Mechanism of Ribonuclease Inhibition by Ribonuclease Inhibitor Protein Based on the Crystal Structure of its Complex with Ribonuclease A

J. Mol. Biol. (1996) 264, 1028–1043

Mechanism of Ribonuclease Inhibition by Ribonuclease Inhibitor Protein Based on the Crystal Structure of its Complex with Ribonuclease A Bostjan Kobe1,2* and Johann Deisenhofer2 1

St. Vincent’s Institute of Medical Research 41 Victoria Parade, Fitzroy Victoria 3065, Australia 2

Howard Hughes Medical Institute and Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9050 USA

We describe the mechanism of ribonuclease inhibition by ribonuclease inhibitor, a protein built of leucine-rich repeats, based on the crystal structure of the complex between the inhibitor and ribonuclease A. The structure was determined by molecular replacement and refined to an Rcryst of 19.4% at 2.5 Å resolution. Ribonuclease A binds to the concave region of the inhibitor protein comprising its parallel b-sheet and loops. The inhibitor covers the ribonuclease active site and directly contacts several active-site residues. The inhibitor only partially mimics the RNasenucleotide interaction and does not utilize the p1 phosphate-binding pocket of ribonuclease A, where a sulfate ion remains bound. The 2550 Å2 of accessible surface area buried upon complex formation may be one of the major contributors to the extremely tight association (Ki = 5.9 × 10−14 M). The interaction is predominantly electrostatic; there is a high chemical complementarity with 18 putative hydrogen bonds and salt links, but the shape complementarity is lower than in most other protein–protein complexes. Ribonuclease inhibitor changes its conformation upon complex formation; the conformational change is unusual in that it is a plastic reorganization of the entire structure without any obvious hinge and reflects the conformational flexibility of the structure of the inhibitor. There is a good agreement between the crystal structure and other biochemical studies of the interaction. The structure suggests that the conformational flexibility of RI and an unusually large contact area that compensates for a lower degree of complementarity may be the principal reasons for the ability of RI to potently inhibit diverse ribonucleases. However, the inhibition is lost with amphibian ribonucleases that have substituted most residues corresponding to inhibitor-binding residues in RNase A, and with bovine seminal ribonuclease that prevents inhibitor binding by forming a dimer. 7 1996 Academic Press Limited

*Corresponding author

Keywords: ribonuclease inhibitor; RNase A; leucine-rich repeats; crystal structure; interaction

Introduction The formation of specific protein-protein complexes is of fundamental importance in most cellular processes. Certain structural motifs, for example the immunoglobulin fold (Williams & Abbreviations used: LRR, leucine-rich repeat; RI, ribonuclease inhibitor; RNase, ribonuclease; 3-D, three-dimensional; BS-RNase, bovine seminal RNase; DTT, dithiothreitol; Ki , inhibition constant; Hepes, N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]; EDN, eosinophil-derived neurotoxin; PC, Patterson correlation. 0022–2836/96/501028–16 $25.00/0

Barclay, 1988), the epidermal growth factor-like module (Campbell & Bork, 1993) and the kringle domain (Furie & Furie, 1988), are widely used in diverse molecular recognition processes and appear to have particularly useful characteristics for involvement in protein-protein interactions. These and many other such motifs have been recruited as modules in mosaic proteins by exon shuffling and duplication (Patthy, 1991). The recently characterized leucine-rich repeat (LRR) is likely to be another structural motif widely used in molecular recognition. LRRs have been found in over 60 different proteins with diverse functions and cellular locations that all appear to be 7 1996 Academic Press Limited

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RNase Inhibitor Protein Mechanism

Table 1. Refinement statistics ˚) Resolution (A Number of reflections used (F > s(F)) Rcryst (%) Total number of non-hydrogen atoms Number of non-hydrogen protein atoms Number of putative water molecules Number of putative sulfate molecules ˚ 2) Average B-factors (A RNase inhibitor All Main-chain ˚ 2) Side-chain (A RNase A All Main-chain Side-chain ˚) r.m.s. bond length deviation (A r.m.s. bond angle deviation (deg.)

Free RI

RNase A-RI

40-2.3 24,468 20.0 3478 3414 64 —

40-2.5 18,859 19.4 4416 4365 46 1

30.4 27.7 33.6

46.7 43.3 50.6

— — — 0.012 1.676

58.6 58.2 59.0 0.013 1.826

Rcryst = Shkl (>Fobshkl = − =Fcalchkl >)/=Fobshkl =, where =Fobshkl = and =Fcalchkl = are the observed and calculated structure factor amplitudes, respectively.

involved in protein-protein interactions and participate in biological processes such as signal transduction, cell adhesion, DNA repair, RNA processing and plant defence (Kobe & Deisenhofer, 1994, 1995a). One of the best characterized members of the LRR superfamily is ribonuclease inhibitor (RI), a cytoplasmic protein that tightly binds and inhibits ribonucleases (RNases) from the pancreatic superfamily (Lee & Vallee, 1993). Its primary structure is built of 15 LRRs, alternately 28 and 29 residues long (Hofsteenge et al., 1988). The crystal structure of porcine RI (Kobe & Deisenhofer, 1993a) revealed that individual LRRs represent structural b-a hairpin units. In individual units, the b-strand and the a-helix are approximately parallel; the units are all aligned nearly parallel with a common axis. The resulting structure has a non-globular shape resembling a horseshoe with an inner diameter ˚ and an outer diameter of about 67 A ˚. of about 21 A The concave face of the parallel b-sheet is exposed to the solvent. The curvature of the horseshoe is determined by the difference in the distances between the strands and the helices. The ribonucleases inhibited by RI all belong to the pancreatic ribonuclease superfamily. It is unusual that RNase A (Blackburn & Moore, 1982), angiogenin (Fett et al., 1985), eosinophil-derived neurotoxin (EDN; identical with hepatic alkaline RNase, human placental RNase and RNase US , Hofsteenge et al., 1989; Shapiro & Vallee, 1991), and tumor-derived RNase (Shapiro et al., 1986), enzymes with very limited sequence similarities (there is only 24% identity between angiogenin and EDN (Shapiro & Vallee, 1991)), are all inhibited with Ki values between 10−14 and 10−16 M (Lee et al., 1989a,b; Vicentini et al., 1990; Shapiro & Vallee, 1991). The affinities of RI for these ribonucleases are among the highest reported for non-covalent binding of proteins. The binding occurs with 1:1 stoichiometry (Blackburn & Moore, 1982). The

potent inhibitory activity of RI is thought to be utilized in RNA processing (Shortman, 1962), angiogenesis (Fett et al., 1985), and protection of the cell from toxic ribonucleases (Roth, 1958). RI inhibits only a subset of the pancreatic ribonuclease superfamily; amphibian ribonucleases like frog liver ribonuclease (Roth, 1962), sialic acid-binding lectin (Nitta et al., 1993), and P-30 protein (Wu et al., 1993) are not inhibited. Various biochemical studies (Lee & Vallee, 1993) jointly with the known three-dimensional (3-D) structures of porcine RI (Kobe & Deisenhofer, 1993a) and the ligand ribonucleases (Kartha et al., 1967; Acharya et al., 1994) shed light on the binding sites of the enzyme and the inhibitor. The exact location of the sites, however, remained unclear. To define the atomic basis of the extremely tight interaction between RI and ribonucleases and the specificity of this association, we determined the crystal structure of the complex between porcine ribonuclease inhibitor and bovine ribonuclease A (Ki = 5.9 × 10−14 M; Vicentini et al., 1990) and refined ˚ resolution. The structure illustrates the it at 2.5 A general properties of the ligand-binding functions of LRR-containing proteins; we have discussed such implications elsewhere (Kobe & Deisenhofer, 1995b). Here, we focus on the mechanism of the inhibition of ribonuclease activity by the proteinaceous inhibitor and the properties of the interface, and examine the interaction in the light of other biochemical studies.

