclease superfamily; nuclear translocation

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: [email protected] 0022-2836/99/031209±25 $30.00/0

# 1999 Academic Press

1210

Introduction Human angiogenin (Ang), a 14.1 kDa protein in the pancreatic RNase superfamily (Kurachi et al., 1985; Strydom et al., 1985), is a potent inducer of new blood vessel formation in vivo (Fett et al., 1985). Ang exhibits a characteristic ribonucleolytic activity that is several orders of magnitude weaker than that of bovine pancreatic RNase A in standard assays (Shapiro et al., 1986, 1988a; Harper & Vallee, 1989) but nonetheless seems to be essential for angiogenic activity. The strongest evidence for this association is provided by the parallel decreases in enzymatic and biological activities produced by mutations of catalytic (His13, Lys40 and His114) and substrate-binding (Thr44) residues (Shapiro et al., 1989; Shapiro & Vallee, 1989; Curran et al., 1993a). The natural RNA substrate for Ang is yet to be identi®ed. However, recent ®ndings suggest that it may reside in the nucleolus of endothelial cell targets, where exogenous Ang accumulates. Translocation of Ang to the nucleolus is an indispensable step in the mechanism by which Ang induces angiogenesis (Moroianu & Riordan, 1994b), and an oligonucleotide inhibitor of both the enzymatic and angiogenic activities of Ang has been shown to co-localise with Ang in the nucleus (Nobile et al., 1998). Ang was ®rst isolated from medium conditioned by human adenocarcinoma cells (Fett et al., 1985) and was subsequently demonstrated to play a critical role in tumour formation. A non-cytotoxic antiAng monoclonal antibody prevents establishment of primary tumours in athymic mice following subcutaneous injection of human colon, lung, ®broblast, and prostate cancer cells (Olson et al., 1994, 1995; Olson & Fett, 1996); it also diminishes local metastatic spread of prostate cancer cells that have been implanted in the prostate gland (Olson & Fett, 1996). Moreover, in vivo administration of an antisense oligonucleotide directed against human Ang mRNA was recently shown to inhibit formation of both metastases and subcutaneous tumours (Olson & Fett, 1998). These ®ndings identify Ang as an attractive target for new anticancer drugs. One approach for the development of clinically useful Ang antagonists is to design small-molecule inhibitors of the ribonucleolytic activity or cellular interactions of Ang based on structural inforÊ ) crystal mation. The medium-resolution (2.4 A structure of recombinant Met-1 human Ang reported previously (Acharya et al., 1994) provided key insights into the structure-function relationships of the protein and a starting point for design efforts (Russo et al., 1996a; Leonidas et al., 1997). Most strikingly, it revealed an unexpected feature of Ang that attenuates its RNase activity: the space that corresponds to the open pyrimidine-binding site of RNase A is obstructed by Gln117. This blockage, which exists in solution as well as in the crystalline state (Russo et al., 1994; Lequin et al., 1997), is associated with a markedly different orientation and secondary structure for the C-term-

High-resolution Crystal Structures of Human Angiogenin

inal segment of Ang compared to that of RNase A. Since RNA substrates must occupy the pyrimidinebinding site in order to be cleaved, it can be inferred that Ang undergoes a conformational change as part of its normal catalytic pathway. The other major difference between the structures of RNase A and Ang involves the region encompassing residues 59-73(RNase)/58-70(Ang), which contributes part of the purine-binding site in RNase A but functions primarily as a cell-binding site in Ang (Hallahan et al., 1991, 1992). Ê resolution crystal strucWe now report a 2.0 A Ê ture for the Met-1 form of human Ang and a 1.8 A resolution structure for the natural Pyr1 form. These high-resolution structures provide a more detailed understanding of the molecular forces that underlie the distinctive topology and unique functional properties of Ang, and are utilised here to interpret the results of extensive mutational studies performed on this protein. Importantly, the availability of these structures has also allowed a more rigorous assessment of the relationship between the ribonucleolytic and angiogenic activities of Ang. For this purpose, structures of the catalyticsite variants K40Q and H13A were determined at Ê and compared with the native structures. 2.0 A 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 changes in some regions beyond the ribonucleolytic site, but the cell-binding site is essentially unaffected by the amino acid replacement.

Results and Discussion Native proteins ``Native'' Ang has been produced in two forms that differ only with respect to their N termini: the natural Pyr1 form contains a pyroglutamic acid residue at position 1, whereas the Met-1 form has glutamine at position 1 and an additional residue, methionine, at position ÿ1. The enzymatic and angiogenic activities of the two proteins are indistinguishable (Shapiro et al., 1988a). The original structural study (Acharya et al., 1994) was performed with the Met-1 form and achieved a resolÊ ; this has now been extended to ution of 2.4 A Ê 2.0 A resolution (Tables 1 and 2). Examination of the Pyr1 form showed that it crystallised in a related but different space group (P21212 versus C2221). Pyr1 Ang crystals are more stable in the X-ray beam and better suited for high-resolution structural studies. The Pyr1 structure presented Ê resolution (Figure 1, here was re®ned to 1.8 A Tables 1 and 2). Overall structures Ang has a kidney-shaped tertiary fold, divided into two lobes (Figure 2). Lobe I comprises four

1211

High-resolution Crystal Structures of Human Angiogenin

Ê Figure 1. Stereoview of a portion of the ®nal 1.8 A 2jFoj ÿ jFcj electron density map for Pyr1 Ang around Gln117. The map is contoured at the 1.0s level.

antiparallel b-strands, B2, B3, B6 and B7, formed by residues 62-65, 69-72, 104-108 and 111-115, respectively. This b-core structure is sandwiched by two helices H1 and H3 (formed by residues 3-13 and 50-57, respectively). Lobe II consists of a b-sheet formed by three antiparallel b-strands, B1, B4 and B5 (residues 42-46, 76-84 and 93-101, respectively), packed between helix H2 (residues 23-32) and a 310 helix (residues 117-121). There are three disulphide bonds, as opposed to four in pancreatic RNases. One is located in lobe I and connects b-strand B6 to helix H3; the other two are in lobe II and connect b-strand B4 to helix H2 and the two large H2/B1 and B4/B5 loops. Both Ang structures are well de®ned in the electron density map (Figure 1), with the exception of the ®rst two (Pyr1) or three (Met-1) residues at the N terminus and the last two C-terminal residues. These residues have high temperature factors and

appear to be disordered; hence they were excluded from all structural comparisons. The two native structures superimpose closely with an r.m.s. deviÊ for 119 equivalent Ca atoms (resiation of 0.26 A dues 3-121), and there are no signi®cant differences. The high resolution of the structures has enabled identi®cation of 53 water molecules in the Pyr1 Ang structure and 43 in Met-1 Ang; 25 of these, including seven in the catalytic site, are conserved in the two structures. A citrate molecule from the crystallisation buffer was found in both structures in similar positions, interacting with the main-chain atoms of Phe100 and the side-chain atoms of Arg101 from a symmetry-related molecule. The structure of Pyr1 Ang appears to be more rigid than that of Met-1: the average value for the temperature factors is signi®cantly lower than that of Met-1 Ang (Table 2). This may be related to the solvent content in the crystals, which is unusually high (63 %) for Met-1 Ang (compared to 48 % for Pyr1 Ang). In the crystal, Pyr1 Ang makes 104 van der Waals contacts and 12 hydrogen bonds. Similarly, Met-1 Ang is involved in 95 van der Waals contacts and 12 hydrogen bonds. Most of the contacts are the same, even though the two proteins crystallise in different space groups. The ribonucleolytic active site 0

Like RNase A, Ang cleaves RNA at P ± O5 bonds on the 30 side of pyrimidines via a transphosphorylation mechanism to form 20 ,30 -cyclic nucleotides, and subsequently hydrolyses the cyclic nucleotides to generate 30 phosphate groups (Shapiro et al., 1988a; Russo et al., 1994). However, Ang is 104 to 106-fold less active than RNase A toward polynucleotide, dinucleotide and cyclic nucleotide substrates, and its speci®city differs signi®cantly (Shapiro et al., 1986, 1988a; Rybak & Vallee, 1988; Harper & Vallee, 1989; Russo et al., 1996a). The

Table 1. X-ray data collection statistics Dataset Space group Unit cell Ê) a (A Ê) b (A Ê) c (A Ê) Resolution range (A Ê) Highest resolution shell (A Reflections measured Unique reflections Rmerge (%)a All data Highest resolution shell Completeness (%) All data Highest resolution shell I/sI All data Highest resolution shell a b

Pyr1 Ang

Met-1 Ang

K40Q

H13A

P21212

C2221

C2221

C222

84.6 37.8 42.4 30.0-1.8 1.9-1.8 86,423 11,357

82.9 119.0 37.4 40.0-2.0 2.1-2.0 141,072 13,170

83.7 120.0 37.6 30.0-2.0 2.1-2.0 104,187 13,155

67.2 104.5 39.0 30.0-2.0 2.1-2.0 21,688 5,493

4.7 37.8

8.8 37.4

5.5 24.8

9.3 24.2

87.8 72.1

99.5 99.4

98.3 94.9

58.3b 31.8

9.3 2.6

13.5 5.2

4.9 1.3

4.7 1.3

Rmergeˆhi jI(h) ÿ Ii(h)j/hi Ii(h), where Ii(h) and I(h) are the ith and the mean measurements of the intensity of re¯ection h. Ê. 99.1 % for resolution range 30.0-2.9 A

1212

High-resolution Crystal Structures of Human Angiogenin

Table 2. Structure re®nement statistics Ê) Resolution (A Rcryst (%)a Rfree (%)b Number of reflections Number of protein atoms Number of solvent molecules Number of citrate molecules Deviations from ideality (rms) Ê) Bond lengths (A Bond angles ( ) Dihedrals ( ) Impropers ( ) Ê 2) Average B-factor (A Main-chain atoms Side-chain atoms All protein atoms Solvent atoms

Pyr1 Ang

Met-1 Ang

K40Q

H13A

20.0-1.8 22.2 27.3 11,348 992 53 1

20.0-2.0 21.7 28.6 12,139 993 43 1

20.0-2.0 22.9 28.6 13,031 993 33 1

20.0-2.0 19.8 31.9 5482 988 38 -

0.011 1.7 28.0 0.9

0.010 1.8 27.7 0.8

0.010 1.9 28.0 0.8

0.011 1.6 28.8 0.8

28.3 33.0 30.6 44.6

39.6 42.5 41.0 43.5

38.7 42.4 40.6 40.9

28.0 31.3 29.6 37.1

a Rcryst ˆ hjFo ÿ Fcj/hFo, where Fo and Fc are the observed and calculated structure factor amplitudes of re¯ection h, respectively. b Rfree is equal to Rcryst for a randomly selected 5 % subset of re¯ections not used in the re®nement (BruÈnger, 1992a).

low enzymatic potency of Ang re¯ects both its weak af®nity for substrates and its slow turnover: Km and kcat values with the dinucleotide CpA (the only substrate for which individual parameters rather than kcat/Km values have been reported) are 62 mM and 0.7 sÿ1, respectively, i.e. 120-fold

higher and 6500-fold lower than for RNase A (Russo et al., 1994). Although Ang shares with RNase A a preference for 30 cytidine versus uridine nucleotides and for 50 adenosine nucleotides, its 30 selectivity is several-fold stronger, whereas its 50 discrimination is much less stringent.

Figure 2. A representation of the Ang structure. The disulphide bonds are shown in ball-and-stick representation. The inset presents the details of the Ang ribonucleolytic active site including water molecules (in blue). The amino acid residues are shown in standard colour. Broken lines represent hydrogen bonds.

