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Structural basis for recognition of 2′,5′-linked oligoadenylates by human ribonuclease L

2004; Springer Nature; Volume: 23; Issue: 20 Linguagem: Inglês

10.1038/sj.emboj.7600420

1460-2075

Nobutada Tanaka, Masayuki Nakanishi, Yoshio Kusakabe, Yoshikuni Goto, Yukio Kitade, Kazuo Nakamura,

Viral gastroenteritis research and epidemiology

Article23 September 2004free access Structural basis for recognition of 2′,5′-linked oligoadenylates by human ribonuclease L Nobutada Tanaka Corresponding Author Nobutada Tanaka School of Pharmaceutical Sciences, Showa University, Hatanodai, Shinagawa-ku, Tokyo, Japan Search for more papers by this author Masayuki Nakanishi Masayuki Nakanishi Department of Biomolecular Science, Faculty of Engineering, Gifu University, Yanagido, Gifu, Japan Search for more papers by this author Yoshio Kusakabe Yoshio Kusakabe School of Pharmaceutical Sciences, Showa University, Hatanodai, Shinagawa-ku, Tokyo, Japan Search for more papers by this author Yoshikuni Goto Yoshikuni Goto Department of Biomolecular Science, Faculty of Engineering, Gifu University, Yanagido, Gifu, Japan Search for more papers by this author Yukio Kitade Yukio Kitade Department of Biomolecular Science, Faculty of Engineering, Gifu University, Yanagido, Gifu, Japan Search for more papers by this author Kazuo T Nakamura Kazuo T Nakamura School of Pharmaceutical Sciences, Showa University, Hatanodai, Shinagawa-ku, Tokyo, Japan Search for more papers by this author Nobutada Tanaka Corresponding Author Nobutada Tanaka School of Pharmaceutical Sciences, Showa University, Hatanodai, Shinagawa-ku, Tokyo, Japan Search for more papers by this author Masayuki Nakanishi Masayuki Nakanishi Department of Biomolecular Science, Faculty of Engineering, Gifu University, Yanagido, Gifu, Japan Search for more papers by this author Yoshio Kusakabe Yoshio Kusakabe School of Pharmaceutical Sciences, Showa University, Hatanodai, Shinagawa-ku, Tokyo, Japan Search for more papers by this author Yoshikuni Goto Yoshikuni Goto Department of Biomolecular Science, Faculty of Engineering, Gifu University, Yanagido, Gifu, Japan Search for more papers by this author Yukio Kitade Yukio Kitade Department of Biomolecular Science, Faculty of Engineering, Gifu University, Yanagido, Gifu, Japan Search for more papers by this author Kazuo T Nakamura Kazuo T Nakamura School of Pharmaceutical Sciences, Showa University, Hatanodai, Shinagawa-ku, Tokyo, Japan Search for more papers by this author Author Information Nobutada Tanaka 1, Masayuki Nakanishi2, Yoshio Kusakabe1, Yoshikuni Goto2, Yukio Kitade2 and Kazuo T Nakamura1 1School of Pharmaceutical Sciences, Showa University, Hatanodai, Shinagawa-ku, Tokyo, Japan 2Department of Biomolecular Science, Faculty of Engineering, Gifu University, Yanagido, Gifu, Japan *Corresponding author. School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan. Tel.: +81 3 3784 8200; Fax: +81 3 3782 5635; E-mail: [email protected] The EMBO Journal (2004)23:3929-3938https://doi.org/10.1038/sj.emboj.7600420 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info An interferon-induced endoribonuclease, ribonuclease L (RNase L), is implicated in both the molecular mechanism of action of interferon and the fundamental control of RNA stability in mammalian cells. RNase L is catalytically active only after binding to an unusual activator molecule containing a 5′-phosphorylated 2′,5′-linked oligoadenylate (2-5A), in the N-terminal half. Here, we report the crystal structure of the N-terminal ankyrin repeat domain (ANK) of human RNase L complexed with the activator 2-5A. This is the first structural view of an ankyrin repeat structure directly interacting with a nucleic acid, rather than with a protein. The ANK domain folds into eight ankyrin repeat elements and forms an extended curved structure with a concave surface. The 2-5A molecule is accommodated at a concave site and directly interacts with ankyrin repeats 2–4. Interestingly, two structurally equivalent 2-5A binding motifs are found at