Results Structure determination The model of free RI (Kobe & Deisenhofer, 1993a) ˚ resolution (Rcryst = 20.0%; see was refined at 2.3 A Table 1 for definitions of R-factors) and used as a starting model to determine the disposition of RI in the crystals of RNase A-RI complex by cross-

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RNase Inhibitor Protein Mechanism

(a)

(b) Figure 1. Structure of RNase A-RI complex. (a) A diagram of the 3-D structure of porcine RI, drawn with the program MOLSCRIPT (Kraulis, 1991). RI is blue and RNase A is red. The N and C termini of both molecules are indicated. (b) Same as in (a), but rotated around the horizontal axis for about 90°. After Kobe & Deisenhofer (1995b).

rotational and translational searches. The orientation of the RNase A molecule in these crystals could not be found in cross-rotation functions with available models of RNase A; however, electron density maps based on the properly positioned RI model revealed the location of RNase A in the crystals. The model of the RNase A-RI complex was ˚ resolution (Rcryst = 19.4%; Table 1) refined at 2.5 A and contains all 456 RI residues of RI including the N-acetyl group, all 124 residues of RNase A, 46 water molecules, and one sulfate molecule. Structure of the complex Two views of the complex are shown in Figure 1. RI is a horseshoe-shaped a/b protein with a

long parallel b-sheet lining its inner circumference, and a-helices lining its outer circumference. RNase A is roughly kidney-shaped, predominantly built of antiparallel b-structure and stabilized by four disulfide bridges. The active site is located in a cleft between the two lobes of the structure. RNase A binds to the inhibitor with its active-site cleft covering the C-terminal corner of the horseshoe; one lobe places into the concave cavity of the horseshoe, and the other lobe lies over the face of the horseshoe. RI is a much larger protein than RNase A. It is also a protein of non-globular shape whose surface area exceeds by 10% that expected for a molecule of its mass (Miller et al., 1987). By binding RNase A to a concave surface, RI uses its larger size and non-globular shape to provide an extensive binding area for the enzyme. Even after binding, the accessible surface area of the complex remains 8% above that predicted for oligomeric proteins (Janin et al., 1988). Contact residues on RI are contributed from throughout the sequence, but the bulk of the interaction is concentrated to the C-terminal part of the inhibitor (Table 2). Over 80% of contacting residues are contributed by the C-terminal half of RI, and almost 60% are contributed by the C-terminal quarter of the RI sequence. Out of 28 residues that form close contacts with RNase A, nine are a part of the b-sheet, contributed by seven different b-strands, only two belong to two different a-helices, and the rest come from the loops connecting the C termini of b-strands with the N termini of helices (ba-loops), with 17 residues contributed by ten different loops. Most of the 24 residues of RNase A that form close contacts with the inhibitor are contributed by four loops (16 residues), and of the rest three residues come from two different helices and five from four different b-strands. The mode of binding of the basic RNase A molecule (pI09) to the parallel b-sheet and the ba-loops of the acidic inhibitor (pI04.7) is consistent with the distribution of the negative electrostatic potential on the structure of RI (Figure 2). The crystal packing of RNase A-RI complexes is similar to the packing of RI molecules in the crystals of free RI (Kobe & Deisenhofer, 1993a). However, when compared with the crystals of free RI, the rotation and translation of the inhibitor in the crystals of the complex necessary to accommodate the RNase A molecule changes the crystal contacts considerably. Only one polar contact between symmetry-related molecules (interaction between the side-chains of Cys215 and Gln412 of RI) is conserved in the two crystals. In the crystals of the complex, RNase A forms close contacts with both the symmetry-related RNase molecules and RI molecules. Accordingly, we were unable to bind RNase A to crystals of free RI by soaking these crystals in solutions containing RNase A; such soaking caused disintegration of the original crystals.

Residue RI H6 C7 D31 N89 D117 E202 D228 W257 W259 E283 W314 K316 E397 C404 V405 G406 D407 Q426 V428 Y430 D431 Y433 W434 T435 E436 R453 I455 S456

a1 RNase A contacts 3 2 1 3 5 2 2 2 11 3 4 1 9 3 3 1 2 1 1 14 4 18 1 3 5 4 1 8

a1 K7 7

2 5

1

1

L2 a2 L3 L3 Q11 N24 Q28 K31 1 7 1 4 3 2

L3 S32 2

1

4 1

1 1 3

9

8 1

2 1

2

1

1 3 1

2

2

5

1

1 6 5

2 2

L3 L3 L3 L3 L3 b1 L4 L4 L4 L4 b4 L5 L5 L35 D38 R39 K41 P42 V43 K66 N67 Q69 N71 E86 G88 S89 1 5 14 9 2 1 2 6 4 5 1 7 9

1

3 3

1

2

5 1 3 5

2 3

L5 L5 b6 b6 b7 S90 K91 A109 E111 H119 1 7 2 14 5

7 1 ˚ is shown for each interacting residue. The segments were assigned according to secondary-structure elements The number of atom-atom contacts at distances below 4 A (program DSSP; a, a-helix; b, b-strand; L or ab or ba, loop including any other secondary structure); RI: ba1 (residues 1 to 11), a1 (12 to 22), ab1 (23 to 26), b2 (27 to 29), ba2 (30 to 36), a2 (37 to 48), ab2 (49 to 54), b3 (55 to 57), ba3 (58 to 63), a3 (64 to 75), ab3 (76 to 83), b4 (84 to 86), ba4 (87 to 99), a4 (100 to 106), ab4 (107 to 111), b5 (112 to 114), ba5 (115 to 120), a5 (121 to 133), ab5 (134 to 140), b6 (141 to 143), ba6 (144 to 153), a6 (154 to 163), ab6 (164 to 168), b7 (169 to 171), ba7 (172 to 177), a7 (178 to 190), ab7 (191 to 197), b8 (198 to 200), ba8 (201 to 207), a8 (208 to 220), ab8 (221 to 225), b9 (226 to 228), ba9 (229 to 234), a9 (235 to 246), ab9 (247 to 254), b10 (255 to 257), ba10 (258 to 264), a10 (265 to 277), ab10 (278 to 282), b11 (283 to 285), ba11 (286 to 291), a11 (292 to 304), ab11 (305 to 311), b12 (312 to 314), ba12 (315 to 324), a12 (325 to 334), ab12 (335 to 339), b13 (340 to 342), ba13 (343 to 348), a13 (349 to 361), ab13 (362 to 368), b14 (369 to 371), ba14 (372 to 378), a14 (379 to 391), ab14 (392 to 396), b15 (397 to 399), ba15 (400 to 406), a15 (407 to 417), ab15 (418 to 425), b16 (426 to 428), ba16 (429 to 435), a16 (436 to 448), ab16 (449 to 452), b17 (453 to 455), ba17 (456); RNase A: L1 (1 to 3), a1 (4 to 12), L2 (13 to 24), a2 (25 to 29), L3 (30 to 42), b1 (43 to 47), L4 (48 to 51), a3 (52 to 55), L5 (56 to 60), b2 (61 to 63), L6 (64 to 71), b3 (72 to 74), L7 (75 to 78), b4 (79 to 86), L8 (87 to 96), b5 (97 to 104), L9 (105), b6 (106 to 111), L10 (112 to 115), b7 (116 to 123) and L11 (124). The one-letter amino acid code is used. A complete list of contacts including the atoms involved and the distances can be obtained from the authors upon request.