High-resolution Crystal Structures of Human Angiogenin

The active site of RNase A contains various subsites, designated P0 . . . Pn, B0 . . . Bn and R0 . . . Rn (Richards & Wyckoff, 1971; PareÂs et al., 1991; Raines, 1998) for binding the phosphate, base and ribose moieties of the RNA substrate, respectively. 0 The central sites are P1, where P ± O5 bond clea0 vage occurs; B1, which binds the 3 pyrimidine; and B2, which interacts with the base of the 50 nucleotide. Superposition of the human Ang and RNase A crystal structures shows that only the P1 site is well conserved. The most dramatic differences between the structures are at the B1 site, but other subsites also have distinct architectures in the two proteins. These features and their implications are now considered in detail. Except where noted otherwise, residue positions are indistinguishable in the Pyr1 and Met-1 Ang structures, and distances speci®ed are those in the Pyr1 structure. The P1 subsite. His12, Lys41 and His119 are the RNase A residues directly involved in catalysis, as ®rst proposed on the basis of chemical modi®cation studies (summarised by Richards & Wyckoff, 1971), later supported by crystallography (see Richards & Wyckoff, 1973), and con®rmed by mutagenesis studies (Trautwein et al., 1991; Thompson & Raines, 1994; Messmore et al., 1995). In the most widely accepted chemical mechanism for the transphosphorylation step (Findlay et al., 1962), His12 acts as a general base to deprotonate the 20 oxygen atom, which then attacks the phosphorus atom to form a pentavalent transition state. This species, which is stabilised by Lys41, collapses to yield the cyclic 20 ,30 -phosphate group, with His119 serving as a general acid to protonate the leaving group oxygen atom. The roles of His12 and His119 are reversed in the hydrolysis step. The positions of His12 (Ne2), Lys41 (Nz) and His119 (Nd1) in the crystal and NMR structures of several nucleotide complexes of RNase A or its subtilisincleaved form, RNase S, are consistent with this scenario (Richards & Wyckoff, 1973; Wodak et al., 1977; Borkakoti, 1983; Zegers et al., 1994; Toiron et al., 1996). However, a recent analysis of the Ê resolution structure of the complex of RNase 1.3 A A with uridine vanadate (UVan; Ladner et al., 1997), an analogue of the phosphorane transition state or reaction intermediate, suggests that His12 is well positioned to protonate the non-bridging phosphoryl oxygen atom and thereby stabilise the phosphorane, and that Lys41, rather than His12, may be the catalytic base in certain cases (Wladkowski et al., 1998). Two additional residues contact the P1 substituent of inhibitors in these structures: Phe120 (main-chain N) and Gln11 (Ne2). However, replacement of Gln11 by Ala produces only minor changes in activity toward standard RNase substrates, and this residue appears to prevent non-productive binding of substrate rather than to participate in catalysis (delCardayre et al., 1995). An extensive set of hydrogen bonds maintains the conformations of the catalytic residues in

1213 RNase A. His12 Nd1 interacts with the carbonyl O of Thr45, while hydrogen bonds between the sidechain of Arg33 and the main-chain oxygen atoms of Arg10 and Met13 help to properly orient the a-helix that contains this histidine residue. The Lys41 main chain is ®xed by an interaction of its carbonyl O with OZ of Tyr97; the 3.5-fold decrease in activity that accompanies mutation of Tyr97 to Phe indicates that this contact is functionally signi®cant (Eberhardt et al., 1996). The position of the Lys41 Nz atom is variable in structures of free RNase A and nucleotide, sulphate and phosphate complexes, but in most instances the ammonium group forms a hydrogen bond with Od1 of Asn44. The side-chain of His119 adopts two distinct conformations (A and B) in free RNase A (Borkakoti et al., 1982); only conformation A is compatible with occupation of the B2R2 site. When His119 is in conformation A, its Ne2 hydrogen bonds with the carboxylate group of Asp121. This interaction has recently been demonstrated to play an important role: replacement of Asp121 by Ala diminishes kcat/Km values for transphosphorylation by about 100-fold (Schultz et al., 1998). Detailed structural and functional characterisation of the D121A RNase A variant indicates that Asp121 serves primarily to orient His119 properly to ful®l its catalytic function. Ang residues His13, Lys40 and His114 correspond to the catalytic triad 12/41/119 of RNase A. Replacements of His13 or His114 by Ala (Shapiro & Vallee, 1989) or Lys40 by Gln (Shapiro et al., 1989) decrease ribonucleolytic activity by factors of at least 104, 104 and 2  103, respectively. The conservative substitution of Arg for Lys40 reduces activity by 50-fold (Shapiro et al., 1989), similar to the effect of the Lys41 ! Arg mutation in RNase A (Trautwein et al., 1991). These ®ndings suggest that the roles of the three Ang residues are similar to those of their counterparts in RNase A. Indeed, the orientations of the catalytic residues in the superimposed structures of Ang and the complex of RNase A with UVan (PDB code 6RSA; Borah et al., Ê resolution; PDB code 1RUV; Ladner 1985, at 1.5 A Ê resolet al., 1997; Wladkowski et al., 1998, at 1.3 A ution) are closely similar, and all of the residues that contact the vanadate moiety appear to be maintained (Figure 3). Ne2 of His13 in Ang is only Ê from its RNase counterpart and would be 0.4 A capable of forming the same two hydrogen bonds with O20 and O3V of the inhibitor. His114 of Ang and His119 of RNase are somewhat farther apart Ê for Nd1 atoms), but again the Ang residue is (0.8 A positioned appropriately to engage in the same hydrogen bonds (with O2V and O30 ). His114 in both Ang crystal structures adopts only conformation A, although NMR has revealed a dynamic equilibrium between A and B conformers in solution (Lequin et al., 1997). The electron density for Nz of Lys40 in the Ang structures is poor. However, the other side-chain atoms of this residue are well de®ned and it seems that the ammonium group could extend suf®ciently close to O20 to

1214

High-resolution Crystal Structures of Human Angiogenin

bonds to an isolated water molecule (Figure 2), which occupies a position close to that of Asp121 Od1 in RNase A. The P1 region of the Ang active site contains numerous additional water molecules (six in the Pyr1 structure), forming a network that links His114 Nd1, His13 Ne2, the carbonyl oxygen atoms of Val113 and Leu115, and the main-chain NH of Gln117 (Figure 2, Table 3). Three of these water molecules occupy the same space as uridine vanadate in the UVan-RNase A complex, and are presumably displaced upon binding of substrates or inhibitors.

Figure 3. Superimposed structures of Pyr1 Ang and RNase A-UVan complex showing the P1 subsite. Ang and RNase A residues are shown in standard colours and in green, respectively. Only the ribose vanadate portion of UVan is shown (blue). Broken lines represent hydrogen bonds in the RNase A complex.

form a hydrogen bond similar to that of Lys41 in RNase A. The position of Nz of the catalytic lysine residue is also unclear in some of the free RNase A structures (Borkakoti et al., 1982). The main-chain N of Leu115 in Ang superimposes well with the corresponding atom (N of Phe120) in the RNase A-UVan complex and is therefore well positioned to form the same interaction with O3V of the inhibitor. The side-chain of Ang Gln12 is oriented differently from that of RNase Gln11, and it is unclear whether its Ne2 could hydrogen bond with the inhibitor. Interchange of Ne2 and Oe1 of Gln12 would place the nitrogen atom in a more favourable position. The assignments of N and O in the Gln12 side-chain are based on the proximity of the His8 imidazole Ê hydrogen bond to group, which donates a 2.8 A e1 O that would not exist if the two atoms were interchanged. The Ang structures contain counterparts of most of the hydrogen bonds that hold the catalytic residues of RNase A in place, i.e. those connecting Lys41 with Asn44 and Tyr97 (Lys40 with Asn43 (observed only in Met-1 Ang) and Tyr94 in Ang), His12 with Thr45 (His13 with Thr44 in Ang), and the main chain of a-helix H1 with Arg33 (Thr11 and Tyr14 to Arg33 in Ang; Table 3). Replacement of Arg33 with Ala has been shown to decrease the ribonucleolytic activity of Ang sevenfold (Shapiro & Vallee, 1992), suggesting that the hydrogen bonds involving this residue are important for maintaining the position of His13. Only one of the interactions involving the catalytic triad of RNase A is not replicated in Ang: that between His119 Ne2 and Asp121 Od1. Asp116, the Ang counterpart Ê away from His114, and forms of Asp121, is >5 A deleterious contacts with Ser118, as will be discussed below. In Pyr1 Ang, His114 Ne2 hydrogen

The B1 subsite. The B1 site of RNase A has a small preference for cytosine over uracil, as judged by comparison of kcat/Km values for cytidylyl and uridylyl substrates (Witzel & Barnard, 1962). The three-dimensional structures of RNase-nucleotide complexes reveal that B1 is a pocket formed by His12, Val43, Asn44, Thr45, Phe120 and Ser123. The primary functional component of the site is Thr45, which forms two hydrogen bonds with pyrimidines: its main-chain NH group donates a proton to O2 of either base, and its Og1 can donate to N3 of cytosine or accept from N3 of uracil. In crystal structures of RNase A complexes with uridine nucleotides, the Thr45 side-chain hydrogen bonds also with the carboxylate group of Asp83 (Wlodawer et al., 1983); this contact is not present in complexes with cytidine nucleotides (Lisgarten et al., 1993; Zegers et al., 1994), where the Og1 proton is unavailable for donation to Asp83 and the Ê farther apart. Ser123 Og two side-chains are >1 A forms a water-mediated hydrogen bond with O4 in high-resolution structures of RNase A complexes (Borkakoti, 1983; Gilliland et al., 1994; Zegers et al., 1994). The phenyl group of Phe120 sits on one side of the pyrimidine ring near O2/N3, making van der Waals contacts and perhaps stacking interactions. His12 Ce1 contacts O2 on the same side of the ring. Val43 and Asn44 occupy the space on the opposite side; the main-chain carbonyl and a carbon of Asn44 contact O2, whereas the carbonyl O of Val43 and the side-chains of both residues lie just beyond van der Waals contact distance. Recent mutational studies by delCardayre & Raines (1994, 1995) provide insights into the functional contributions of Thr45 and Asp83 in RNase A that also have relevance for an understanding of Ang, as will be discussed below. The two key ®ndings in this regard are: (i) substitution of Ala for Asp83 decreases activity toward uridylyl nucleotides by tenfold, but barely affects cleavage of cytidylyl substrates; and (ii) replacement of Thr45 by Gly in D83A-RNase lowers activity toward uridylyl and cytidylyl substrates similarly, by 11-fold and sixfold, respectively. These results suggest that the observed hydrogen bond between Thr45 Og1 and N3 of the pyrimidine ring is functionally important, and that its strength is modulated by the additional interaction of the threonine sidechain with Od of Asp83. In the absence of Asp83,

1215

High-resolution Crystal Structures of Human Angiogenin

Table 3. Hydrogen bond interactions of the residues in the ribonucleolytic active site of human Ang in the structures of Pyr1 and Met-1 Ang and variants H13A and K40Q Donor Arg5 NZ1 His8 Ne2 His13 Nd1 His13 Ne2 Arg33 NZ1 Arg33 NZ2 Lys40 N Lys40 Nz Lys40 Nz Water Asn43 Nd2 Thr44 N Thr44 N Thr44 Og1 Ile46 N Thr80 Og1 Tyr94 OZ His114 Nd1 His114 Ne2 Leu115 N Gln117 N Gln117 Ne2 Ser118 N Ser118 Og Phe120 N Arg121 N Water Water Water Water Water Water Water Water

