Artigo Acesso aberto Revisado por pares

Molecular recognition of human angiogenin by placental ribonuclease inhibitor_an X-ray crystallographic study at 2.0Aresolution

1997; Springer Nature; Volume: 16; Issue: 17 Linguagem: Inglês

10.1093/emboj/16.17.5162

ISSN

1460-2075

Autores

Anastassios C. Papageorgiou, Reneé Shapiro, K. Ravi Acharya,

Tópico(s)

Angiogenesis and VEGF in Cancer

Resumo

Article1 September 1997free access Molecular recognition of human angiogenin by placental ribonuclease inhibitor—an X-ray crystallographic study at 2.0 Å resolution Anastassios C. Papageorgiou Anastassios C. Papageorgiou Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath, BA2 7AY UK Search for more papers by this author Robert Shapiro Robert Shapiro Center for Biochemical and Biophysical Sciences and Medicine, Boston, MA, 02115 USA Department of Pathology, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author K.Ravi Acharya Corresponding Author K.Ravi Acharya Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath, BA2 7AY UK Search for more papers by this author Anastassios C. Papageorgiou Anastassios C. Papageorgiou Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath, BA2 7AY UK Search for more papers by this author Robert Shapiro Robert Shapiro Center for Biochemical and Biophysical Sciences and Medicine, Boston, MA, 02115 USA Department of Pathology, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author K.Ravi Acharya Corresponding Author K.Ravi Acharya Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath, BA2 7AY UK Search for more papers by this author Author Information Anastassios C. Papageorgiou1, Robert Shapiro2,3 and K.Ravi Acharya 1 1Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath, BA2 7AY UK 2Center for Biochemical and Biophysical Sciences and Medicine, Boston, MA, 02115 USA 3Department of Pathology, Harvard Medical School, Boston, MA, 02115 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1997)16:5162-5177https://doi.org/10.1093/emboj/16.17.5162 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Human placental RNase inhibitor (hRI), a leucine-rich repeat protein, binds the blood vessel-inducing protein human angiogenin (Ang) with extraordinary affinity (Ki <1 fM). Here we report a 2.0 Å resolution crystal structure for the hRI–Ang complex that, together with extensive mutagenesis data from earlier studies, reveals the molecular features of this tight interaction. The hRI–Ang binding interface is large and encompasses 26 residues from hRI and 24 from Ang, recruited from multiple domains of both proteins. However, a substantial fraction of the energetically important contacts involve only a single region of each: the C-terminal segment 434–460 of hRI and the ribonucleolytic active centre of Ang, most notably the catalytic residue Lys40. Although the overall docking of Ang resembles that observed for RNase A in the crystal structure of its complex with the porcine RNase inhibitor, the vast majority of the interactions in the two complexes are distinctive, indicating that the broad specificity of the inhibitor for pancreatic RNase superfamily proteins is based largely on its capacity to recognize features unique to each of them. The implications of these findings for the development of small, hRI-based inhibitors of Ang for therapeutic use are discussed. Introduction The molecular basis for protein–protein recognition, a critical issue in structural biology, recently has come under intense scrutiny. A large number of crystal structures of protein–protein complexes have now been determined, and considerable progress has been made toward an understanding of their general characteristics (Janin and Chothia, 1990; Jones and Thornton, 1996). Nonetheless, it has not yet become possible to deduce the energetic contributions of individual interface residues based solely on crystallography. Moreover, in the few instances where detailed functional information on a