ba1 ba1 ba2 ba4 ba5 ba8 b9 b10 ba10 b11 b12 ba12 b15 ba15 ba15 ba15 a15 b16 b16 ba16 ba16 ba16 ba16 ba16 a16 b17 b17 ba17

Segment

Table 2. Contacts between RNase A and RI in the complex

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RNase Inhibitor Protein Mechanism

(a)

(b) Figure 2. Molecular surfaces of RNase A (right) and RI (left). Associated complexes are formed by rotating each molecule by 90° around the vertical axis (clockwise for the left molecule, anticlockwise for the right molecule). The ˚ . (a) Surface color-coded according to electrostatic potential mapped to the surface of the probe radius was 1.4 A molecules. Lys and Arg were assigned a single positive charge, Glu, Asp and the C terminus were assigned a single negative charge, and all other residues were considered neutral. A uniform dielectric constant of 80 for the solvent and 2 for the protein interior were assumed; the ionic strength was set to zero. Coloring is continuous going from blue (potential +10 kt/e; 1 kt = 0.6 kcal, e is the charge of an electron) through white to red (potential −10 kt/e). (b) Surface color-coded according to surface complementarity. Red, Sc > 0.76; yellow, 0.76 > Sc > 0.3; green, 0.3 > Sc > −0.3; light blue, Sc < −0.3. See the text for more information about the calculation. Selected interface residues with high complementarities are labelled.

Structural changes upon complex formation RI bound to RNase A has a slightly different conformation from that found in the crystals of free RI (Kobe & Deisenhofer, 1993a). The r.m.s. deviation of the superimposed Ca atoms of the free

˚ . The cavity in the and bound forms is 1.46 A structure is somewhat enlarged; the shortest distance between the N-terminal and C-terminal ˚ in the free form and repeats of RI is about 12 A ˚ in the complex. The increases by more than 2 A conformational change can best be described as a

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RNase Inhibitor Protein Mechanism

Figure 3. Conformational changes in RI. The models of RI from the RNase A-RI complex (continuous lines) and RI from the lithium sulfate crystals (broken lines) were superimposed on the high-resolution model of free RI with the program INSIGHT (using only the Ca atoms). The r.m.s. deviations of the Ca atoms are shown as thin lines, and the fourth-order polynomial curves fitted to the r.m.s. deviations curves are shown as thick lines.

continuous accumulation of small shifts along the chain, resulting in changes of the curvature and the twist of the horseshoe (Figure 3). Unlike most other conformational changes observed in nature, there is no obvious hinge responsible for the movement of separate domains relative to each other; it is a plastic adaptation of the entire structure. There is no significant difference in either the accessible surface area or the molecular volume of the free and bound forms of RI. The observed conformational change was not unexpected; the superhelical organization of the protein fold without any long-range stabilizing interactions must allow a substantial amount of flexibility of the structure and a substantial movement of the two ends of the molecule relative to each other in solution (Kobe & Deisenhofer, 1993a). Although exceeding it in magnitude, the nature of the conformational change between free and RNase-bound forms of RI resembles the

Table 3. Hydrogen-bonding and salt link interactions between RNase A and RI in the complex RNase A Lys7 NZ Gln11 NE2 Asn24 OD1 Asn24 ND2 Leu35 O Asp38 OD1 Arg39 NH1 Arg39 NH2 Val43 O Lys66 O Asn67 ND2 Asn71 ND2 Glu86 OE1 Gly88 O Ser89 OG Ser89 OG Glu111 OE1 Glu111 OE2

RI Ser456 OG Ser456 O Asn89 ND2 Asp117 OD2 Tyr430 OH Arg453 NH1 Glu397 OE2 Glu397 OE2 Asp431 OD2 Cys404 SG Val405 O Tyr433 OH Lys316 NZ Trp257 NE1 Glu202 OE1 Trp259 NE1 Tyr433 OH Glu436 OE2

˚) Distance (A 3.1 3.3 3.2 3.0 3.4 2.8 2.9 2.7 3.2 2.8 3.1 2.7 3.2 3.5 3.1 3.4 3.2 3.5

conformational differences between the molecules of free RI in the crystals from two different crystallization solutions, one containing ammonium sulfate and the other containing lithium sulfate as the precipitant (Figure 3; and see Kobe, 1994). Overall, the deviations are the largest in the loop regions and the smallest in the b-sheet; such behavior is usual in proteins. Because of the larger conformational differences between RI in the crystals of its complex with RNase A and free RI than between RI molecules in different crystals of free RI, the observed conformational change is more likely a result of complex formation than crystal contacts or properties of the protein solution. In addition to the continuous rearrangement of the backbone of RI, there are several local conformational changes caused by the complex formation. The side-chain of Tyr433 swings around to prevent a steric clash and to form a specific polar contact with the enzyme. Several other side-chains need to adjust to form specific contacts with ribonuclease, among them Cys404, Glu436 and Arg453. Most of the side-chains that exhibit different conformations in the complex and in the free inhibitor, and are not apparently influenced by the complex formation or differences in crystal contacts, belong to arginine, lysine and glutamate residues, and reflect inherent flexibility of these side-chains. The structure of RNase A in the complex does not substantially differ from the structure of free RNase A. The r.m.s. deviations of the Ca atoms after ˚ for both the sulfate-bound superposition is 0.54 A (Howlin et al., 1989) and sulfate-free forms (Wlodawer et al., 1988). Six pairs of atoms have ˚ discrepancies in the Ca atoms greater than 1 A (residues 1, 91, 93, 94, 112 and 113). Of these residues, only Lys91 is involved in a close contact with the inhibitor. The changes may therefore be caused by the neighboring residues involved in

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RNase Inhibitor Protein Mechanism

˚ sphere around the sulfate ion Figure 4. An annealed omit electron density in the active site of RNase A. An 8 A ˚ shell were restrained in the RNase active site was omitted and the immediate surrounding atoms consisting of a 3 A to avoid artificial movement into the omitted region. Simulated annealing was run with a starting temperature of ˚ . The stereo view of the electron density calculated with the phases derived from the omitted model and 1000 A ˚ , was contoured at 3s and plotted with a program written by coefficients =Fobs = − =Fcalc = for data between 40 and 2.5 A D. Xia. Superimposed is the model of the RNase A-RI complex. RNase A residues are indicated with E, and RI residues are indicated with I.