Acceptor

Pyr1 Ang

Met-1 Ang

K40Qa

H13Ab

Glu108 Oe1 Gln12 Oe1 Thr44 O Water Thr11 O Tyr14 O Leu35 O Ile42 O Asn43 Od1 Lys40 Nz Gln12 O Gln117 Oe1 Water Thr80 Og1 His13 O Water Lys40 O Water Water Water Water Thr44 Og1 Asp116 Od1 Asp116 Od1 Gln117 O Ser118 O Asn43 Od1 Thr44 O Val113 O Leu115 O Asp116 Od2 Asp116 O Ile119 O Phe120 O

2.9 2.9 3.0 2.7 2.9 3.3 2.8 2.9 2.8 2.8 2.7 2.8 2.8 2.9 3.3 3.2 3.1 3.1 2.5 3.0 3.1 3.0 2.8 2.9 2.7 2.8 3.0

2.8 2.7 3.2 3.0 3.1 3.2 2.6 2.7 2.9 2.7 2.6 2.7 2.8 2.7 3.2 3.1 3.1 3.0 2.6 3.0 3.2 3.1 3.0 3.0 2.9 2.7 -

2.9 2.7 3.0 2.7 2.9 3.3 2.9 3.0 2.8 2.7 2.6 2.8 2.5 3.2 3.1 3.0 2.4 3.1 3.2 3.0 2.8 2.8 -

3.2 2.5 2.6 3.1 3.2 3.2 3.1 2.8 2.7 2.4 3.0 2.8 2.4 3.2 2.6 3.0 3.3 2.4 2.8 -

Ê . Hydrogen bonds are listed if the distance between a donor (D) and an acceptor (A) is Numbers in columns are distances in A Ê and if the angle D-H-A is greater than 120 . Hydrogen bond parameters were calculated with the program shorter than 3.3 A HBPLUS (McDonald & Thornton, 1994).aIn K40Q residue 40 is glutamine instead of lysine.bIn H13A residue 13 is alanine instead of histidine.

the hydrogen bonds with N3 of cytosine and uracil are nearly comparable in avidity; Asp83 selectively improves the interaction with uracil, probably by increasing the partial negative charge on Thr45 Og1 and by aligning the hydroxyl group appropriately. If the hydrogen bond between the pyrimidine N3 and Thr45 Og1 in wild-type RNase A favours uracil, then what compensatory interactions account for the overall preference of the enzyme for cytosine? One possibility is that the hydrogen bond between the pyrimidine O2 and Thr45 NH is stronger for cytosine; the cytosine O2 should carry a larger partial negative charge, making it a better hydrogen acceptor. Cytosine might also engage in more favourable stacking interactions with Phe120, due to the greater delocalisation of electrons throughout its ring. There may also be subtle differences in base interactions with Val43 and/or Asn44 that increase relative af®nity for cytosine. The water-mediated hydrogen bond between N4/ O4 and Ser123 apparently in¯uences speci®city in the opposite direction: replacement of Ser123 with Ala by fragment complementation diminishes activity toward uridine cyclic 20 ,30 -phosphate by

fourfold but does not affect hydrolysis of the cytidine cyclic nucleotide (Hodges & Merri®eld, 1975). The region of Ang that corresponds to the B1 subsite of RNase A contains many identical or similar elements, Thr44 (for Thr45), Ile42 (Val43), Asn43 (Asn44), Thr80 (Asp83) and Leu115 (Phe120), but is blocked by Gln117, which occupies the position where the pyrimidine binds in RNase (Figure 4). This obstruction was ®rst noted in the Ê resolution Met-1 Ang structure (Acharya 2.4 A et al., 1994), and was later observed in the bovine Ang crystal structure (where Glu118 replaces Gln117; Acharya et al., 1995) and in the NMR structures of both human and bovine Ang (Lequin et al., 1996, 1997). A modelling study indicated that no alternative conformation of RNA substrates would allow productive binding to Ang (Russo et al., 1994) and it was inferred that native Ang exists primarily or entirely in an inactive state that must reorient as part of the normal catalytic pathway. It has not been determined whether the transition occurs prior to substrate binding, i.e. whether major ``B1 closed'' and minor ``B1 open'' structures exist in equilibrium, or whether it is induced by interaction with substrate.

1216

High-resolution Crystal Structures of Human Angiogenin

Figure 4. Superimposed structures of Pyr1 Ang and RNase A-UVan complex showing the B1 subsite. Ang and RNase A residues are shown in standard colours and in green, respectively. Only the uracil portion of UVan is shown (blue). Broken lines represent hydrogen bonds in the RNase A complex.

Figure 5. Ang residues stabilising the inactive conformation of Gln117. The C-terminal segment 115-121 is shown with a ®lled backbone. Other residues are shown in brick colour. Only the uracil portion of UVan is shown in blue for clarity. Broken lines represent hydrogen bonds.

Superpositions of the high-resolution Ang structures with those of RNase A-inhibitor complexes show that the positions of all side-chain and mainchain atoms of Gln117 are incompatible with that of the pyrimidine (Figure 4). Most dramatically, the side-chain passes directly through the pyrimidine ring and forms interactions with Thr44 that mimic those made by O2 and N3 of the pyrimidine with Thr45 of RNase A, i.e. Oe1 and Ne2 of Gln117 hydrogen bond to Thr44 NH and Og1, respectively Ê and 3.1 A Ê ). These two hydrogen (distances of 2.9 A bonds seem well ®xed in the crystal structures, Ê 2) for all four with relatively low B-factors (20-30 A components. The glutamine side-chain is more ¯exible in the NMR structure of Ang, which was determined at 40  C (Lequin et al., 1997), but all of its observed positions are obstructive. Because each of the Gln117 main-chain atoms clashes with the pyrimidine ring in the superpositions (distances to Ê ), it is C4/C5/C6 of UVan range from 1.3 to 2.5 A clear that opening of the B1 site cannot be accomplished simply by movement of the Gln side-chain. An additional Ang residue, Phe120, partially obstructs the putative B1 pocket. The Phe120 sidechain occupies a position between those of uracil/ cytosine O4/N4 and Ser123 Og in the RNase comÊ from O4 and Ce1 is plexes, e.g. its Cd2 atom is 1.8 A g Ê 1.1 A from the serine O in the superimposed Ang and UVan-RNase A structures. Ser118, the residue that corresponds to Ser123 of RNase A in the Ang primary structure, is well removed from its Ê for NH atoms), Ê for Og and 6.5 A counterpart (9.2 A and forms two hydrogen bonds with Asp116 Od1 Ê to Og and 3.1 A Ê to NH) that stabilise the (2.5 A obstructive position of the intervening Gln117 residue (Figure 5). The tight 310 helix that contains residues 117-121 is maintained by 117/120 and

118/121 main-chain hydrogen bonds, and its orientation appears to be determined in part by the packing of the Ile119 and Phe120 side-chains against hydrophobic residues at the convergence of b-strands B4 (Val 78), B6 (Val103/Val104) and B7 (Leu115). Results of mutational studies have revealed the importance of various C-terminal residues and interactions in suppressing the RNase activity of Ang. Replacement of Gln117 by Ala or Gly enhances activity 18 to 30-fold (Russo et al., 1994); the Gly substitution increases af®nity for the pyrimidine nucleotide inhibitor 20 -CMP ®vefold but has no effect on the binding of 50 -AMP, whose base is expected to occupy the B2 subsite. Preliminary structural data on the Q117G derivative (Acharya et al., 1998) indicate that truncation of the Gln side-chain is not by itself suf®cient to open the B1 site. Therefore, the increased activity of Gln117 variants may re¯ect destabilisation of the native ``inactive'' state due to loss of the hydrogen bonds with Thr44. It is possible that the Gln117 sidechain physically impedes the conformational rearrangement or is involved in deleterious interactions even after it has moved from its native position. Mutagenesis at Asp116, performed prior to the determination of the Ang crystal structure, had produced unexplained increases in activity toward RNA, in some cases as large as 15 to 18-fold (for D116A, D116S and D116H; Harper & Vallee, 1988; Curran et al., 1993b). The enhancements can now be rationalised as consequences of disruption of the two hydrogen bonds between 116 Od1 and Ser118. Consistent with this view, replacement of Ser118 by Ala, which eliminates one of the bonds, increases activity sevenfold (Shapiro, 1998). Thus,

High-resolution Crystal Structures of Human Angiogenin

the 116/118 interactions play an important role in stabilising the inactive conformation of Ang. At the same time, some of the effects of the Asp116 substitutions are complex and cannot be explained solely in terms of facilitation of the opening of the B1 site. For example, the activity increases are less substantial with NpN0 dinucleotide substrates than with tRNA, and most of the replacements alter base speci®city at the N and/or N0 position (Harper & Vallee, 1988; Curran et al., 1993b). These observations suggest that Asp116 may have additional positive or negative functions not apparent from the structure of the free protein. The importance of other C-terminal residues and interactions for maintaining the obstructive conformation has also been investigated by mutagenesis studies. The variant I119A/F120A-Ang is fourfold more active than Ang (Russo et al., 1996b). This increase was originally interpreted as evidence that the buried hydrophobic residues Ile119 and Phe120 contribute signi®cantly to establishment of the inactive state of Ang. However, the high-resolution Ang structure now reveals a possible direct impingement of the B1 site by the Phe120 side-chain (see above), and this may also be a factor. The roles of the main-chain 117/120 and 118/121 hydrogen bonds were explored by characterising deletion mutants. The effects of removing residues 121-123 (Russo et al., 1996b) and 119-123 (Shapiro, 1998) are complex, but suggest that these hydrogen bonds have only a small role. Mutational studies have provided information on how the B1 site of Ang functions once it opens. This site, like that of RNase A, prefers cytosine over uracil, but its selectivity is considerably stronger. The major determinants of this speci®city are Thr44 and Thr80. These residues correspond to the important B1 site components Thr45 and Asp83 of RNase A, respectively, and form an analogous hydrogen Ê ). However, the changes in bond (Og1 to Og1, 2.8 A activity produced by mutations of the RNase A (see above) and Ang residues differ markedly. In Ang, substitution of Thr44 with Ala decreases activity toward cytidylyl substrates 24 to 40-fold, but reduces activity toward uridylyl nucleotides only three to ®vefold (Curran et al., 1993a). Consequently, the variant no longer discriminates between the two pyrimidines. These results suggest that the Thr44 side-chain, although inaccessible in free Ang, interacts directly with the pyrimidine moiety in Ang-substrate complexes and makes a stronger contact with cytosine than with uracil. Replacement of Thr80 by Ala selectively enhances activity toward cytidylyl substrates by factors of 11 to 14, so that the cytidine over uridine preference increases to 120-fold (Shapiro, 1998). In RNase A, mutation of Asp83 to Ala also ampli®es cytidine speci®city, but in this case it is through a selective tenfold decrease in activity { Free energy changes (1 cal ˆ 4.186 J) are calculated as RT ln[(kcat/Km)wild-type/(kcat/Km)variant].