protein–protein interaction has also been collected by site-specific mutagenesis, some residues 'observed' to form strong contacts in the crystal structures have been found to have little functional importance, and other residues have been shown to make strong contributions that were not apparent from the structures (as discussed by Clackson and Wells, 1995; Schreiber and Fersht, 1995; Chen and Shapiro, 1997). Examination of additional complexes by an approach that combines structural and functional data should lead to a more predictive understanding of the physicochemical basis for protein–protein recognition. The complex of human placental RNase inhibitor (hRI), a 50 kDa leucine-rich repeat protein, with human angiogenin (Ang), a 14.1 kDa blood vessel-inducing protein in the pancreatic RNase superfamily (Fett et al., 1985; Strydom et al., 1985), is among the tightest on record (Ki <1 fM; Lee et al., 1989a). Binding of hRI inhibits both the enzymatic and angiogenic activities of Ang (Shapiro and Vallee, 1987); indeed this finding provided one of the first indications that the ribonucleolytic action of Ang is required for its biological activity, as later confirmed by mutational studies (Shapiro and Vallee, 1989; Shapiro et al., 1989). The detailed mechanism of Ang action and the identity of its in vivo substrate in particular remain to be determined. However, Ang has been demonstrated to interact with endothelial cells in a variety of ways pertinent to angiogenesis: it (i) binds cell surface receptors, producing a mitogenic response (Hu et al., 1997); (ii) induces second messengers (Bicknell and Vallee, 1988, 1989); (iii) stimulates cell-associated proteolytic activity and invasiveness (Hu et al., 1994); (iv) mediates cell adhesion (Soncin, 1992); and (v) organizes cells into tubular structures (Jimi et al., 1995). Moreover, Ang undergoes nuclear translocation in these cells (Moroianu and Riordan, 1994), bringing it in direct apposition to potential RNA substrates. Ang originally was isolated from human adenocarcinoma cell conditioned medium, and recent studies indicate that it is critically involved in the establishment and growth of a wide range of human tumour types in athymic mice (Olson et al., 1994, 1995; Olson and Fett, 1996). These results identify Ang as a potentially useful target for new anticancer drugs. hRI itself is unlikely to have therapeutic utility, primarily because of its sensitivity to oxidation (Blackburn et al., 1977) and its broad specificity for proteins in the pancreatic RNase superfamily (see Lee and Vallee, 1993; Hofsteenge, 1997). hRI binds human RNase-2 [also known as placental RNase and eosinophil-derived neurotoxin (EDN)] almost as avidly as it binds Ang (Shapiro and Vallee, 1991), and Ki values for pancreatic-type RNases are still in the mid to upper femtomolar range [44 fM for bovine (Lee et al., 1989a) and 200 fM for human (Boix et al., 1996)]. The interactions of hRI with yet other RNases [human RNase-4 (Shapiro et al., 1986) and RNase-6 (R.Shapiro, unpublished observations)] are also very tight, although in these cases the dissociation constants have not been quantitated. The high affinity of hRI for all of its ligands is remarkable in view of the fact that these proteins share only ∼25–35% sequence identity. We report now a crystal structure for the hRI–Ang complex at 2.0 Å resolution. This structure, in combination with extensive functional data on the complex available from earlier studies, provides a detailed view of how Ang and hRI form their extremely strong association. Although the overall docking of Ang resembles that observed for RNase A in the crystal structure of its complex with the closely related porcine RNase inhibitor (pRI) (Kobe and Deisenhofer, 1995), the specific interactions at the interface differ substantially. Indeed, the vast majority of the hydrogen bonds and van der Waals contacts in the two complexes are distinctive, indicating that inhibitor