contacts with the inhibitor, crystal contacts (residue 1), or inherent flexibility of these loop regions. The only major local movement caused by the complex formation is the 180° swing of the side-chain of Arg39 to form specific polar contacts with the inhibitor; smaller side-chain motions to make contacts are seen for residues 7, 67 and 69. Again, there are some small changes in the conformations of side-chains not caused by the complex formation, mainly involving residues with long side-chains and reflect inherent flexibility of these. Out of 46 water molecules included in the model, only nine are found in equivalent positions in either free RI or RNase A. One of these interacts with both the enzyme and the inhibitor. The enzyme-inhibitor interface ˚ The accessible surface area buried from a 1.4 A probe in the interface between RI and RNase A in ˚ 2. This is over 70% the complex amounts to 2550 A more than the usual number for formation of protein-protein complexes (Janin, 1995); the only example of a similarly large interface between two folded heterologous proteins is found in the complex between TEM-1 b-lactamase and the inhibitor BLIP (Strynadka et al., 1996). Of the buried ˚ 2 and RI for surface, RNase A accounts for 1316 A ˚ 2; upon complex formation, 6.6% of the RI 1235 A surface and 19% of the RNase A surface get buried. Most protein-protein interfaces have a chemical character similar to that of the accessible surfaces of proteins in general, being 55% non-polar, 25% polar and 20% charged (Janin & Chothia, 1990). The buried surface in the RNase A-RI complex is 49% non-polar, 27% polar and 24% charged, slightly more charged than usual. Interestingly, the binding surface on the RI molecule is 47% non-polar, 23%

polar and 29% charged, with an extremely high percentage of charged atoms; 79% of these are negatively charged (Figure 2). The countersurface on RNase A is less charged (52% non-polar, 30% polar and 18% charged); oddly, 54% of the charged surface is again negatively charged. In the complex, the surfaces of 50 residues of RI and 40 residues of RNase A become partially buried ˚ probe. There are 117 contacts between from a 1.4 A ˚ RI and RNase A at distances shorter than 4 A (Table 2); 28 residues (71 atoms) of RI and 24 residues (63 atoms) of RNase A are involved in these contacts. There are 18 hydrogen bonds and salt links connecting RI and RNase A (Table 3). Of these, 11 involve at least one charged partner, and four of them are salt links. Seven interactions therefore do not have the charge compensated, and one is a ˚ between two glutamate side-chains. contact at 3.5 A The latter is most likely mediated through a positively charged solvent ion. Fourteen residues of RNase A and 15 residues of RI contribute to the polar contacts. Main-chain atoms are utilized six times, twice contributed by RI and four times by RNase A; all are carbonyl oxygen atoms. From the entropy point of view, interactions involving main-chain atoms are more favorable than interactions involving side-chain atoms. Of the surface area of RI buried upon complex formation, only 8.6% is contributed by main-chain atoms. The number is slightly higher for RNase A, 15%. Many RI residues involved in the interaction are contributed by the parallel b-sheet and adjacent residues, where the main-chain atoms are effectively shielded from solvent by the side-chains due to the curvature of the sheet. The degree of shape complementarity between the interacting protein surfaces was estimated by

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RNase Inhibitor Protein Mechanism

Figure 5. The structural similarity between the interaction of Tyr433 of RI and the thymine base of the thymidilic acid tetramer (d(pT)4 ) with RNase A. The structure of RNase A (yellow) from the complex with d(pT)4 (green; only the T4 nucleotide is shown) was superimposed on the structure of the RNase A (red) from the complex with RI (blue). Only selected residues in the vicinity of the T4 nucleotide from d(pT)4 are shown; RNase A residues are indicated with E, RI residues are indicated with I, the T4 nucleotide is denoted T4, and the sulfate ion (red) from the RNase A-RI complex is denoted SO4.

calculating the shape correlation statistic, Sc (Lawrence & Colman, 1993). Sc would be equal to 1.0 for two perfectly complementary surfaces, and around zero for surfaces with no complementarity. Sc is usually between 0.70 and 0.76 for oligomeric and protease-inhibitor interfaces, between 0.64 and 0.68 for antibody-antigen interfaces (Lawrence & Colman, 1993), and 0.70 for barnase-barstar interface, another ribonuclease-inhibitor interface (Buckle et al., 1994). For the RNase A-RI interface, Sc = 0.58 (Figure 2), poorer than usually observed in protein-protein complexes, although numbers as low as 0.57 are observed for antibody-antigen interfaces and 0.49 for oligomeric interfaces (Lawrence & Colman, 1993). In the RNase A-RI interface, the poorer complementarity is apparently compensated by a larger interface; this provides an opportunity for many interactions between the two molecules and is thus probably one of the major factors for the observed tightness of the complex. The value of Sc does not increase substantially after including solvent molecules in the calculation. In ˚3 addition, there is a cavity with a volume of 151 A present in the interface, lined by residues 1 to 5, 7, 8, 11, 12, 41, 111, 112, 118 and 119 of RNase A, and 431 to 436, 439, 442, 443, 446, 455 and 456 of RI (Kleywegt & Jones, 1994). A part of this cavity is filled by a sulfate ion (see below), and we found no ordered water molecule in the rest of the cavity, although water molecules are present on the corresponding surfaces of both free RNase A and free RI. The energy gain for burial of non-polar surfaces ˚ 2 (Chothia, 1974). has been estimated to be 25 cal/A

Burying the surface in the RNase A-RI interface should therefore provide −64 kcal/mol. The observed free energy of association is only −18 kcal/ mol; the difference must come from the polar nature of the interacting surface, the lack of surface complementarity, and the conformational changes of the inhibitor. Four water-mediated hydrogen bonds were identified in the interface. Three more water molecules contact both the enzyme and the ˚ . Due to the inhibitor at a distance under 4 A moderate resolution of the crystal structure, only a limited number of solvent molecules could be identified.