1217 toward uridylyl substrates (delCardayre & Raines, 1995). In both cases, the activity changes are mediated by Thr44/45, and are abolished when this residue is mutated. These ®ndings indicate that Thr80 in Ang plays a role quite different from its RNase A counterpart: rather than improving the effectiveness of uracil recognition by Thr44/45, it undermines the capacity of this residue to recognise cytosine. Interestingly, introduction of Asp at position 80 increases activity toward uridylyl substrates 17-fold and thereby converts Ang into a uridine-preferring enzyme. Thus an Asp to Ala replacement at position 80 in Ang has largely the same effect as the Asp83 to Ala mutation in wildtype RNase A. A complete understanding of the functional roles of Thr44 and Thr80 must await the determination of structures of Ang complexes with pyrimidine nucleotides. Nonetheless, analysis of the superimposed high-resolution structures of free Ang and the complexes of RNase A or RNase S with UVan (PDB code 1RUV; Ladner et al., 1997), d(CpA) (PDB code 1RPG; Zegers et al., 1994), 20 -CMP (PDB code 1ROB; Lisgarten et al., 1993), and UpcA (coordinates from G. Gilliland; Gilliland et al., 1994) already suggests some potential ways in which these two residues might act. Thr44 NH occupies a space nearly identical with that of Thr45 NH in the RNase A complexes, and therefore seems capable of interacting with O2 of both pyrimidines. In contrast, the position of Thr44 Og1 in Ang deviates by Ê from those of Thr45 Og1 in the RNase up to 1.3 A A structures, which form a relatively tight group in the superpositions. As a consequence, the Ang atom is signi®cantly farther from the pyrimidine N3 atoms than are its counterparts in the RNase Ê versus 2.6-2.8 A Ê ). This shift, if complexes (3.3-3.6 A present when substrate binds, might cause the Og1/N3 hydrogen bond to be weaker than in RNase A complexes, or it might cause the pyrimidine ring to orient less optimally. The position of Thr44 in free Ang appears to be due to the hydrogen bond between this atom and Thr80, a bond that could not form if Thr44 were oriented like Thr45 in RNase (the Og1/Og1 distance would be Ê ). In RNase A, where Thr80 is replaced by the 4 A longer Asp83 residue, Thr45 can engage in both interactions (with Asp83 and N3 of uracil) simultaneously. The 44/80 interaction in Ang is stabilised by a water-mediated hydrogen bond between Thr80 and the carbonyl O of Thr97. These observations suggest that the increased activity of T80D-Ang toward uridylyl substrates is due to the introduction of an Asp80/Thr44/N3 network similar to the Asp83/Thr45/N3 network that improves uracil recognition by RNase A. It is less straightforward to explain (i) why the suppressive role of Thr80 is limited to cleavage of cytidylyl substrates, (ii) why Thr44 Og1, despite its interaction with Thr80, seems to make an even larger contribution (1.9 kcal/mol) to stabilising the transition state for cytidylyl substrates than does Thr45 Og1 in RNase A (1.0 kcal/mol){ (this contribution

1218 becomes greater still (3.7 kcal/mol) when Thr80 is removed), and (iii) why Ang prefers cytosine over uracil so strongly. The ®rst result indicates that the 44/80 hydrogen bond is not present when uridine binds; the second and third suggest that Ang forms an interaction with cytosine that RNase A does not. At least two plausible models that incorporate these features can be envisioned. In one model, the 44/80 hydrogen bond in complexes with cytidine nucleotides is stabilised by an additional strong interaction between Thr44 Og1 and N4, and the Thr44 side-chain forms, at best, a weak contact with N3. In T80A, Thr44 Og1 becomes free to interact with both N3 and N4 of cytosine, hence accounting for the enormous effect of mutating Thr44 in T80A. Such a double interaction would be sterically feasible if one of the two components undergoes a minor reorientation. This model does not by itself explain the absence of the 44/80 hydrogen bond when uracil binds; the basis for this would presumably become apparent when structures of Ang-nucleotide complexes are determined. In an alternative model, there is no 44/80 hydrogen bond even when cytosine is bound, and the increased activity of T80A toward cytidylyl substrates is due to formation of a new hydrogen bond with cytosine N4 that becomes possible only when Thr80 is removed. The open B1 site of wildtype Ang is then structurally ``equivalent'' to D83A-RNase A, consistent with the similar cytosine preferences of the two proteins. If the Ang component that hydrogen bonds to cytosine N4 in T80A is an obligate acceptor, then it would not interact with O4 of uracil. This would explain why replacement of Thr80 by Ala does not affect activity toward uridylyl substrates. In the preceding discussion, it has been assumed that the conformational rearrangement that opens the B1 site of Ang does not alter the locations of the P1-site components (which place constraints on the position of the pyrimidine ring) or the structure of the b-sheet that contains both Thr44 and Thr80. The strong similarity of the putative P1 site of Ang to the observed P1 site of RNase A provides considerable support for the ®rst assumption. The second seems reasonable in view of the large number of hydrogen bonds that de®ne the b-sheet and the extensive perturbation of the overall structure of the protein that would be likely to result from altering it. The remaining three putative B1-site components of Ang, Ile42, Asn43 and Leu115, occupy spaces similar to those of their RNase A counterparts (Val43, Asn44 and Phe120, respectively). The superposition is particularly close for Asn43/44. The additional methyl group of Ile42 versus Val43 points away from the site where the pyrimidine would be expected to bind. Leu115 follows Phe120 of RNase A fairly well out to Cg, and its Cd1 atom is within van der Waals contact distance of the pyrimidine ring.

High-resolution Crystal Structures of Human Angiogenin

The B2 site. This subsite of RNase A exhibits a strong base preference in the order A > G > C > U: relative kcat/Km values for cleavage of NpA, NpG and NpC compared to NpU are 400, 23 and 8, respectively (Witzel & Barnard, 1962). Thus far, only the interactions of adenine in the B2 site have been examined by crystallography or NMR complexes with d(CpA) (Toiron et al., 1996; Zegers et al., 1994), UpcA (UpA in which the 50 oxygen atom of the phosphodiester group is replaced by a methylene group; Gilliland et al., 1994; Richards & Wyckoff, 1973), 20 ,50 CpA (Toiron et al., 1996; Wodak et al., 1977) and d(ApTpApA) (FontecillaCamps et al., 1994)). Even here, considerable variability in the participation of RNase residues has been observed. The residues that contact adenine in some or all of the structures are Cys65, Asn67, Gln69, Asn71, Ala109, Glu111 and His119. In the two NMR structures (Toiron et al., 1996), Asn67, Gln69, Asn71 and Glu111 all form transient hydrogen bonds with the base. In some of the crystal structures (Fontecilla-Camps et al., 1994; Wodak et al., 1977), both Gln69 and Asn71 hydrogen bond to the base, and in others (Gilliland et al., 1994; Zegers et al., 1994), only Asn71 hydrogen bonds to adenine (Od1 to N6 and Nd2 to N1). In virtually all of the RNase A-dinucleotide complexes, the imidazole group of His119 engages in face-to-face stacking interactions with the ®ve-membered ring of Ê apart and nearly adenine; the rings are 3.6-4.0 A parallel. This is a highly favourable arrangement that may contribute signi®cantly to binding of purines. In addition, Cys65 Sg and Ala109 Cb are within van der Waals contact distance of the base. In the single published mutagenesis study on the B2 site (Tarragona-Fiol et al., 1993), N71A-RNase A was shown to have 46-fold decreased activity toward CpA, whereas Q69A- and E111Q-RNase have unchanged or slightly diminished activity toward this substrate. The activity of N71A toward CpG, CpC and CpU is also considerably lower than that of RNase A; although quantitative data were not reported, these changes do not seem to be as dramatic as with CpA. Thus, Asn71 apparently does interact with the other bases, as suggested from structural considerations (Zegers et al., 1994), but not as strongly. This may account, in part, for the preference of the enzyme for adenine. The replacement Glu111 ! Gln produces a modest decrease in activity toward CpG, suggesting that Glu111 also participates in guanine recognition. Examination of the RNase A structures indicates that the glutamate residue could potentially accept a hydrogen bond from either N1 or N2 of this base: the N2 interaction is guaninespeci®c and a hydrogen bond with N1 of adenine can be formed only if either the nitrogen atom or the carboxylate group is protonated. The B2 subsite of Ang shows the same order of selectivity as that of RNase A (A > G > C > U), but discriminates among the bases much less effectively: relative kcat/Km values for NpA, NpG and NpC compared to NpU are only 20, 7 and 2,

High-resolution Crystal Structures of Human Angiogenin

respectively (Russo et al., 1996a). These functional differences are associated with substantial deviations in the primary and tertiary structures of the two proteins in this region. RNase A residues 67, 69 and 71 lie on a loop that is stabilised by the 6572 disulphide bridge; Ang lacks this disulphide and contains no direct structural counterpart to the loop. Moreover, the sequences of Ang and RNase A in this segment are almost completely distinct (see Figure 8). The signi®cance of the sequence differences for the enzymatic activity and speci®city of Ang was tested by regional mutagenesis prior to determination of the Ang structure (Harper & Vallee, 1989). An Ang/RNase hybrid protein in which the RNase A segment 59-73 replaces Ang 58-70 was shown to have increased activity and a much stronger preference for adenine in B2. Superposition of the high-resolution Ang structures and those of RNase A complexes with dinucleotide analogues (Figure 6) now reveals that no Ang component occupies the position of the key RNase A residue Asn71. The d atoms of Asn68 of Ang lie near Ca/Cb of RNase Gln69, beyond hydrogen-bonding range of the adenine in every superposition except for that of Met-1 Ang and the d(CpA)-RNase A complex. Recent kinetic data demonstrate that Asn68 does not play an important role in enzymatic function: the activities of an N68A variant toward CpA and CpG are 40 % and 80 %, respectively, of that of Ang (R. Shapiro, unpublished results). No other Ang residue appears to be situated appropriately to form hydrogen bonds with the base. Glu108 is even farther away than is Glu111 of RNase A, consistent with the earlier ®nding that mutation of this residue has only a minor effect on activity (Curran et al., 1993a). Ala106 is positioned similarly to Ala109 of RNase A, and might form van der Waals

Figure 6. Superimposed structures of Pyr1 Ang and RNase A-d(CpA) complex showing the B2 subsite. Ang and RNase A residues are shown in standard colours and in green, respectively. Only the adenine portion of d(CpA) is shown (blue). Broken lines represent hydrogen bonds in the RNase A complex.

1219 contacts with adenine. The side-chain of Leu69, which is near Cys65 of RNase A in the superposition, also might interact with the base. The major element of commonality between the B2 sites of Ang and RNase A appears to be His114/119, which in both cases seems to be well placed to form stacking interactions. It is possible that His114 is the primary B2 site component of Ang; the base preference at this site would then re¯ect the different binding energies realised through stacking of the imidazole ring against the various ®ve-membered (purine) and six-membered (pyrimidine) rings. Alternatively, the bases might ®t into this site in a manner different from that observed in the superpositions (e.g. see Leonidas et al., 1997), and the B2 site of Ang might include residues that have not been considered here. In this regard, the changes in B2-site speci®city that accompany some mutations of Asp116 (Curran et al., 1993b), a residue that appears to lie outside B2 in the superposition, are perhaps signi®cant. The P2 subsite. The P2 site of RNase A has not been investigated as extensively as P1, B1 or B2 from either a functional or a structural point of view. The existence of the site has been demonstrated kinetically: 30 ,50 -ADP binds 10 to 17-fold more tightly than 50 -AMP, and addition of a 20 ,30 cyclic phosphate group to the substrate UpU increases the kcat/Km several-fold (Irie et al., 1984; Russo et al., 1997). Lys7 and Arg10 were originally proposed to be constituents of P2 on the basis of chemical modi®cation results and molecular modeling (Irie et al., 1984; Richardson et al., 1988). In the complex of RNase A with d(ApTpApA) determined subsequently, only Lys7 interacts with the P2 phosphate group, whereas the guanidino group Ê away (Fontecilla-Camps et al., of Arg10 is 8 A 1994) and no movement of this side-chain can bring it signi®cantly closer. Nevertheless, the activity decreases produced by Gln substitutions of Lys7 and Arg10 have been interpreted as evidence that both residues are involved in forming a cationic cluster for phosphate binding (Boix et al., 1994); perhaps Arg10 contributes to substrate binding through non-speci®c electrostatic effects. Surprisingly, replacements of both Lys7 and Arg10 diminish catalytic ef®ciency with substrates that have no P2 phosphate group, suggesting that these residues in¯uence events at P1 as well (Boix et al., 1994; Neumann & Hofsteenge, 1994; Nogues et al., 1995). Ang also has a kinetically demonstrable P2 site: it binds 30 ,50 -ADP sixfold more tightly than 50 -AMP and cleaves CpAp ninefold more ef®ciently than CpA (Russo et al., 1996a). The structural analogues of the RNase A residues Lys7 and Arg10 in Ang are His8 and Thr11. Although His8 occupies a posÊ ition similar to that of its counterpart, it is 4.5 A away from the phosphate group in the superimposed structures of Ang and the RNase A-d(ApTpApA) complex (Figure 7) and cannot move much closer unless the nucleotide binds differently.