versatility is based in large part on its capacity to recognize features unique to each of its ligands. This information should now provide a basis for efforts to design small, active hRI derivatives or mimics suitable for therapeutic use. Results Crystallization of the hRI–Ang complex Ionic interactions have been shown to play an important role in the binding of both Ang and RNase A to hRI (Lee et al., 1989a,b; Chen and Shapiro, 1997). Therefore, relatively low ionic strength was maintained in crystallization solutions and non-ionic precipitants were used. Concentrations of phosphate and sulfate in particular were held low since these bind to the active sites of pancreatic RNase family proteins (Richards and Wyckoff, 1973; Shapiro et al., 1987; Howlin et al., 1989; Mosimann et al., 1996). Crystals of the hRI–Ang complex were grown by pre-forming the complex in HEPES buffer, and then adding an equal volume of a reservoir solution containing citrate, a small amount of ammonium sulfate and polyethylene glycol (PEG) as the precipitant (see Materials and methods). The stability of the complex under these conditions [but with 0.1 mM rather than 25 mM dithiothreitol (DTT)] was evaluated by measuring the rate constant for its dissociation. For this purpose, [14C]Ang was used and the reservoir solution contained a large molar excess of RNase A to act as a scavenger for any free RI dissociating from the complex (Chen and Shapiro, 1997). At various times, free and RI-bound [14C]Ang were separated and quantitated by scintillation counting. After 4 days at 25°C, <2% of the Ang had been released, indicating that the rate of complex dissociation was similar to that measured under the standard conditions used for kinetic studies (t1/2 = 70 days). Description of overall structure The three-dimensional crystal structure of the hRI–Ang complex was determined at 2.0 Å resolution (Figure 1, Table I). The complex crystallizes as a dimer (Figure 2A), although in solution it probably exists as a monomer, as has been shown for free hRI and its complex with RNase A (Blackburn et al., 1977). The two monomers have nearly identical structures [the r.m.s. deviations are 0.26 Å (Cα atoms), 0.56 Å (main-chain) and 0.96 Å (side-chain)]. The interactions detailed below are those in molecule 1, and can be assumed to be replicated in molecule 2 except where noted otherwise. The dimer contains 133 ordered water molecules (76 in molecule 1 and 57 in molecule 2); the majority either lie at the monomer–monomer interface or mediate interactions between Ang and hRI. No sulfate or other salt molecules from the crystallization solution are observed in the structure. Figure 1.A view of a slice through the electron density map calculated using 2|Fobs|−|Fcalc| coefficients and calculated phases onto the refined coordinates of the hRI–Ang complex. Download figure Download PowerPoint Figure 2.(A) Overall view of the hRI–Ang dimer (perpendicular to the plane of the horseshoe), drawn with the program MOLSCRIPT (Kraulis, 1991). The hRI molecules are in cyan and yellow, and the Ang molecules are shown in red and green. (B) Stereo view of a Cα trace of the hRI–Ang complex superimposed on the pRI–RNase A complex (Kobe and Deisenhofer, 1995). The colour codes are: green, hRI; red, pRI; blue, Ang; and cyan, RNase A. Drawn with the program MOLSCRIPT (Kraulis, 1991). (C and D) Molecular surfaces of hRI (C) and pRI (D) drawn with the program GRASP (Nicholls and Honig, 1991). Surface complementarity at the interface in hRI–Ang and pRI–RNase complexes is colour coded based on calculations using the program SHAPE (Lawrence and Colman, 1993). Red, yellow and light blue colours correspond to Sc values of 1.0, 0.5 and 0.0 respectively. The horseshoe was rotated by ∼20° from the standard orientation in order better to show the inner surface of the C-terminal half, where many of the contacts are. For identification of hRI residues in regions of high