Discussion Mechanism of inhibition of ribonuclease activity RI inhibits RNase by preventing the access of substrates to the active site by steric hindrance and does not mimic the RNase-RNA interaction to a major extent. Several active-site residues (Borkakoti, 1983; Birdsall & McPherson, 1992; Fontecilla-Camps et al., 1994) do contact the inhibitor, including the catalytic residues Lys41 and His119; His12 does not interact with the inhibitor. Other RI-contacting RNA-binding residues of RNase A include Lys7, Gln11, Arg39, Val43, Cys65, Lys66, Gln69, Asn71, Glu111, but not Cys40, Asn44, Thr45, Asp83, Phe120 and Ser123. We found strong electron density in the active site of RNase A in the complex that we contributed

1036

Figure 6. Alignment of the primary sequences of RNase A, angiogenin and P-30 protein. Alignments are based on the structural information. The one-letter amino acid code is used. The elements of secondary structure are shown above the sequence. RNase A residues ˚ ) with RI in the RNase involved in close contacts (<4 A A-RI complex are shown bold and underlined.

to a sulfate ion, because ammonium sulfate was the precipitating agent in the crystallization (Figure 4). Similarly, a sulfate or phosphate ion is always found in the crystals of free RNase A if present in the crystallization medium. The sulfate ion in RNase A-RI complex is bound in a manner similar to that in free RNase A (Howlin et al., 1989), coordinated by Gln11, His12, Lys41 and His119 at ˚ . The electron density is most distances below 3.5 A consistent with His119 in the A conformer, as in the complex between RNase A and d(CpA) (Zegers et al., 1994). In addition, there is a close contact between a sulfate oxygen atom and the carboxyl ˚ ). The pKa of group of Asp431 of the inhibitor (2.9 A the aspartate residue must be perturbed by the structural environment to allow such interaction, and/or mediated through a positively charged solvent ion. It is plausible that in the absence of sulfate (or phosphate), Asp431 of the inhibitor might interact directly with Lys41 or other residues of RNase A. The presence of sulfate could even have an effect on the size of the cavity observed in the RNase A-RI interface, possibly resulting in a rearrangement of the residues lining the cavity and considerable differences in RNase A-RI contacts. The presence of salt substantially influences the affinity of the two molecules (Lee et al., 1989b; Blackburn et al., 1977). The only major structural similarity between RNase-RI and RNase-nucleotide binding appears to be the aromatic ring of Tyr433 resembling the aromatic ring of a base in the B2 binding pocket of RNase A (Birdsall & McPherson, 1992; FontecillaCamps et al., 1994). The ring of Tyr433 superimposes closely with the ring of the T4 thymine base in the complex of RNase A with thymidilic acid tetramer d(pT)4 (Birdsall & McPherson, 1992; entry 1RTA in the Protein Data Bank, Bernstein et al., 1977), with the hydroxyl group of the phenol ring of the tyrosine residue pointing in the direction of the methyl group at position 5 of the base (Figure 5). As mentioned above, the p1 pocket of RNase A is occupied by a putative sulfate ion from the solvent; B1 and R2 pockets are also not filled with any inhibitor atom. Asp431 is close to the R1 pocket,

RNase Inhibitor Protein Mechanism

but the interactions are different from those of nucleotides. The following atoms of RNase A form similar interactions with RI and nucleotides: Arg39 NH2 (contacts O1P and O3P of T1 in the complex of RNase A and d(pT)4 (Birdsall & McPherson, 1992) and Glu397 OE2 in RI); Asn67 ND2 (contacts O4 of deoxythymidine in the complex of RNase A covalently bound to deoxythymidine (Nachman et al., 1990), and Val405 O in RI); Lys7 NZ (contacts O2P of A3 in the RNase A-d(ApTpApApG) complex (Fontecilla-Camps et al., 1994) and Ser456 OG in RI); and Gln69 OE1 (contacts N6 of A3 in RNase A-d(ApTpApApG) complex (FontecillaCamps et al., 1994) and Asp-407 N in RI). Another example of inhibition of ribonuclease activity by a proteinaceous inhibitor comes from Bacillus amyloliquefaciens, where the extracellular ribonuclease barnase forms a 1:1 complex (Ki110−14 M) with its natural inhibitor barstar (Hartley, 1989). The primary structures of barnase and barstar are unrelated to those of RNase A and RI, respectively; barnase is related to a series of other bacterial and fungal ribonucleases (Hill et al., 1983). The mechanism of RNA cleavage is identical for the barnase-related and RNase A-related enzymes. The active site of barnase is formed by residues His102, Glu73 and Arg87, which are strictly conserved in its family of ribonucleases; these residues correspond to His12, His119 and Lys41 in RNase A. There seem to be both similarities and differences in the fundamental mechanism of ribonuclease inhibition between the barnase-barstar and RNase A-RI systems. The crystal structure of the complex between barnase and an active Cys40Ala, Cys82Ala mutant of barstar (Guillet et al., 1993; Buckle et al., 1994) showed that the interactions between these proteins are similar to those between barnase and nucleotides (Buckle & Fersht, 1994); the anchor point for the recognition seems to be the binding of an aspartate residue in the phosphate-binding site of barnase. The phosphate-binding site is not even used in the interaction between RI and RNase A, allowing a sulfate ion to remain bound in this site. The association and dissociation rate constants are of a similar magnitude for barnase-barstar (Schreiber & Fersht, 1993) and RNase A-RI interactions (Vicentini et al., 1990), suggesting similar electrostatic steering effects for both interactions. The kinetics of binding for both RNase-inhibitor pairs suggest a two-step binding mechanism (Lee et al., 1989a; Schreiber & Fersht, 1993). In addition to the electrostatic steering effects, the conformational changes of the inhibitors for both complexes may contribute to the similar behavior; barstar responds to binding of barnase by a subtle conformational change involving rigid body movements of the barstar helices. Solvent plays an important role in barnasebarstar binding (Guillet et al., 1993; Buckle et al., 1994); the base-recognition site of barnase, for which barstar has no equivalent, is filled by nine water molecules in the complex. In RNase A-RI

RNase Inhibitor Protein Mechanism

interaction, water does not seem to play such an important role; the moderate resolution of the RNase A-RI crystal structure, however, allows the localization of only a limited number of water molecules in the complex.

Agreement of the structure with biochemical studies The location of the RI-ribonuclease interface has been studied for both partners by chemical modification, proteolysis and mutagenesis. Although human placental RI was used in many of these studies, it shares 77% sequence identity with porcine RI and has most RNase A-binding residues conserved, and we assume that the qualitative conclusions of these studies apply equally to porcine RI. Carboxymethylation of the catalytic residues His12, His119 and Lys41 of RNase A showed that the latter is important for the interaction, while the former two are not (Blackburn & Jailkhani, 1979; Blackburn & Gavilanes, 1980). Lys41 indeed forms nine close contacts with the inhibitor, while the two histidine residues interact mostly with the putative sulfate ion in the active site and not directly with the inhibitor, with His119 forming only a few van der Waals contacts with the inhibitor. Amidination of the available lysine residues of the RNase A-human RI complex identified lysine residues 7, 31, 37, 41, 61 and 91 to be protected, and lysine residues 1, 66, 98 and 104 not to be protected (Blackburn & Gavilanes, 1982). Residues 7, 31, 37, 41 and 91 in fact get buried upon complex formation, and 1, 98 and 104 do not. Lys66 contacts the inhibitor, but only through its main-chain atoms. Lys61, however, is not involved in the interaction. The involvement of Lys61 has been questioned, because dinitrophenylation of its counterpart Lys60 of angiogenin did not affect the interaction (Lee & Vallee, 1989), and neither did the substitution of residues 58 to 70 in angiogenin with the sequence 59 to 73 of RNase A (Harper & Vallee, 1989). Studies using derivatives of RNase A modified at Lys1 and Lys7 have shown that these two residues, especially Lys7, provide most of the binding energy contributed by the N-terminal region (Neumann & Hofsteenge, 1993). Lys7 was proposed to form an ion-pair; in the structure, it indeed interacts with the C-terminal residue serine of the inhibitor. Lys1 does not form any specific contact with the inhibitor, but the elimination of its charge may perturb the general charge complementarity in that region. A mixture of truncated forms of porcine RI lacking the N-terminal 90 or 93 residues binds to RNase A only 2.3-fold weaker than the intact inhibitor (Hofsteenge et al., 1991b). In agreement with this result, residues from that region contribute only a few contacts and only one H-bond in the complex (Tables 2 and 3), with most of the