1220

Figure 7. Superimposed structures of Pyr1 Ang and RNase Ad(ApTpApAp) complex showing the P2 subsite. Ang and RNase A residues are shown in standard colours and in green, respectively. Only the ribose 30 -phosphate moiety of ApA from d(ApTpApAp) is shown (blue). Broken lines represent hydrogen bonds in the RNase A complex.

Thr11 of Ang is even more distant than Arg10 of RNase. Mutagenesis, kinetic and modelling results have implicated Arg5, a residue unique to Ang (its RNase A counterpart is Ala4), as a component of the P2 site in this protein. Replacement of Arg5 by Ala decreases activity toward polynucleotides by fourfold, but has little effect on cleavage of small substrates (Shapiro & Vallee, 1992), indicating that this residue is involved in binding some peripheral substrate component. The decreased preference of R5A for CpAp versus CpA as substrate and 30 ,50 ADP versus 50 -AMP as inhibitor identi®es the peripheral component as the P2 phosphate group (Russo et al., 1996a). In both high-resolution native Ang structures, Arg5 is involved in crystal packing interactions and makes hydrogen bonds with the carbonyl oxygen atoms of Trp89 and Pro91 from a symmetry-related molecule. Perhaps because of these contacts, the Arg side-chain in the superimposed structures of Ang and the RNase A-d(ApTpApA) complex lies close to the R3 ribose moiety Ê from the nearest oxygen atom of the P2 and 4.3 A phosphate group. In the absence of crystal packing constraints, this residue would presumably be free to reorient, and modelling shows that it can readily move into hydrogen-bonding range of the phosphate group. At least one additional Ang residue, not apparent from the superimposed Ang and RNase A structures, seems to be involved in the P2 site, since the R5A variant can still ``sense'' the phosphate group that would bind at this site (Russo et al., 1996a). The cell-binding site Several structural modi®cations of Ang abolish angiogenic activity but have no appreciable effect

High-resolution Crystal Structures of Human Angiogenin

on enzymatic activity; these include (i) proteolytic cleavage at the 60/61 or 67/68 peptide bond or at both (producing the ``clipped'' species, Ang-K and Ang-E, and des(61-67)Ang, respectively; Hallahan et al., 1991); (ii) deamidation of Asn61 or Asn109 to isoAsp or Asp (Hallahan et al., 1992); and (iii) mutation of Arg66 to Ala (Shapiro & Vallee, 1992). In addition, replacement of Ang residues 58-70 by the corresponding RNase A residues 59-73 eliminates angiogenic activity but, as noted above, increases ribonucleolytic activity (Harper & Vallee, 1989). Ang-K, Ang-E, the Ang/RNase hybrid protein, and the two isoAsp angiogenins have been shown to lack the capacity to inhibit Ang-induced neovascularisation, in contrast with the angiogenically inactive catalytic mutants H13A and H114A, which are effective inhibitors (Shapiro & Vallee, 1989). Based on these ®ndings, the residues altered in the various derivatives were proposed to constitute a critical cell-binding site distinct from the catalytic centre (Hallahan et al., 1991, 1992). One of the residues replaced in the Ang/RNase A hybrid, Arg70, has since been demonstrated not to be involved, since replacement by Ala does not affect angiogenic activity (Shapiro & Vallee, 1992). Ang interacts with cultured endothelial cells in numerous ways that may contribute to the angiogenesis process: it induces second messengers (Bicknell & Vallee, 1988, 1989), promotes cell migration and invasiveness (Hu et al., 1994), organises the formation of tubular structures (Jimi et al., 1995), and stimulates cell proliferation (Hu et al., 1997). Initial attempts to identify a cell-surface receptor for Ang instead resulted in the isolation of a dissociable Ang-binding protein (AngBP; Hu et al., 1991) that is a member of the actin family (Hu et al., 1993). Skeletal muscle actin also binds tightly to Ang, and an anti-actin antibody has been shown to inhibit Ang-induced angiogenesis (Hu et al., 1993). The latter ®nding indicates that AngBP/actin plays an essential part in the angiogenic mechanism. The subsequent discovery that the Ang-actin complex activates tissue plasminogen activator (Hu & Riordan, 1993) suggests that this role may be to enhance proteolytic degradation of the basement membrane, a necessary step in the genesis of new blood vessels. More recently, a 170 kDa putative receptor was isolated from human endothelial cells grown in sparse culture, which proliferate in response to Ang and do not contain actin on their surface (Hu et al., 1997). The preceding observations raise the question as to whether the ``cell-binding site'' of Ang corresponds to the site for binding AngBP/actin, the receptor, or both. The evidence thus far implicates Ang residues 60-68 in the interaction with actin: the proteolytic derivatives Ang-K and Ang-E reduce cross-linking of AngBP/actin to wild-type Ang much less effectively than do the catalytic site variants H13A and H114A (Hu et al., 1991, 1993). However, the full extent of the region that binds actin has not been determined. The receptor-binding site is largely unde®ned. Preliminary mapping

1221

High-resolution Crystal Structures of Human Angiogenin

with Ang derivatives suggests that it may overlap the actin-binding site, but it is at least partially distinct (G.-F. Hu & R.S., unpublished results). It is unclear which site contains the ``cell-binding'' residue Asn109. Neither site involves the catalytic core of Ang. Ang residues 60-68 are part of an external region that includes strand B2 and the loops on either side of it (Figure 2). The entire segment, apart from the side-chain of Arg66 beyond Cg, is well de®ned in the electron density maps and its structures in the Met-1 and Pyr1 native proteins are nearly identical. The main-chain conformation appears to be stabilised by seven hydrogen bonds with helix H3 and strand B3 (Table 4). The side-chain of the critical residue Asn61 forms three hydrogen bonds (Od1 to N of Ser74; Nd2 to the carbonyl O of Ser52 and Og1 of Ser74) and can potentially form only a single additional bond (using Od1) with actin or the receptor. This intermolecular contact might not be affected adversely by replacement of Asn61 with Asp; the loss of angiogenic activity associated with the mutation may then re¯ect a structural perturbation due to removal of the interaction with Ser52. Alternatively, the hydrogen bonds in the native structure may normally be replaced with ligand interactions upon binding, and these may no longer be possible in the Asp61 protein. Deamidation of Asn61 to isoAsp would probably weaken or eliminate all interactions of the side-chain, which is shortened by one methylene group. In addition, the extra methylene group introduced into the backbone by this conversion would most likely disrupt the main-chain hydrogen bonds of

neighbouring residues. Indeed, deamidation of Asn61 to isoAsp appears to have an even more severe impact on actin/receptor binding than does the Asp substitution (Hallahan et al., 1992). NZ1 of Arg66, another important residue, interacts with the carboxylate group of Glu67 in Pyr1 Ang, but, as already noted, this portion of the Arg side-chain is highly ¯exible. The only other inter-residue hydrogen bond of the 60-68 segment is between Nd2 of Asn63 and the main chain of Gly62. Overall, the side-chains in this region are available to form numerous interactions with ligands. The other residue implicated in cell binding, Asn109, lies on the 108-111 loop that connects strands B6 and B7, adjacent to residues 60-68. This loop, which is well de®ned in the electron density maps of both the Pyr1 and Met-1 Ang structures, is exposed to solvent and its side-chains do not form any intramolecular contacts. Inspection of the loop structure suggests that isoAsp replacement of Asn109 would be less disruptive than for Asn61, but might affect main-chain interactions between B6 and B7 to some extent. There is virtually no similarity between the cellbinding site of Ang, as presently de®ned, and the analogous region of RNase A in terms of amino acid sequence (Figure 8) or three-dimensional structure (Figure 9). The RNase A segment that corresponds to Ang 60-68 from an evolutionary point of view, 61-71, contains 0-2 identical residues (depending on the alignment), two additional residues, and a disulphide that is absent from Ang. Part of this segment constitutes one face of the B2 subsite of the enzymatic active site, as discussed

Table 4. Hydrogen bond interactions of the residues in the cell-binding site of human Ang in the structures of Pyr1 and Met-1 Ang and variants H13A and K40Q Donor Arg5 NZ1 Asn61 N Asn61 Nd2 Asn61 Nd2 Gly62 N Asn63 N Asn63 Nd2 Asn63 Nd2 His65 N His65 Ne2 His65 Nd1 Arg66 N Arg66 NZ1 Arg66 NZ2 Leu69 N Ile71 N Lys73 N Ser74 N Glu108 N Gly110 N Leu111 N Water

Acceptor

Pyr1 Ang

Met-1 Ang

K40Qa

H13Ab

Glu108 Oe1 Ala55 O Ser52 O Ser74 Og Glu58 O Ile71 O Gly62 O Water Leu69 O Water Water Water Glu67 Oe2 Ser118 Og His65 O Asn63 O Asn61 O Asn61 Od1 Leu111 O Water Glu108 O Asn68 O

3.1 3.1 3.0 3.0 2.9 2.9 2.9 2.6 2.9 2.9 3.1 3.1 2.8 3.2 2.9 2.7

3.2 3.2 2.9 3.2 2.9 2.6 3.0 2.9 3.1 2.9 2.7 2.7 2.9 3.2 3.2 2.3

3.0 3.3 3.1 3.2 2.9 3.1 2.7 2.8 2.9 2.9 2.9 2.9 2.7 3.2 2.6

3.2 2.7 3.1 2.9 3.3 2.9 2.7 2.9 3.0 3.0 2.7 3.0 2.6 3.2

Ê . Hydrogen bonds are listed if the distance between a donor (D) and an acceptor (A) is Numbers in columns are distances in A Ê and if the angle D-H-A is greater than 120 . Hydrogen bond parameters were calculated with the program shorter than 3.3 A HBPLUS (McDonald & Thornton, 1994). a In K40Q residue 40 is glutamine instead of lysine. b In H13A residue 13 is alanine instead of histidine.