repeats 2 and 4. The structural basis for 2-5A recognition by ANK is essential for designing stable 2-5As with a high likelihood of activating RNase L. Introduction In mammals, viral infections initiate an innate immune response predominantly mediated by type I interferons (IFNs) (Sen, 2001). Type I IFNs regulate the transcription of a number of genes that inhibit or block viral replication by diverse mechanisms (Stark et al, 1998). There are three well-established antiviral pathways for IFN action: these three pathways are associated with the double-stranded RNA (dsRNA)-dependent protein kinase PKR (Williams, 1995), the Mx proteins (Pavlovic and Staeheli, 1991), and the 5′-triphosphorylated, 2′,5′-phosphodiester-linked oligoadenylate (2-5A) system (Player and Torrence, 1998). In the 2-5A system, treatment of cells with IFN activates genes encoding several 2′,5′-linked oligoadenylate synthetases (OASs) (Chebath et al, 1987) and a single gene encoding ribonuclease L (RNase L) (Zhou et al, 1993). The OASs are activated by binding to dsRNA (Hovanessian et al, 1977), a frequent byproduct of viral infection. The activated OASs generate 2-5A from ATP. The crystal structure of porcine OAS1 was recently reported (Hartmann et al, 2003). The mechanisms of its activation induced by dsRNA and its synthesis of 2-5A were discussed based on its structure and structure-based mutagenesis studies, although the structure contained neither activator nor substrate analogs. RNase L is known to be activated by binding to 2-5A, changing from an inactive monomer to a catalytically active homodimer (Dong and Silverman, 1995). The activated RNase L cleaves RNA containing dyads of UU, UA, AU, AA, and UG (Floyd-Smith et al, 1981; Wreschner et al, 1981). The RNA degradation inhibits protein synthesis and thus inhibits viral replication. RNase L is an unusual ribonuclease because it requires the activator molecule 2-5A to catalyze the hydrolysis of single-stranded RNA. 2-5A is itself very unusual, consisting of a type of oligoadenylates with 2′,5′ internucleotide linkages, in contrast to the typical 3′,5′ linkages found in RNA and DNA. 2-5A is unstable in cells due to the activities of phosphodiesterases and phosphatases. Early studies revealed the requirement of at least two 5′-phosphoryl groups and at least three 2′,5′-linked adenylyl residues for the efficient activation of murine RNase L (Kerr and Brown, 1978). Thereafter, it was shown that only a single 5′-phosphoryl group on the trimer of 2′,5′-linked oligoadenylate was required for the full activation of human RNase L (Haugh et al, 1983; Yoshimura et al, 2002). The human form of RNase L is a 741-amino-acid protein with a molecular mass of 83 543 Da (Zhou et al, 1993). RNase L consists of three domains, namely the N-terminal ankyrin repeat domain, the protein kinase homology domain, and the C-terminal ribonuclease domain. The N-terminal ankyrin repeat domain, a region containing nine ankyrin-like macromolecular recognition repeats (the ninth ankyrin repeat is incomplete), is responsible for 2-5A binding, and the C-terminal domain is responsible for catalytic activity (Dong and Silverman, 1997; Nakanishi et al, 2004). The binding of 2-5A to RNase L is thought to induce a conformation change in the enzyme or result in the unmasking of an interaction domain, permitting dimerization and activation of RNase L. Recently, the enzyme was proposed as a candidate risk factor for hereditary prostate cancer (Casey et al, 2002; Xiang et al, 2003). Among the naturally occurring mutants of RNase L that have been examined, only the Arg462Gln variant showed low RNase activity, and this variant was also shown to be significantly associated with prostate cancer. Therefore, elucidation of all of the amino-acid residues that might influence RNase L activity (i.e., 2-5A binding, dimerization, and catalysis) remains necessary. Here, we present the crystal structure at 1.8 Å resolution of the N-terminal ankyrin repeat domain of human RNase L complexed with 2-5A. To our knowledge, this is the first structural view of