1037 interaction being concentrated at the C-terminal part of the inhibitor. Chemical labelling of cysteine residues showed that Cys371 and Cys404 become more than 90% protected in the presence of RNase A, while Cys57 does not (Hofsteenge et al., 1991a). In the structure, Cys404 is involved in several contacts with the enzyme, including a polar contact through its side-chain, and Cys57 is not involved in the interaction. Cys371 is at the edge of the interface and its side-chain does get partially buried upon complex formation. A series of internal repeats of human RI were deleted by mutagenesis (Lee & Vallee, 1990a,b) mutants with residues 139 to 252 or 311 to 367 deleted (the numbering of porcine RI is used) inhibited RNase A, although with reduced affinities for both RNase A and angiogenin. The deleted regions contribute only minor contacts, as seen in the structure, and the studies correctly suggested that the main contact area resides outside of the deleted regions. A possible explanation for the retention of inhibitor activity is that owing to the strict deletion of complete repeats the protein could fold in a conformation similar to that of intact RI. The part of the structure that had not been perturbed could still bind RNase in the same manner as the intact inhibitor; the rest might not be involved in binding or might interact in new ways. In agreement with this interpretation, the majority of the interaction is concentrated in the C-terminal part of the inhibitor (16 of 28 interacting residues are in the region 368 to 456). The deletion mutagenesis experiment bears general implications for the structure of proteins with leucine-rich repeats, as it suggests that individual repeats can be deleted or inserted during evolution to modulate the structure and function of these proteins (Kobe & Deisenhofer, 1994, 1995a). Implications for inhibition of other pancreatic ribonucleases by RI RI binds with high affinity to several ribonucleases from the pancreatic superfamily that share very limited sequence similarities. By contrast, sequences of RIs are highly conserved; human (Lee et al., 1988; Schneider et al., 1988; Crawford et al., 1989) and rat (Kawanomoto et al., 1992) RIs are very similar to pig RI (77% and 76% identical, respectively) with no insertion or deletion except for a short insertion at the N terminus of human RI. Most binding residues are strictly conserved in all species; the only contact residues that do show variation among the three species (residues 6, 228, 405 and 436 in porcine RI) are either not involved in hydrogen-bonding interactions through their side-chains, or, as is the case for Glu436, conservatively substituted by Asp in rat RI. The RNases known to be inhibited by RI include RNase A (Blackburn & Moore, 1982), angiogenin (Fett et al., 1985), EDN (Shapiro & Vallee, 1991; Hofsteenge et al., 1989), and tumor-derived RNase

1038 (Shapiro et al., 1986). RNase A is a secreted enzyme that should not normally come into contact with the cytoplasmic inhibitor; RI may have a role in inhibiting traces of secretory RNases if they illegitimately formed in the cytoplasm or leaked into the cytoplasmic compartment (Roth, 1958; Lee & Vallee, 1993). Angiogenin is endocytosed and translocated into the nucleus during the process of angiogenesis (Moroianu & Riordan, 1994). RI was proposed to be involved in the regulation of angiogenesis (Fett et al., 1985; Lee & Vallee, 1993), and was shown to be a potent inhibitor of tumor growth (Polakowski et al., 1993). Angiogenin binds RI even more tightly than RNase A (Ki = 7.1 × 10−16 M; Lee et al., 1989a). The 3-D structure of angiogenin (Acharya et al., 1994) is similar to that of RNase A, but differs particularly at the ribonucleolytic active site and the putative receptor binding site. Many RI-binding residues of RNase A are either conserved (residues 11, 35, 41, 88, 90, 109, 111 and 119) or substituted by similar amino acids (7, 31, 43 and 69) in angiogenin (Figure 6). However, more than half of the interacting residues are substituted non-conservatively or do not have an amino acid in an equivalent position; several of these are involved in hydrogen-bonding interactions in the RNase A-RI complex. To better understand the angiogenin-RI interaction, we superimposed angiogenin (entry 1ANG in the Protein Data Bank) on the RNase A molecule in the complex with RI, and subjected the corresponding model of the RI-angiogenin complex to energy minimization. Even before the energy minimization, the complex showed a high degree of complementarity and no severe steric clashes, suggesting a similar mode of binding of RNase A and angiogenin to RI. Most of the RI-binding residues of RNase A that are conserved or substituted conservatively in angiogenin appear to be able to form similar contacts. The exception is His8 (corresponding to Lys7 of RNase A), whose side-chain seems too short to directly interact with the C terminus of the inhibitor. The non-conservatively substituted residues 24, 38, 41, 86 and 88 of angiogenin may still be able to form favorable contacts with RI. In addition, several unique regions of angiogenin may contribute to the interaction; these include the N-terminal sequence (residues 1 and 5), residue 84, the sequence including residues 93 and 95, and the C-terminal sequence (residues 113, 117 and 121 in the C-terminal extension). Overall, angiogenin appears to be able to form even more contacts with RI than RNase A, consistent with its higher affinity. Mutagenesis and chemical modification studies agree with our observations, demonstrating roles in the interaction for Arg5 (Shapiro & Vallee, 1992), Lys40 (Lee & Vallee, 1989) and His114 (Lee & Vallee, 1989). In addition, the deletion of sequence 315 to 371 of human RI weakens the binding of angiogenin 100-fold more than the binding of RNase A (Lee & Vallee, 1990a), suggesting more extensive interactions of this region with angio-