1222

High-resolution Crystal Structures of Human Angiogenin

Figure 8. Structure-based sequence alignment of Ang, RNase A (Wlodawer et al., 1988), EDN (Mosimann et al., 1996) and ONC (Mosimann et al., 1994) as determined using the program SHP (Stuart et al., 1979). Every tenth residue in the Ang sequence is numbered. The dots indicate gaps inserted for optimal alignment. Residues that are identical between Ang and other proteins are highlighted in bold letters. Residues that form the P1 subsite of RNase A in complex structures are boxed; those in B1 are boxed and shaded; and residues associated with B2 are shaded. His119 of RNase A and its counterparts in the other proteins should be considered part of both P1 and B2. Secondary structural elements (b-strands and helices) as determined by DSSP (Kabsch & Sanders, 1983) are also shown in cyan and red, respectively.

above. The putative cell-binding Ang residue Asn109 is replaced by Gly in RNase A, and the loop on which this Gly lies is two residues larger. Again, the RNase A loop includes part of the B2 site (Glu111). Thus it appears that this region has evolved to ful®l largely different functions in the two proteins: cell binding in Ang and purine recognition in RNase A.

and observations: (i) NLSs are typically very rich in basic amino acids; (ii) R33A-Ang is not transported to the nucleolus; (iii) R31A- and R32A-Ang are transported much less ef®ciently than the native protein; and (iv) coupling of Ang residues 31-35 targets non-nuclear proteins to the nucleolus

The nuclear translocation site In addition to the ribonucleolytic active site and the cell-binding site of Ang, a third region has been implicated as necessary for angiogenic activity. This site, which contains 31RRRGL35, constitutes a nuclear localisation signal (NLS) that is involved in transport of Ang to the nucleolus following uptake by endothelial cells (Moroianu & Riordan, 1994a,b). Studies with Ang derivatives have shown that translocation is an essential step in the induction of angiogenesis: i.e. Ang variants that are not transported to the nucleolus are not angiogenic. In the nucleolus, Ang is in direct contact with potential RNA substrates, suggesting that the nucleolus might be where Ang exerts its enzymatic activity, e.g. participating in the processing of pre-rRNA into the new ribosomes required for cell proliferation. The identi®cation of Ang residues 31-35 as the NLS is based on several ®ndings

Figure 9. Stereo view of a superposition of the cell binding site of Pyr1 Ang (residues 60-68 and 108-110; dark) onto its structural counterpart in RNase A (residues 61-71 and 111-115; light). Residues are drawn as ball-and-stick models and the Ang residues are labelled.

1223

High-resolution Crystal Structures of Human Angiogenin

of permeabilised endothelial cells. The involvement of Arg33 in nuclear translocation presumably accounts for the extensive loss of angiogenic activity associated with mutation of this residue to Ala (Shapiro & Vallee, 1992); the replacement causes an only sevenfold decrease in enzymatic activity. Ang residues 31 and 32 lie at the C-terminal end of helix H2, and residues 33-35 are part of the loop connecting the helix with strand B1 (Figures 2 and 8). The entire 31-35 segment is well de®ned in the electron density map. Arg31, Arg32 and Arg33 form several interactions with other residues but are largely accessible to the solvent. Atom NZ1 of Arg31 makes a water-mediated hydrogen bond with the main-chain NH of Cys92. NZ1 of Arg32 hydrogen bonds to the hydroxyl group of Ser28. The Ne atoms of Arg32 and Arg33 interact with each other via water, while NZ1 and NZ2 of Arg33, as noted above, hydrogen bond with the mainchain oxygen atoms of Thr11 and Tyr14. NLS sequences have been identi®ed in a large number of other proteins, and the three-dimensional structures of several of these have been determined. NLS sequences display enormous diversity, even with respect to whether they lie on one contiguous segment or are multipartite (Dingwall & Laskey, 1991). The major type of NLS, like that of Ang, contains a cluster of basic amino acids and is exposed on the protein surface. Most of those whose crystal or solution structures have been determined tend to be part of ¯exible loops. For example, the NLS of the POU domain protein Tst-1/Oct6 is part of a relatively unstructured region preceding the ®rst a-helix of the homeodomain (Sivaraja et al., 1994; Sock et al., 1996); that of HIV-1 p17 is on an extruded loop that connects two b-strands (Massiah et al., 1994); and the NLS of lymphoid enhancer factor-1 is in a region of b- and g-turns following a-helix 3 (Prieve et al., 1996). However, the C-terminal portion of the NLS of the basic-helix-loop-helix domain of MyoD shares some similarity to the NLS of Ang: i.e. its basic residues are at the end of an a-helix (Ma et al., 1994).

Figure 10. Stereo view of the superimposed Ca backbones of native Ang (Pyr1 form, black) onto those of K40Q (green) and H13A (red).

K40Q Met-1 K40Q-Ang crystallised in the same space group as the Met-1 native protein (C2221) and its structure was determined to the same resolution Ê ). The overall structures of the native and (2.0 A mutant proteins are nearly identical (Figure 10): Ê for 119 equivalent Ca the r.m.s. deviation is 0.17 A atoms, and the only differences between side-chain positions involve ¯exible residues on the surface

Structures of angiogenin variants The most convincing evidence that the ribonucleolytic activity of Ang is required for angiogenic activity has been the simultaneous loss of both activities that results from replacements of His13 by Ala or Gln, Lys40 by Gln or Arg, Thr44 by His, and His114 by Ala or Asn (Shapiro et al., 1989; Shapiro & Vallee, 1989; Curran et al., 1993a). In each case, however, the possibility must be considered that the amino acid substitution produces structural perturbations beyond the enzymatic site and that these are the true cause of reduced biological activity. Here, we have examined this question by determining the structures of the K40Q and H13A variants.

Figure 11. Superimposed structures of the ribonucleolytic active sites of Pyr1 Ang (cyan), H13A (red) and K40Q (green). Residues are drawn as ball-and-stick models.

1224 (Arg51, Lys82 and Arg95). The mutation site is clearly visible in the electron density map of K40Q and all atoms of the glutamine 40 residue are well de®ned. The conformation of the side-chain of Gln40 from atom Cb onwards differs from that of the lysine residue in native Ang (Figure 11); hence the hydrogen bond to Asn43 is lost (Table 3) and the glutamine residue instead hydrogen bonds to a water molecule. The hydrogen bond between the Lys40 main chain and Tyr94 that appears to help position this residue in the native protein (see above) is retained in the variant structure (Table 3). The interactions of the other residues in the catalytic site and the placement of water molecules are similar to those in the unmodi®ed protein (Table 3). Thus, the loss of enzymatic activity in K40Q can be attributed unequivocally to the removal of the lysine residue, which is thought to participate in stabilisation of the pentavalent transition state. Lys40 was also shown by mutagenesis to play an important role in the interaction of Ang with human placental RNase inhibitor (RI), a 50 kDa leucine-rich repeat protein that binds Ang with an extraordinarily low Ki value of 0.7 fM (Lee et al., 1989; Lee & Vallee, 1989). In the crystal structure of the complex determined recently (Papageorgiou et al., 1997), Lys40 forms two hydrogen bonds with Asp435 of the inhibitor. Thus, the 1300-fold decrease in binding af®nity for the K40Q variant was proposed to re¯ect the loss of these interactions. In support of this conclusion, the K40Q structure shows no change in the RI-contact region of Ang other than at residue 40. Importantly, the structures of the cell-binding site and NLS in K40Q versus native Ang are virtually indistinguishable. The r.m.s. deviations for all atoms in segments 60-68, 108-110 and 31-35 are Ê , 0.2 A Ê and 0.2 A Ê , respectively, and all of the 0.5 A side-chain and main-chain interactions of these residues in native Ang are retained in the variant. H13A H13A-Ang did not crystallise under the conditions (citrate/tartrate/PEG, pH 5.2) used sucessfully for the native proteins and K40Q, and the crystals used for structure determination were grown using a different buffer and precipitant (Hepes/ammonium sulphate, pH 7.5; see Materials and Methods for details). Moreover, the space group of the H13A-Ang crystal was different from that for the Pyr1 native protein (C222 versus P21212). The resolution of the ®nal structure is Ê , but the data were only 55 % comnominally 2.0 A Ê resolution. Therefore, plete between 2.9 and 2.0 A the H13A-Ang structure is in fact at lower resolution than those for native Ang and K40Q. The r.m.s. deviation between 119 equivalent Ca atoms in the superimposed structures of H13A and Ê , much greater than the Pyr1 native Ang is 0.69 A that between the Pyr1 versus Met-1 native Ang or between Met-1 native versus K40Q (Figure 10). The r.m.s. deviations are similar when H13A is com-

High-resolution Crystal Structures of Human Angiogenin

Ê for native pared to the two Met-1 proteins (0.71 A Ê for K40Q). The largest differences are and 0.69 A in helix H1, the loop region connecting helix H2 to b-strand B1, and the loop between b-strands B4 and B5, where the r.m.s. deviations from the corresponding regions in the native structures are Ê (Ca positions). 1.5-2.0 A In the crystal structure of H13A Ang, the substitution of His13 by Ala is very clear in the electron density map, where there is no density beyond atom Cb. This replacement, in contrast with that of Lys40 by Gln, has extensive repercussions throughout the active-site region. In the native structure, His13 Nd1 forms a hydrogen bond with the mainchain O of Thr44 and Ne2 makes a water-mediated interaction with the amide nitrogen atom of Leu115 (Table 3). In the variant, the His13 Nd1 atom is replaced by a water molecule, which forms hydrogen bonds with the carbonyl O of Thr44 and the side-chain of Asn43. Atom Ne2 of Gln12 has Ê away from its position in the native moved 1.7 A structure towards the empty space left by the removal of the imidazole ring, and the hydrogen bond between the carbonyl O of Gln12 and the side-chain of Asn43 is lost (Table 3). The imidazole group of His114 has rotated slightly and shifted Ê closer to where the P1 phosphate moiety is 1.2 A expected to bind (Figure 11). Furthermore, atoms Ê , 1.2 A Ê, Cb, Cg, Cd, Oe1 and Ne2 of Gln117 are 0.7 A Ê , 1.9 A Ê and 0.9 A Ê respectively, from their pos0.4 A itions in native Ang and, as a result, Oe1 no longer interacts with the main-chain NH group of Thr44. The reorganisation of the active site in the H13A structure appears to affect other parts of the proÊ from tein as well. Helix H1 is approximately 1.3 A its position in the native structure, possibly due to loss of the two hydrogen bonds of His13. The shift of H1, together with the removal of 12/43 and 44/ 117 hydrogen bonds, may be responsible for the perturbation of the disulphide-linked loops 33-41 Ê and 85-92, whose positions differ by up to 2.3 A from those in native Ang. The ®rst of these loops includes part of the NLS. Overall, the NLS residues 31-35 of H13A and native Pyr1 Ang have r.m.s. Ê and 0.69 A Ê for Ca and all deviations of 0.17 A atoms, respectively, and the three Arg side-chains are oriented similarly. The conformation of the cell-binding site in H13A is largely the same as in native Ang. The r.m.s. deviations for Ca atoms and all atoms (resiÊ and 0.96 A Ê, dues 60-68 and 108-110) are 0.29 A and only the side-chains of the highly ¯exible residues 66, 67 and 109 are oriented differently in the two proteins. The positions of the critical component Asn61 in the superimposed structures Ê for Od1 and Nd2). are particularly close (0.2 A Comparison of the native Ang structure with those of eosinophil-derived neurotoxin and onconase Ang is not the only member of the pancreatic RNase superfamily that has been shown to be a