complementarity, compare (C) with Figure 4A. Download figure Download PowerPoint Table 1. Crystallographic data processing and refinement statistics Space group monoclinic, P21 Cell dimensions a = 66.55 Å, b = 105.60 Å, c = 93.52 Å, β = 107.09° Two hRI–hAng complex molecules/a.u. (solvent content ∼51%) Resolution (Å) 40.0−2.0 Nma 158 593 Nub 72 355 Overall completeness (%) 86.9 (average) 4.5 (1.6 between 2.07−2.0Å) Completeness of outer shell 65.4% (2.07−2.0 Å) Rsymc (%) 9.4 No. of reflections used in refinement (20−2 Å) 72 308 Rcrystd (F>0σ) (%) 19.3 Rfreee (%) 28.6 No. of protein atoms 8806 No. of water molecules 133 R.m.s deviation in bond lengths (Å) 0.009 bond angles (°) 1.40 Average B-factors (Å2) Overall B-factor (Wilson plot) 40.4 hRI–hAng molecule 1 41.2 hRI–hAng molecule 2 42.6 hRI all atoms 40.4 mol 1 41.2 mol 2 main-chain atoms 37.5 mol 1 39.2 mol 2 side-chain atoms 43.7 mol 1 45.3 mol 2 Ang all atoms 44.1 mol 1 45.1 mol 2 main-chain atoms 41.7 mol 1 42.5 mol 2 side-chain atoms 46.5 mol 1 47.6 mol 2 Water molecules 42.2 a Number of measurements. b Number of unique reflections. c Rsym = ΣhklΣi|Ii(hkl)− |/ΣhklΣiIi(hkl) where is the averaged intensity of the i observations of reflection hkl. d Rcryst =Σ||Fo|−|Fc||/Σ|Fo| where Fo and Fc are the observed and calculated structure factor amplitudes, respectively. e Rfree is equal to Rcryst for a randomly selected 5% subset of reflections not used in the refinement (Brünger, 1992b). The topology of the hRI molecule is closely similar to that of pRI (Kobe and Deisenhofer, 1993, 1995) (Figure 2B). The two proteins exhibit 77% amino acid sequence identity and one-third of the substitutions are conservative (Figure 3). Compared with the porcine protein, hRI has a four residue extension at the N-terminus, and no insertions or deletions. The secondary structural features of the two proteins are nearly identical. Briefly, the 15 alternating 29 and 28 residue leucine-rich repeat units that comprise most of the hRI molecule form β–α hairpin units and are arranged in the non-globular, symmetric shape of a horseshoe (Figure 2A and B). The inner and outer diameters of the horseshoe are ∼20 and 68 Å, respectively, and its thickness is 35 Å (the corresponding values for the pRI complex are 21, 67 and 32 Å). The molecular fold is generated by an extended right-handed superhelix with alternating β-strands and α-helices. All of the 17 short β-strands, which form a solvent-exposed parallel β-sheet, line up on the inner surface of the horseshoe (except for the elongated N-terminal strand that is involved in dimerization; see below); the outer surface is decorated by 16 helices that are in most instances 10–13 residues in length. These secondary structural elements are connected by loops that contain 4–11 residues. The horseshoe structure is stabilized by a large number of intra- and inter-repeat interactions, in the same manner as described previously for free pRI (Kobe and Deisenhofer, 1993). All of the 32 hRI cysteine residues are in the reduced form and are well ordered in the complex. Figure 3.Amino acid sequence alignment for (i) hRI (Lee et al., 1988) and pRI (Hofsteenge et al., 1988), and (ii) Ang (Strydom et al., 1985), RNase A and EDN (Beintema et al., 1988a). The contact residues are shown in boxes: for hRI and Ang as shown in Table III, for pRI and RNase as described by Kobe and Deisenhofer (1995, 1996). The figure was produced using ALSCRIPT (Barton, 1993). The secondary structure elements [based on the program DSSP (Kabsch and Sander, 1983)] contain the following residues: for hRI: β1 (1–10), βα1 (11–15), α1 (16–22), αβ1 (23–30), β2 (31–35), βα2 (36–43), α2 (44–52), αβ2 (53–58), β3 (59–61), βα3 (62–67), α3 (68–76), αβ3 (77–87), β4 (88–90), βα4 (91–100), α4 (101–110), αβ4 (111–115), β5 (116–118), βα5 (119–124), α5 (125–136), αβ5 (137–144), β6 (145–147), βα6 (148–157), α6 (158–167), αβ6 (168–172), β7 (173–175), βα7 (176–181), α7 (182–195), αβ7 (196–201), β8 (202–204), βα8 (205–213), α8 (214–224), αβ8 (225–229), β9 (230–232), βα9 (233–238), α9 (239–250), αβ9 (251–258), β10 (259–261), βα10 (262–268), α10 (269–279), αβ10 (280–286), β11 (287–289), βα11 (290–295), α11 (296–307), αβ11 (308–315), β12 (316–318), βα12 (319–328), α12 (329–338), αβ12 (339–343), β13 (344–346), βα13 (347–352), α13 (353–364), αβ13 (365–372), β14 (373–375), βα14 (376–385), α14 (386–395), αβ14 (396–400), β15 (401–403), βα15 (404–410), α15 (411–422), αβ15 (423–429), β16 (430–432), βα16 (433–439), α16 (440–452), αβ16 (453–456), β17 (457–458), βα17 (459–460); and for Ang: L1 (1–4), α1 (5–13), L2 (14–22), α2 (23–33), L3 (34–40), β1 (41–46), L4 (47–49), α3 (50–56), L5 (57–68), β2 (69–71), L6 (72–75), β3 (76–83), L7 (84–93), β4 (94–107), L8 (108–111), β5 (112–116), 310 (117–123). Abbreviations: α, α-helix; β, β-sheet; L, αβ or βα loops. Download figure Download PowerPoint Structural information is not yet available for the free hRI molecule, and it is therefore not possible to gauge what conformational changes, if any, occur upon Ang binding. However, comparison of the hRI structure in the complex with those of free and RNase A-bound pRI suggests that the type of global structural alterations associated with binding of RNase A may not occur with Ang. Kobe and Deisenhofer (1995, 1996) reported an accumulation of small shifts along the polypeptide chain in bound versus free pRI, resulting in a widening of the horseshoe opening from 12 to 14.4 Å. The corresponding distance in the hRI–Ang complex is 12.7 Å, much closer to that in free pRI. Moreover, a least-squares superposition of the hRI structure (Cα atoms) with those of free and bound pRI results in r.m.s. deviations of 1.24 and 1.88 Å, respectively (compared with 1.46 Å for free versus bound pRI), indicating that the hRI backbone structure in the Ang complex is more similar to free than to RNase A-bound pRI. Alignment of hRI and bound pRI in segments of ∼150 residues reduces the r.m.s. deviation in Cα positions to 0.63–0.87 Å. Comparison of the side-chain orientations within these segments reveals that several contact residues for Ang on hRI (see Table III below) adopt positions that differ significantly from those of the corresponding residues in the pRI complex. The largest changes are seen for Trp261 and Trp318: the indole ring of Trp261 occupies a space similar to that of the corresponding Trp in pRI (257) but is rotated by ∼180°; the indoles of Trp318 and pRI Trp314 are nearly perpendicular to each other. In addition, the positions of the side-chain OH and carboxylate of the C-terminal Ser are interchanged in hRI and pRI. Potentially significant shifts of 1–4 Å are also observed for Tyr150, Lys320, Glu344, Gln346, Glu440 and Tyr437. Numerous RI side chains, primarily flexible surface residues, outside the interface region also have different conformations in the two proteins. The structure of Ang in the complex is similar to those of free <Glu-1 (D.D.Leonidas, S.C.Allen and K.R.Acharya, unpublished results) and Met-(−1) Ang (Acharya et al., 1994). All three disulfide bridges are present and well ordered, even though the crystals were grown in the presence of 25 mM DTT. Superposition of 118 Cα atoms in free and complexed 120° are shown (D, hydrogen bond donor; A, hydrogen bond acceptor). Bond angles are not given for hydrogen bonds involving NZ atoms since the hydrogen position is ambiguous. Hydrogen bond parameters were calculated with the CCP4 program CONTACT (CCP4, 1994). Table 5. Water-mediated hydrogen bonds between hRI and Ang in the complex structure Water hRI residue Distance (Å) hAng residue Distance (Å) Solvent accessibilitya (Å2) B-factor (Å2) 1703 Asp435 OD1 2.6 Leu115 O 2.9 0.0 37.1 1704 Asp435 N 2.6 Gln12 OE1 3.4 1.8 24.6 1734 Glu206 OE2 3.3 Gly86 N 2.7 14.9 37.2 Trp263 NE1 3.4 1745 Asp36 OD2 3.4 Arg24 NH1 2.7 8.6 58.1 Ser64 O 3.4 1749 Asn178 OD1 3.4 Gln93 NE2 3.1 1.1 53.3 S

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