RNase Inhibitor Protein Mechanism

genin than RNase A; this sequence most likely interacts with residues 41, 85 to 88 and 121 of angiogenin. As angiogenin, EDN must engage in numerous additional interactions to compensate for the loss of non-conserved RNase A-type contact points and achieve the Ki of 9 × 10−16 M (Shapiro & Vallee, 1991; Shapiro et al., 1995; Boix et al., 1996). Two properties of the RNase A-RI interaction help to explain how RI can bind several diverse RNases. First, the inhibitor is capable of small conformational adjustments to accommodate different ligands. Second, the unusually large surface area buried in the interface appears to compensate for a lower surface complementarity, retaining a very high affinity of binding. These same properties may be utilized in ligand binding of other proteins with leucine-rich repeats (Kobe & Deisenhofer, 1995a,b). Several amphibian ribonucleases are not inhibited by RI (Wu et al., 1993; Roth, 1962; Nitta et al., 1993). The 3-D structure of P-30 protein has been determined (Mosimann et al., 1994; entry 1ONC in the Protein Data Bank); in spite of having a structure very similar to RNase A, which would most likely not cause any severe steric clashes if P-30 bound to RI in a mode similar to that of RNase A, there does not appear to be sufficient complementarity between P-30 and RI to allow these two proteins to interact. Only three residues corresponding to RI-binding residues of RNase A are conserved, and only three are substituted by similar amino acids in this protein (Figure 6). Likewise, very few such residues are conserved in other amphibian RNases (Boix et al., 1996). As the amphibian RNases seem to have diverged enough to become resistant to inhibition by the mammalian RI, we expect these organisms to contain an amphibian RNase-specific inhibitor. Indeed, there is evidence for an amphibian analogue of RI (Nagano et al., 1976). Bovine seminal RNase (BS-RNase) is the only mammalian RNase A homologue not inhibited by RI in its native, covalently linked dimeric form. The carboxymethylated or DTT-induced monomeric forms, however, are inhibited by RI (Murthy & Sirdeshmukh, 1992). The structure of BS-RNase (Capasso et al., 1983; entry 1BSR in the Protein Data Bank) clearly shows that the presence of the second molecule in the dimer sterically prevents RI from binding. The resistance of BS-RNase to inhibition by RI may be the principal reason for its unusual biological properties such as antitumor, antispermatogenic and immunosuppressive activities (Kim et al., 1995). P-30 may be cytotoxic for similar reasons. Conclusions The crystal structure of the RNase A-RI complex shows that RNase A uses the unusual features of the structure of RI for its tight binding to this protein. RI exhibits the flexibility of its structure by conformationally adapting to the RNase A

RNase Inhibitor Protein Mechanism

molecule upon binding. The interface is large, and dominated by electrostatic interactions. RI inhibits RNase activity by sterically blocking the active site, but mimics the interaction between RNase and nucleotides to a very limited extent. RI is the only proteinaceous inhibitor of mammalian RNases thus far identified; by contrast, the number of protease inhibitors is similar to the number of proteases (Laskowski & Kato, 1980). Accordingly, RI can inhibit many diverse RNases. The high affinity of binding to diverse RNases is most likely achieved by conformationally adjusting to a particular RNase in the binding site, and by compensating a lower degree of complementarity with an unusually large contact area. The structure of RNase A-RI complex enriches our basic understanding of protein-protein recognition, and offers practical applications. It can serve as a model for other LRR-containing protein-ligand complexes, many of great biological importance (Kobe & Deisenhofer, 1994, 1995a). In addition, it can guide the design of inhibitors of angiogenesis and tumor growth (Polakowski et al., 1993).

Materials and Methods Refinement of free RI We previously described an atomic model of RI ˚ without solvent molecules that was refined at 2.5 A resolution using data with F > 2s(F) (Kobe & Deisenhofer, 1993a). We further refined (program package ˚ X-PLOR (Bru¨nger et al., 1987)) this model at 2.3 A resolution using data with F > s(F). We included the N-acetyl group (Hofsteenge et al., 1988) and solvent molecules in the model. The inclusion of the weak data and data of higher resolution noticeably improved the electron density maps. Completeness in the highest-resol˚ was 62.1%. B-factors ution shell between 2.4 and 2.3 A were restrained for bonded and angle-related atoms; ˚ 2 for target values for B-factor deviations were 1.5 A ˚ 2 for bonded side-chain bonded main-chain atoms, 2.0 A ˚ 2 for angle-related main-chain atoms, and atoms, 2.0 A ˚ 2 for angle-related side-chain atoms. The program 2.5 A package O (Jones et al., 1990) was used for inspection of atomic models and electron density maps and for model building. We selected peaks in the electron density map calculated with model-derived phases and coefficients =Fobs = − =Fcalc = as candidate water molecules with the program WATER (written by S. R. Sprang); only water ˚ of a polar atom and B-factors molecules within 3.5 A ˚ 2 were kept in the model. Several cycles of below 80 A positional refinement, refinement of B-factors, simulated annealing (Bru¨nger et al., 1987) and manual rebuilding resulted in an Rcryst of 20.4% and Rfree of 29.0% for data ˚ resolution (Rfree is equivalent to between 40 and 2.3 A Rcryst but calculated with the 10% of reflections omitted from the refinement process). Subsequently, the reflections thus far omitted from the refinement process for calculation of Rfree were included in further refinement; that caused further improvement of electron density maps. Refinement by simulated annealing and refinement of individual isotropic B-factors resulted in the final model of RI; refinement statistics are shown in Table 1. To evaluate Rfree after completion of refinement, we again randomly omitted 10% of the data and subjected the

1039 model to a round of simulated annealing with an initial temperature of 3000 K, followed by 120 cycles of positional refinement. This resulted in an Rcryst of 19.9% ˚ and Rfree of 26.1% for data between 40 and 2.3 A resolution and F > s(F). Contoured at 1s, the final electron density map showed continuous density in the backbone of the protein; it was poorly defined only for the side-chains of 33 (out of 456) residues; 1, 2, 6 to 14, 16, 17, 29, 38, 41, 59, 81, 84, 105, 130, 135, 136, 165, 168, 212, 393, 423, 436, 437, 443, 446 and 450. In these regions, the B-factors are high and reflect the disorder of the side-chains. All non-glycine residues except Cys7 have (F, C) angles within allowed hard-sphere limits. The mean coordinate ˚ on the basis of the Luzzati error is estimated to be 0.29 A ˚ based on the SIGMAA plot (Luzzati, 1952) and 0.48 A method (Read, 1986). The diffraction data collected from RI crystals soaked for two days in a solution containing 1.8 M lithium sulfate, 20 mM Hepes buffer (pH 7) and 20 mM dithiothreitol (269,249 measurements; 19,020 unique reflections; Rmerge = 9.99% for data with F > 3s(F); completeness = 68.4% for data with F > 2s(F) between 20 ˚ ) showed a substantial change in cell and 2.8 A ˚ , c = 84.1 A ˚ ) and a large mean parameters (a = 136.2 A fractional isomorphous difference (26.1% for data ˚ and F > 3s(F)) when compared with between 10 and 4 A ˚, the crystals grown in ammonium sulfate (a = 134.8 A ˚ ; Kobe & Deisenhofer, 1993a). To assess the c = 83.6 A differences between these two crystal forms, we refined the final model of RI (without the solvent molecules) ˚ and using the lithium sulfate data between 40 and 2.9 A F > s(F) by positional refinement, refinement of individual B-factors and simulated annealing using a bulk solvent correction (Bru¨nger, 1992). This resulted in Rcryst = 18.0% and Rfree = 30.0% (10% of reflections omitted for the calculation of Rfree ). In the final cycle of refinement, ˚ with F > s(F) all 13,899 reflections between 40 and 2.8 A were included in the refinement, resulting in the final lithium sulfate model (Rcryst = 19.8%, r.m.s. bond ˚ , r.m.s. bond angles = 2.0°, mean coordilengths = 0.014 A ˚ based on the Luzzati plot (Luzzati, nate errors are 0.32 A ˚ based on the SIGMAA method (Read, 1952) and 0.49 A 1986)). The average r.m.s. deviation of the Ca atoms after superposition with the high-resolution model of RI is ˚ , above the estimated coordinate errors of both 0.64 A models. Detailed comparison of the structures of RI and the crystal packing in the two crystal forms are described elsewhere (Kobe, 1994). Structure determination of the RNase A-RI complex The crystallization and the structure determination of the RNase A-RI complex have been described (Kobe et al., 1994; Kobe & Deisenhofer, 1995b). Therefore, we only briefly mention here the methods used, certain interesting details and the quality of the final model. RI was purified from porcine liver (Kobe & Deisenhofer, 1993b); RNase A from bovine pancreas was purchased from Sigma. The crystals have the symmetry of the tetragonal ˚ and c = 86.7 A ˚ . Because space group I4 with a = 133.3 A free RI and the RNase A-RI complex crystallized in a unit cell of the same symmetry and similar cell dimensions, we expected a similar packing of the inhibitor molecules in the two crystals. However, using data between 10 and ˚ resolution and F > 3s(F), the RI model had an Rcryst 4A of 53.1% in its original position from the crystals of free RI, and Rcryst did not drop after rigid body refinement. Therefore, the real-space Patterson search method in