High-resolution Crystal Structures of Human Angiogenin

potent biological effector: eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein are toxic to neurons and certain parasites (Ackerman et al., 1985; Gleich et al., 1986), onconase (ONC) inhibits tumour growth (Mikulski et al., 1990), and bovine seminal RNase has both anti-tumour and immunosuppressive activity (MatousÏek, 1973; Soucek et al., 1986). Three-dimensional structures have been determined for three of these proteins: EDN (Mosimann et al., 1996), ONC (Mosimann et al., 1994) and bovine seminal RNase (Mazzarella et al., 1993). The seminal RNase is unique, in that its native form is a dimer, and this enzyme will not be considered further here. EDN shares 26 % and 36 % sequence identity with Ang and RNase A, respectively (using the structure-based alignment shown in Figure 8, with only the number of residues in the smaller of the two proteins compared), and its enzymatic activity toward RNA is nearly equivalent to that of RNase A (Slifman et al., 1986; Sorrentino et al., 1988). ONC, an amphibian protein with 27 % sequence identity with both Ang and RNase A, is about 100-fold less active toward RNA than is RNase A (Boix et al., 1996). As with Ang, the unusual physiological activities of EDN and ONC appear to require retention of their enzymatic action (Sorrentino et al., 1992; Newton et al., 1994). It seems likely that the cytotoxic effects of EDN and ONC are due to general destruction of RNA following speci®c uptake by target cells (Wu et al., 1993). Superposition of the Pyr1 Ang structure with those of EDN (provided by S. Mosimann and M.N.G. James) and ONC (PDB code 1ONC) gives Ê and 2.8 A Ê for 106 and 98 r.m.s. deviations of 2.6 A Ê for common Ca atoms, respectively (versus 1.9 A 111 equivalent Ca atoms of Ang and RNase A; PDB code 7RSA; Figures 8 and 12). Residues in helices H1-H3 and strands B1 and B4-B6 account for nearly all the equivalent residues in the four structures. Strand B2 of Ang and RNase A is absent from EDN and ONC. The only non-loop residues of Ang that do not superimpose with their counterparts in EDN and ONC are on helix H2 and strands B2 and B7. In EDN, helix H2 is three residues longer than that of Ang, while in ONC it is ®ve residues shorter. Strand B7 is quite different within the four structures and is shortest (®ve residues) in Ang. Most of the insertions, deletions and non-conservative replacements are in loop regions; the three-dimensional structures of virtually all of these loops are distinct in the four proteins. The other region where there are striking structural differences between Ang and the other proteins involves the C-terminal segment. In RNase A and EDN, these residues are mostly part of strand B7, and the a-carboxylate group approaches the end of strand B5. In ONC, strand B7 is followed by a short turn that places the carboxylate group near strand B6, and the C-terminal residue (Cys104) forms a disulphide bond with Cys87 that is unique to the amphibian RNases (Mosimann et al., 1994). The C-terminal segment of

1225 Ang is oriented in a markedly different direction from those in the other proteins and is composed largely of 310 structure. The cell-binding site of Ang is among the regions most poorly conserved in the EDN and ONC structures (Figures 8 and 12). The 60-68 segment includes the B2 strand that is missing from the other two proteins. The corresponding residues of EDN and ONC lie on loops that connect helix H3 and strand B3. In EDN, the loop is stabilised by a disulphide bridge not present in Ang, and bears some similarity to the 65-72 disulphide loop that forms part of the active site of RNase A. The H3/ B3 loop in ONC is oriented much differently from its counterparts in the other three proteins. The B6/B7 loop, which contains another of the cellbinding residues of Ang, Asn109, has an 11 residue insertion in EDN. The B6/B7 loop of ONC has the same length as that in Ang, and Asn109 is conserved (as Asn92). However, access to this region by AngBP/actin or the Ang receptor would most likely be prevented by the H3/B3 loop. These observations provide an explanation for the distinctive physiological properties of Ang vis-a-vis EDN and ONC. Conversely, the unusual antitumour activity of ONC has been shown to involve a region, the N terminus, that differs dramatically in the other three homologues (Boix et al., 1996). The structural features of EDN responsible for its cytotoxic action have not yet been determined. The P1 subsite of the ribonucleolytic active site and parts of B1 and B2 have similar structures in the four proteins. The catalytic triad of RNase A and Ang is present in EDN and ONC (as His15, Lys38 and His129 in EDN, and as His10, Lys31 and His97 in ONC). His15/10 of EDN/ONC superimpose well with His13/12 of Ang/RNase A. His129 of EDN and His97 of ONC occupy the Bconformation seen in some RNase A structures (see above), but should be free to reorient to the productive A-conformation. Lys38 of EDN is positioned much as its Ang and RNase A counterparts, Ê away and would need but Lys31 of ONC is 3 A to reorient for its ammonium group to interact with a phosphate group at P1 (Mosimann et al., 1996). Gln14 of EDN and Gln12/11 of Ang/RNase A have similar positions. This residue is replaced in ONC by Lys9, which forms an interaction with Pyr1 that has been demonstrated to be important for enzymatic activity (Boix et al., 1996). The NH group of EDN Leu130 and Phe98 of ONC are well positioned to interact with the phosphate group in the same manner as those of Leu115 of Ang and Phe120 of RNase A. The B1 sites of EDN and ONC, like those of Ang and RNase A, are selective for pyrimidines. EDN shows a small preference for cytosine, similar to that of RNase A, whereas ONC prefers uracil (Ardelt et al., 1994). Both EDN and ONC have unobstructed B1 sites. Thus, the blockage in Ang appears to be associated with the unique C-terminal conformation of this protein. The key B1 residue Thr44/45 of Ang/RNase A is present in EDN

1226 and ONC as Thr42 and Thr35, respectively, which superimpose reasonably well with their counterparts in the other proteins. The important modulating residue Thr80/Asp83 of Ang/RNase A is His82 in EDN and Asp67 in ONC. Both form interactions with Thr41/35 similar to those in the other two proteins. Two additional residues associated with the B1 sites of Ang and RNase A, Asn43/44 and Leu115/Phe120, are conserved in the EDN and ONC structures. The hydrophobic residues

High-resolution Crystal Structures of Human Angiogenin

Ile42 (Ang) and Val43 (RNase A), which form part of one face of the site, are replaced by Gln40 in EDN and by Lys33 in ONC. It has been proposed that these residues may form hydrogen bonds with O4/N4 of uracil/cytosine moieties of substrates and inhibitors (Mosimann et al., 1996). Ser123 of RNase A is replaced by hydrophobic residues in both EDN (Ile133) and ONC (Val101). The B2 site of EDN exhibits a strong selectivity for adenine, at least one order of magnitude great-

Figure 12. Representations of (a) Pyr1 Ang, (b) RNase A (PDB code 7RSA; Wlodawer et al., 1988), (c) EDN (Mosimann et al., 1996) and (d) ONC (PDB code 1ONC; Mosimann et al., 1994).

High-resolution Crystal Structures of Human Angiogenin

er than for RNase A (Shapiro & Vallee, 1991), whereas that of ONC prefers guanosine (Ardelt et al., 1994). As discussed above, the primary residues that appear to be involved in base recognition by RNase A are Asn71 and Glu111 (hydrogen bonds) and His119 (stacking). Superposition with the RNase A-d(CpA) complex (PDB code 1RPG) indicates that the most important of these residues, Asn71, has a direct structural counterpart in EDN (Asn70), but not in ONC. The side-chain of Arg68 of EDN might also be close enough to interact with the base (Mosimann et al., 1996). Glu111 of RNase A is replaced in EDN by Asp112 and is conserved in ONC as Glu91; both are oriented differently from Glu111 and are farther from the base. The side-chain of Asn56 in ONC is near N6 of adenine in the superposition, and could potentially interact with this amine group (or the corresponding 6-oxy of guanine). A minor reorientation could bring Thr89 within hydrogen-bonding distance of N2 of a guanine base; such an interaction might contribute to the guanine-preference of ONC. The EDN and ONC counterparts of His119, His129 and His97, respectively, both adopt the B-conformation that is incompatible with occupation of the B2 site, but could rotate to form the stacking interactions with the ®ve-membered ring of purines observed in RNase A complexes and proposed to exist in Ang complexes (see above). It seems likely that this stacking is a universal feature of the B2 site in pancreatic RNase superfamily enzymes, in the same way as are the hydrogen bonds of the phosphate group with the catalytic triad at P1 and the interactions of pyrimidine O2/N3 with Thr45 (RNase A numbering) at B1.

Conclusions Structural basis for the characteristic low ribonucleolytic activity of Ang The most obvious and unique feature of the native Ang structures that suppresses enzymatic potency is the positioning of Gln117 precisely where the pyrimidine base must ultimately bind for substrate transphosphorylation or hydrolysis to occur. The placement of the glutamine residue at this site appears to be accomplished through an extensive set of interactions: hydrogen bonds of the residue itself with Thr44, additional hydrogen bonds of Asp116 with Ser118 and of main-chain atoms in the C-terminal 310 helix, plus the hydrophobic burying of Ile119 and Phe120 (Figure 5). Mutagenesis results indicate that all of these factors, except for the backbone interactions within the helix, make important contributions to attenuation of activity. In the pancreatic RNases, the presence of Lys66 at the position occupied by Ser118 in Ang most likely precludes a similarly obstructive conformation for the corresponding segment (Shapiro, 1998). It has not yet been possible to assess the full impact of the B1 blockage in energetic terms. The

1227 effects of disrupting the Gln117 and Asp116 sidechain interactions are only slightly additive (Russo et al., 1994) and some of the residues that help stabilise the inactive native conformation may have additional roles once the pyrimidine site has opened. Nonetheless, the available evidence suggests that the obstruction of the pyrimidine site accounts for no more than two of the approximately six orders of magnitude difference between the ribonucleolytic potencies of Ang and RNase A. It is striking that the B1 site is weakened by a second mechanism: i.e. through Thr80, which interacts with the Thr44 side-chain on the side opposite Gln117. The almost full additivity of the effects of the Gln117 and Thr80 mutations indicates that the suppressive role of Thr80 is largely or entirely independent of the B1 blockage (Shapiro, 1998). The negative role of Thr80 appears to account for an additional factor of 10 in the difference between the catalytic ef®ciencies of Ang and RNase A. The presence of a threonine residue at this position has a direct effect on cleavage of cytidylyl substrates, but is also a consideration in the decreased activity of Ang toward uridylyl substrates (i.e. replacement by Asp, the corresponding residue in RNase A, enhances activity toward UpA). Yet another structural feature of the B1-site of Ang may contribute to its enzymatic weakness: the presence of Leu at position 115, where RNase A has Phe (120). This replacement eliminates potentially important interactions with the pyrimidine ring, and has been demonstrated to decrease activity by eightfold in an RNase A fragment complementation system (Lin et al., 1972). However, EDN contains Leu rather than Phe at this position, and yet is nearly as potent as RNase A. Moreover, this residue is Phe in bovine and mouse Ang (Bond et al., 1993), both of which are even less active than the human protein (Nobile et al., 1996; Strydom et al., 1997). Of course it is possible that other amino acid differences more than compensate for the positive role of the Phe in the nonhuman angiogenins and for the absence of the Phe in EDN. Kinetic studies showed previously that the B2 site of Ang is also much less effective than that of RNase A (Harper & Vallee, 1989; Russo et al., 1996a), and the present structural observations suggest that this may re¯ect the absence of any precise counterparts for two of the three RNase residues that form important interactions with purines (Asn71 and Glu111; the third residue, His119, does appear to be structurally conserved). Introduction of the RNase A 65-72 disulphide loop into Ang imparts a more RNase A-like B2 speci®city (A4G) and increases overall activity toward RNA by several-hundred-fold (Harper & Vallee, 1989). The improved base discrimination and part of the activity enhancement are most likely due to formation of the same contacts with adenine that are made by Asn71 of RNase A. However, structural and functional evidence indicates that much of the