an ankyrin repeat structure directly interacting with a nucleic acid, rather than with a protein. The crystal structure of the ANK/2-5A complex clearly shows that the bound 2-5A molecule directly interacts with ankyrin repeats 2–4. We have re-evaluated the structure–function relationship studies of RNase L and 2-5A in terms of the present crystal structure analysis. The structural basis for 2-5A recognition by ANK is essential for designing stable 2-5As with a high likelihood of activating RNase L. Results Structure determination The crystallization trials have to date not successfully obtained the crystals of full-length human RNase L, as only a minute amount of the full-length recombinant human RNase L has been obtainable; however, crystals of the N-terminal ankyrin repeat domain (ANK) of human RNase L (Figure 1A) were successfully obtained. Addition of 2-5A to the ANK solution was essential for obtaining crystals. For this study, a 2-5A trimer with 5′-monophosphate (p5′(A2′p5′)2A) was used for crystallization (Figure 1B). We determined the crystal structure of the ANK/2-5A complex by the molecular replacement (MR) method using the coordinate of a 'consensus ankyrin repeat protein' (Kohl et al, 2003) (PDB code: 1MJ0) as the template for constructing a search model, and we refined the resulting model to an R-factor of 0.202 (Rfree of 0.230) at 1.8 Å resolution. The final model consisted of residues 21–305, 220 water molecules, and one 2-5A molecule. The refinement statistics are summarized in Table I. Figure 1.Crystal structure of ANK complexed with 2-5A. (A) Structural and functional domains of RNase L. Ankyrin repeats are shown starting with blue at repeat 1 and ending with red at repeat 8. (B) Structure of the predominant trimeric species of 2-5A ((pp)p(A2′p5′)2A) (Kerr and Brown, 1978). (C, D) Surface (top) and ribbon (bottom) representations of the ANK/2-5A complex. Ankyrin repeats (R1–R8) are shown as in (A). The bound 2-5A molecule is shown as a ball-and-stick model. The view in (D) was obtained by rotating the view in (C) by 90°. Download figure Download PowerPoint Table 1. Data collection and refinement statistics of ANK Data collection statistics X-ray source PF-AR NW12 Wavelength (Å) 0.978 Resolution range (outer shell) (Å) 40–1.8 (1.9–1.8) Observed reflections (no sigma cutoff) 154 126 Unique reflections 35 922 Multiplicity 4.3 (3.7) Mean 〈I/σ(I)〉 5.5 (2.1) B-factor (Wilson plot) (Å2) 27.3 Rsym (%) 8.4 (33.8) Completeness (%) 99.7 (99.5) Refinement statistics Resolution range (Å) 40–1.8 No. of reflections Working set 34 084 Test set 1791 R-factor 0.202 Free R-factor 0.230 No. of protein atomsa (average B-factors (Å2)) 2184 (28.3) No. of water molecules (average B-factors (Å2)) 220 (37.0) No. of 2-5A atoms (average B-factors (Å2)) 67 (23.5) R.m.s. deviations Bond distances (Å) 0.008 Bond angles (deg) 1.119 B-factors related by main-chain bonds (Å2) 0.669 B-factors related by side-chain bonds (Å2) 1.825 Ramachandran plot Most favored (%) 92.6 Additional allowed (%) 7.4 aHis tag, N-terminal residues (1–20), and C-terminal residues (306–333) are disordered. Overall fold of ANK ANK folds into eight ankyrin repeat elements and forms an extended curved structure with a groove running across the long concave surface (Figure 1C and D). Previous primary structure analysis suggested that RNase L had nine ankyrin repeats, but the ninth ankyrin repeat is incomplete (Hassel et al, 1993). However, this prediction for RNase L differs from the present crystal structure of ANK, which consists of eight ankyrin repeats (R1–R8 in Figure 1C and D). Residues 306–333, corresponding to the incomplete ninth repeat (Hassel et al, 1993), are disordered. As in other ankyrin repeat proteins (Sedwick and Smerdon, 1999), each repeat is formed by ∼33 amino-acid residues and consists of pairs of antiparallel α-helices stacked side by side and which are connected by a series of intervening β-hairpin motifs. In general, the structure is shaped similar to a cupped hand (Jacobs and Harrison, 1998). There is a noticeable curvature across the 