1040 Eulerian space (Huber, 1985) was used for cross-rotation against the diffraction data of the RNase A-RI complex of the atomic models of porcine RI (entry 1BNH in the Protein Data Bank) and ribonuclease A (Wlodawer et al., 1988; entry 7RSA in the Protein Data Bank). The model of RI was arbitrarily rotated (u1 = −17°, u2 = −27°, u1 = −37°) before cross-rotation functions were calculated. ˚ resolution and a radius of 20 A ˚ Data between 15 and 4 A were used for rotation searches of the RI model. The top 150 maxima of the rotation function were filtered by Patterson correlation (PC) refinement (Bru¨nger, 1990) consisting of ten steps of conjugate gradient minimization (Powell, 1977) of the orientation of the molecule. One outstanding peak was found with a correlation coefficient of 0.124 (signal to noise ratio 1.4), corresponding to a rotation of u1 = 59.5°, u2 = 32.5°, u1 = 347.5° with a height of 3.6 s in the cross-rotation function; the resulting rotation after PC refinement was u1 = 52.9°, u2 = 32.2°, u1 = 352.1°. A translation function search of the RI model in this orientation yielded the top solution of 17.3 s at (0.118, 0, 0). RI in this position produced an Rcryst ˚ resolution and F > 2s(F). of 49.1% between 10 and 3.5 A Cross-rotation and translation searches in various resolution ranges using RNase A as the search model did not yield a solution. Therefore, 2=Fobs = − =Fcalc = and =Fobs = − =Fcalc = electron density maps phased by only the properly positioned RI molecule were inspected. Electron density resembling the RNase A molecule was clearly visible, and the model of RNase A was positioned into this electron density on the graphics display. The resulting model containing both RI and RNase A had an ˚ resolution Rcryst of 49.0% using data between 10 and 3.5 A and F > 2s(F). The inhibitor and the enzyme were first refined as rigid bodies resulting in an Rcryst of 42.3% and Rfree of 43.0% ˚ resolution and F > 2s(F) (10% of between 10 and 3.5 A data randomly omitted for the calculation of Rfree ). Further refinement of the inhibitor treating initially two halves, then four quarters and finally eight eighths of the sequence as rigid bodies resulted in an Rcryst of 33.3% and ˚ resolution Rfree of 34.2% for data between 10 and 3.5 A and F > 2s(F). Several cycles of positional refinement (Powell, 1977), refinement of individual isotropic B-factors, manual rebuilding, and addition of solvent molecules, resulted in an Rcryst of 19.4% and Rfree of 28.6% ˚ resolution with a bulk using data between 40 and 2.5 A solvent correction and F > s(F). Although the inclusion of the low-resolution data and the weak reflections increased the R-factors, there was a substantial improvement of the electron density maps. B-factors were restrained and water molecules were selected as in the refinement of free RI. Refinement by simulated annealing (Bru¨nger et al., 1987) caused an increase in Rfree and was therefore abandoned. In the final stage of refinement we included the data thus far omitted from refinement for calculation of Rfree , which resulted in a further improvement of the electron density maps. The completeness in the highest-resolution shell between 2.61 ˚ was 31.4%. Final refinement statistics are and 2.5 A shown in Table 1. To evaluate Rfree after the completion of refinement, 10% of data were again randomly omitted and the model was subjected to a round of simulated annealing with an initial temperature of 3000 K, followed by 120 cycles of positional refinement. This resulted in an Rcryst of 19.0% and Rfree of 26.1% for data between 40 and ˚ resolution and F > s(F). 2.5 A Despite the high average B-factors of the molecules in the crystals, the final electron density was of high quality. Contoured at 1s, the electron density contained no region

RNase Inhibitor Protein Mechanism

of discontinuous density in the backbone of the protein; it was poorly defined only for the side-chains of 25 residues; 9, 23, 36, 41, 48, 62, 73, 81, 105, 126, 136, 155, 168, 187, 212, 213, 217, 252, 269, 273, 336, 342, 347, 436 and 453 of RI; and seven residues; 1, 7, 9, 37, 98, 113 and 121 of RNase A. In these regions, the B-factors are high and reflect the disorder of the side-chains. All non-glycine residues have (F, C) angles within allowed hard-sphere limits. The mean coordinate error is estimated to be ˚ based on the Luzzati plot (Luzzati, 1952) and 0.32 A ˚ based on the SIGMAA method (Read, 1986). 0.57 A Structure analyses Structures were analyzed on a graphics workstation using program packages O (Jones et al., 1990) and INSIGHT (Biosym). Interactions were analyzed with the program package X-PLOR (Bru¨nger et al., 1987) and the program CONTACT from the CCP4 program package (CCP4, 1994). Accessible surface areas were calculated with the program SURFACE from the CCP4 program package (CCP4, 1994); group radii were used and hydrogen atoms were excluded from the calculations (Chothia, 1975). Surfaces were graphically analyzed and molecular volumes were calculated with the program GRASP (Nicholls et al., 1991). Surface complementarity was analyzed by the method of (Lawrence & Colman, 1993). Cavities were analyzed with the program VOIDOO (Kleywegt & Jones, 1994); only cavities larger ˚ 3 were considered. Secondary structure was than 1.25 A assigned with the program DSSP (Kabsch & Sander, 1983). The energy minimization of the RI-angiogenin model was performed with the program X-PLOR (Bru¨nger, 1992); both molecules were treated as rigid bodies and minimized for 100 cycles. The coordinates and the structure factors of the high-resolution structures of RI and RNase A-RI complex have been deposited in the Brookhaven Protein Data Bank (Bernstein et al., 1977; ID codes 2BNH, R2BNHSF, 1DFJ and R1DFJSF, respectively); until processed, they can be obtained from the authors upon request.

Acknowledgements We thank M. Lawrence for the surface complementarity program and the referees for useful suggestions. B.K. on leave from Jozef Stefan Institute, Department of Biochemistry and Molecular Biology, Jamova 39, 1000 Ljubljana, Slovenia.

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RNase Inhibitor Protein Mechanism

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Edited by R. Huber (Received 5 July 1996; received in revised form 23 September 1996; accepted 27 September 1996)

Supplementary material, comprising one Table, is available from JMB Online.