1228 increased potency of the hybrid protein derives from an indirect effect: disruption of the inactive native conformation of the B1 site (Shapiro, 1998). The P1 site of Ang is closely analogous to its RNase A counterpart in structure, but has one obvious de®ciency that might attenuate ribonucleolytic activity: the absence of a hydrogen bond corresponding to that between His119 and Asp121 of RNase A, which enhances catalytic ef®ciency by a factor of 100 (Schultz et al., 1998). This difference may underlie the several-fold lower reactivity of His114 of Ang versus His119 of RNase A during chemical modi®cation (Shapiro et al., 1988b). It should also be noted that even the small variations observed in the positions of catalytic residues in the two proteins can potentially have large effects. In this regard, it may be signi®cant that Lys40, like His114, is much less chemically reactive than its RNase A counterpart (Shapiro et al., 1987, 1989). His13 is modi®ed as rapidly as His12 of RNase A (Shapiro et al., 1988b). Finally, a more complete understanding of the structural basis for the weakness of Ang as an RNase will undoubtedly be provided by structures of Ang-nucleotide complexes, which are not yet available. These structures may well reveal novel features of the ``active conformation'' that cannot be imagined at this stage. Moreover, they might yield information regarding the natural physiological substrate(s) of Ang and mechanisms by which Ang might be activated at the appropriate time and location in vivo to become a more potent enzyme. Structural basis for the inactivity of K40Qand H13A-Ang Comparison of the K40Q and native Ang structures has revealed no detectable differences in either backbone or side-chain atoms, except for a small number that involve ¯exible surface residues. Indeed, the r.m.s. deviation for all atoms in the K40Q versus Met-1 structures is even lower than that for the Met-1 versus Pyr1 native structures Ê versus 0.84 A Ê ). Thus, it is entirely clear that (0.51 A the loss of both enzymatic and angiogenic activity for the K40Q variant is solely due to the replacement of the lysine side-chain. This ®nding now constitutes the strongest available evidence that the ribonucleolytic action of Ang is absolutely necessary for angiogenicity, but still falls short of ®nal proof. Although Lys40 lies outside the cell-binding site and NLS, we cannot exclude the possibility that this residue is involved in some other nonenzymatic function of Ang, yet to be discovered, that is required for angiogenic activity. It also remains possible that Ang induces neovascularisation through binding some RNA or RNA-like molecule without cleaving it. These questions will be answered de®nitively when the full details of the molecular mechanism of Ang are elucidated. In contrast with the Lys40 to Gln substitution, replacement of His13 by Ala may cause some

High-resolution Crystal Structures of Human Angiogenin

structural perturbations beyond the residue mutated. The superimposed structures of H13A and native Ang differ signi®cantly in the neighbourhood of residue 13, as well as in at least two regions beyond the active site (the H2/B1 and B4/ B5 loops). Any interpretation of these observations is complicated by differences in the space groups of the crystals, the effective resolutions of the structures, and the conditions used to grow the crystals. This last factor may be particularly noteworthy: histidine residues are expected to be largely protonated in the native crystals (pH 5.2), but unprotonated in H13A (pH 7.5). Thus it is unclear whether the observed structural divergence re¯ects true differences. Previous experimental ®ndings suggest that neither of the loops that is perturbed in the crystal structure is altered appreciably in solution: (i) the His13 replacement does not affect binding to placental RNase inhibitor (Shapiro & Vallee, 1989), which interacts with both loops (Papageorgiou et al., 1997); and (ii) the mutation does not prevent translocation of the protein to the nucleolus (Moroianu & Riordan, 1994b), which involves the NLS located on the H2/B1 loop. The cell-binding site of native Ang is well maintained in the H13A crystal structure, and it appears that the variant would be able to form all of the necessary interactions of this region. This is consistent with the results of earlier functional studies with H13A, which showed it to be an effective inhibitor of the angiogenic activity of Ang (Shapiro & Vallee, 1989) and of Ang cross-linking to its binding protein on the endothelial cell surface (Hu et al., 1991). Thus, the combined structural and functional evidence indicates that the loss of angiogenic activity for H13A is most likely a direct consequence of changes in the ribonucleolytic active site, and further bolsters the view (subject to the same quali®cation cited above for K40Q) that induction of angiogenesis by Ang requires the enzymatic action of this protein.

Materials and Methods Crystallisation Human Pyr1 and Met-1 Ang, and the Met-1 variant K40Q were prepared from a recombinant system in Escherichia coli (Shapiro et al., 1988a; 1989). H13A Ang (Shapiro & Vallee, 1989) was produced as the Pyr1 form with a different system (Shapiro & Vallee, 1992). Protein crystals were grown using the vapour diffusion technique. Orthorhombic crystals of Pyr1 Ang, Met-1 Ang and K40Q were grown at 16  C using a modi®cation of an earlier protocol (Acharya et al., 1992). Brie¯y, drops containing proteins (10 mg/ml) at pH 5.2 in 10 mM sodium citrate buffer, 0.1 M sodium potassium tartrate and 5 % PEG 6000 were equilibrated against reservoirs containing 20 mM sodium citrate buffer, 0.2 M sodium potassium tartrate and 10 % PEG 6000 at pH 5.2. Crystals of H13A were grown from drops containing 10 mg/ml protein at pH 7.5 in 50 mM Hepes buffer, 1 M ammonium sulphate and 1 % PEG 400, which were equilibrated against reservoirs containing 100 mM Hepes buffer, 2 M ammonium sulphate and 2 % PEG 400 at

1229

High-resolution Crystal Structures of Human Angiogenin pH 7.5. Single crystals usually appeared after seven to ten days at 16  C. Crystallographic details for all four proteins are presented in Table 1. Data processing and reduction Initial diffraction data for Pyr1 Ang and the H13A and K40Q derivatives were collected using several crystals on a Siemens area detector mounted on a Siemens rotating anode X-ray source with CuKa radiation operating at 45 kV and 80 mA. Additional data to high resolÊ ) for Pyr1 Ang were collected on stations ution (2.0-1.8 A Ê ) and PX 9.6 (l ˆ 0.88 A Ê ) of the PX 9.5 (l ˆ 0.92, 0.80 A Synchrotron Radiation Source (SRS), Daresbury, U.K., using a 30 cm diameter MAR-research image plate. High-resolution data for Met-1 Ang were also collected at SRS (station PX 9.5) from three different crystals. Synchrotron data were processed using DENZO and its companion program SCALEPACK (Otwinowski & Minor, 1997). The in-house area detector data were processed with the XDS program (Kabsch, 1988) and merged with the synchrotron data using the program SCALA (CCP4, 1994). The data processing statistics of these data sets are given in Table 1. Phasing The structures of Pyr1 Ang and the variants were determined by molecular replacement using the program AMoRe (Navaza, 1994) with the coordinates of Met-1 Ê resolution (PDB code 1ANG; Acharya et al., Ang at 2.4 A Ê were 1994) as a search model. Data in the range 20-3.0 A used for both the rotation and the translation function Ê, a searches. Using a Patterson cut-off radius of 18 A single rotation function peak was obtained for every structure with correlation coef®cient values of 34.6, 26.2 and 37.0 for Pyr1 Ang, H13A and K40Q, respectively. The translation function gave a single peak with correlation coef®cients 74.5, 50.6 and 78.1 for the above rotation peaks. These correlation coef®cients increased to 77.7, 59.7 and 80.8 after rigid-body re®nement with AMoRe, and the R factors were 31.2, 39.9 and 31.0 % at this stage for the Pyr1 Ang, H13A and K40Q structures, respectively. Refinement For each structure, the output model from AMoRe was subjected to rigid-body re®nement with X-PLOR 3.851 (BruÈnger, 1992b). For the re®nement of Met-1 Ang, Ê resolution coordinates were used as a starting the 2.4 A point. Alternating cycles of manual building, conventional positional re®nement and the simulated annealing method as implemented in X-PLOR improved each model, while solvent correction as implemented in XPLOR 3.851 (Jiang & BruÈnger, 1994) allowed all measured data to be used in the re®nement. Model Ê resolution rebuilding was initially performed at 3.0 A Ê in the case of Met-1 Ang) using the interactive (2.4 A computer graphics program O version 5.10.3 (Jones et al., Ê to 2.0 A Ê 1991). Extension of re®nement from 3.0 A Ê (Pyr1 Ang) and from (H13A and K40Q), or to 1.8 A Ê to 2.0 A Ê resolution in the case of Met-1 Ang was 2.4 A Ê resolution steps. The quality of performed in 0.1 A each model was monitored using sigma A-weighted 2jFoj ÿ jFcj maps (Figure 1) calculated using the program SIGMAA (Read, 1986), improved by cycles of solvent ¯attening, histogram matching and density modi®cation

using the program DM (Cowtan, 1994). The behaviour of Rfree (BruÈnger, 1992a) was monitored throughout the re®nement. During the ®nal stages of re®nement, water molecules were inserted into the models only if there were peaks in the jFoj ÿ jFcj electron density maps with heights greater than 3s and they were at hydrogen bond forming distances from appropriate atoms. The 2jFoj ÿ jFcj maps were used to check the consistency in peaks. Water molecules with a temperature factor greatÊ 2 were excluded from subsequent re®nement er than 60 A steps. In the case of the H13A variant, the re®nement was Ê to 2.9 A Ê and initially performed using data from 30.0 A at this stage the Rcryst was 0.206 and the Rfree was 0.335. These values were reasonable at this resolution. The Ê resolution re®nement was gradually extended to 2.0 A Ê to 2.0 A Ê resolution were although the data from 2.9 A only 55 % complete. The inclusion of the higher-resolution data in the re®nement allowed individual B-factor re®nement and the addition of several water molecules to the model. A signi®cant improvement of the electron density maps and a drop of approximately 1 % in the Rfree value indicated that the inclusion of the additional data improved the model. The details of re®nement statistics for the four structures are presented in Table 2. The program PROCHECK (CCP4, 1994) was used to assess the quality of the ®nal structures. Analysis of the Ramachandran (f, c) plot for each of the structures showed that all residues lie in the allowed regions. The two peptide bonds connecting residues Ser37 to Pro38 and Pro90 to Pro91 adopt a cis conformation. Structural superpositions were performed using the program SHP (Stuart et al., 1979) and Figures were drawn with MOLSCRIPT (Kraulis, 1991) as modi®ed by R. Esnouf (1997). The atomic coordinates for Met-1 Ang, Pyr1 Ang as well as for the H13A and K40Q derivatives have been deposited with the Brookhaven Protein Data Bank (accession codes 2ANG, 1B1I, 1B2J, and 1B1E, respectively).

Acknowledgements We are grateful to the staff at the Synchrotron Radiation Source at Daresbury, England and to Dr A. C. Papageorgiou for help with X-ray data collection. We thank Drs M. N. G. James and S. Mosimann for EDN coordinates, Dr G. L. Gilliland for the RNase-UpcA complex cooordinates, Drs B. L. Vallee and J. F. Riordan for advice and support, and Dr D. E. Holloway for helpful discussions. This work is supported by the Medical Research Council, UK (Programme grant 9540039 to K.R.A.), the Cancer Research Campaign, UK (Project grant SP2354/0101 to K.R.A.), the Wellcome Trust, UK (Biomedical Research Collaboration grant 044107 to K.R.A. and R.S.), and the National Institutes of Health, USA (grant HL52096 to R.S.). K.V. thanks the Royal Society (UK) and the Indian National Science Academy (India) for a visiting fellowship.

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Edited by R. Huber (Received 24 July 1998; received in revised form 26 October 1998; accepted 28 October 1998)