'palm', such that the surface created by the β-hairpins (fingers) and the α1 'inner' helices is concave, whereas that formed by the α2 'outer' helices, that is, the back of the cupped hand, is convex (Figure 1D). The 2-5A molecule fits in the concavity and directly interacts with ankyrin repeats 2–4. Ankyrin repeats of RNase L A structure-based sequence alignment of the eight ankyrin repeats of RNase L is shown in Figure 2. Definition of the ankyrin repeat consensus is based on the exon boundaries of the ankyrin gene (Lux et al, 1990). This definition separates the two β-strands of the β-hairpin structure and incorporates the most commonly occurring nonconserved elements within the repeating unit (Sedwick and Smerdon, 1999); however, the insertion helix (αI: residues 159–164) of ANK is incorporated between R4 and R5 (Figures 1C, D, and 2). We therefore consider the ankyrin repeat motif of RNase L to be defined as a strand–helix–loop–helix–loop–strand (βααβ) structure. Figure 2.Structure-based sequence alignment of the eight ankyrin repeats (R1–R8) of human, mouse, and rat RNase Ls. Secondary structural elements are shown on top and are colored as in Figure 1. The 2-5A binding motifs, equivalently found in repeats 2 and 4, are boxed in pink. The two GKT motifs are boxed in gray. Download figure Download PowerPoint Residue Gly(2) in β1 is invariant across all repeats (Figure 2), except for R1. Hereafter, the consensus ankyrin repeat numbering will be shown in italics and parentheses. This glycine residue allows a sharp turn within the β-hairpin structure. Since R1 lacks β1, Gly(2) is not conserved in R1. Highly conserved hydrophobic residues (Leu(6) and Ala(9) from the inner helix α1, and Leu(21) and Leu(22) from the outer helix α2) are involved in packing interactions within and between repeats, giving rise to a continuous hydrophobic core. As previously noted (Jacobs and Harrison, 1998), the inner helix α1 contains hydrophobic residues (Ala(9) and Ala/Val(10)) smaller than those in the outer helix α2 (Leu(21) and Leu(22)), causing the stack of ankyrin repeats to curve toward the inner helix α1. The signature Thr(4)–Ala/Pro(5)–Leu(6)–X(7) motif characteristic of ankyrin repeats forms a tight turn that initiates the inner helix α1. Because the inner helix α1 starts at four residues upstream from Thr(4) in R1, Thr(4) is not conserved in R1. Residues Gly(25) and Ala(26) allow a sharp turn at the end of the outer helix α2. The side chain of the highly conserved polar residue Asn(29) is involved in hydrogen bonds with the carbonyl oxygen atom of Gly(25) and the imino nitrogen atom of X(27) from the next repeat. Several insertions are found within and between repeats. Met191 and Glu262 are inserted after the outer helix α2 within R5 and R7, respectively. Each of these insertions allows for a substitution of the Gly(25)–Ala(26) residues in R5 or R7, which enable a sharp turn at the end of α2. Four residues (Ser213–Asp214–Asp215–Ser216) are inserted after the inner helix α1 within R6. The insertion of two acidic residues would be expected to affect the electrostatic properties of the surface. Glu57 is inserted between R1 and R2. A total of 10 residues (Thr157 to Lys166), including the insertion helix αI (residues 159–164), are inserted between R4 and R5. These insertions also exist for mouse and rat RNase Ls. 2-5A binding mode ANK is the first representative of an ankyrin repeat protein for which the structure has been solved in the form of a nucleotide (rather than a protein) complex. Here, the electron density, shown at 1.8 Å resolution, corresponding to the bound 2-5A molecule is very well defined (Figure 3A). Thus, we were able to unequivocally determine the conformations of 2-5A and the surrounding amino-acid residues, as well as the location of water molecules. As shown in Figure 1C and D, only one 2-5A molecule is bound to the concavity of ANK. Since ANK is responsible for 2-5A binding in RNase L (Dong and Silverman, 1997), the present crystal structure is consistent with previous observations demonstrating that the dimerization of RNase L requires the binding of one 2-5A molecule per RNase L monomer and that the enzymatic activity of RNase L is also maximized at a 1:1 2-5A:RNase L stoichiometry (Cole et al, 1996). As shown in Figure 3A, the bound 2-5A molecule adopts an extended conformation in which the first and second adenine rings (Ade1 and Ade2, respectively) are in an anti conformation, whereas the third adenine ring (Ade3) is in a syn conformation. The first and second ribose rings have 3E (C3′-endo) puckering, whereas the third ribose has 4E (C4′-exo) puckering (IUPAC-IUB, 1983). The 2-5A molecule is accommodated in the concavity and directly interacts with ankyrin repeats 2–4 (Figure 1C and D). In the present crystal structure analysis, we used a 2-5A trimer with 5′-monophosphate (p5′(A2′p5′)2A) for crystallization (Figure 1B). The 2-5A molecule is thus considered to be a 2′,5′-linked trimer of 5′-AMP. Figure 3.Recognition of 2-5A by ANK. (A) Stereodiagram showing the mode of 2-5A binding to ANK. The carbon atoms of ANK are colored as in Figure 1, and those of the bound 2-5A molecule are shown in white. Possible hydrogen bonds or salt bridges are indicated by dashed lines, and distances (in Å) are given. The refined model is superimposed on the weighted 2∣Fo∣–∣Fc∣ map (1.5σ, yellow) and the ∣Fo∣–∣Fc∣ omit map of 2-5A (4.5σ, orange), calculated at 1.8 Å resolution. Bound water molecules are shown as spheres (cyan). (B, C) Recognition of the first and third AMP moieties of 2-5A by repeats 4 and 2, respectively, of ANK. Download figure Download PowerPoint The first AMP moiety of the 2-5A directly interacts with the fourth repeat (R4) of ANK (Figure 3A and B). The 5′-phosphate group of the first AMP (Phos1) forms bifurcated salt bridges with the side chain of Arg155. The side chain of Arg155 is fixed by bifurcated salt bridges with the side chain of Asp174 (Figure 3A). The adenine ring of the first AMP (Ade1) is stacked between the side chain of Phe126 and the adenine ring of the second AMP (Ade2), and is fixed by bifurcated hydrogen bonds with the side chain of Glu131, that is, OE1(Glu131)—N6(Ade1) and OE2(Glu131)—N1(Ade1). The side chain of Phe126 also stacks with the guanidino group of the side chain of Arg155, forming a quadruplex (Arg155–Phe126–Ade1–Ade2) of stacking interactions (Figure 3B). The second AMP moiety of 2-5A interacts only slightly with ANK. The 5′-phosphate group of the second AMP (Phos2) is exposed to solvent, and no direct interactions are found between Phos2 and the surface of ANK. The adenine ring of the second AMP (Ade2) is stacked with Ade1 as described above, and is fixed by a single hydrogen bond with the side chain of Tyr135, that is, OH(Tyr135)—N1(Ade2). The 3′-OH group of the second AMP is involved in a hydrogen bond network and is fixed on the protein surface via water molecules. The second AMP appears to be rather weakly fixed on the ANK surface relative to the two ends of the 2-5A molecule. The third AMP moiety of 2-5A directly interacts with the second repeat (R2) of ANK (Figure 3A and C). The 5′-phosphate group of the third AMP (Phos3) forms a salt bridge with the side chain of Lys89. The adenine ring of the third AMP (Ade3) is stacked with the side chain of Trp60, and is fixed by a hydrogen bond network involving the side chains of Gln68 (OE1(Gln68)—N6(Ade3) and NE2(Gln68)—N1(Ade3)) and Asn65 (OD1(Asn65)—N6(Ade3)), as well as a water molecule (O(Wat)—N7(Ade3) and O(Wat)—ND2(Asn65)). The side chain of Trp60 also stacks with the CD–CE–NZ bonds of the side chain of Lys89, forming a triplex (Lys89–Trp60–Ade3) of stacking interactions (Figure 3C). Unlike Ade1 and Ade2, Ade3 adopts a syn conformation. The 2′- and 3′-OH groups of the third AMP do not interact with either the protein surface or with water molecules. Interestingly, the 2-5A binding residues in the R4 and R2 of ANK are located at the structurally equivalent position of the ankyrin repeat (Figure 2, boxed in pink) and these residues play a functionally equivalent role. The side chains of Arg155 in R4 and Lys89 in R2 form salt bridges with Phos1 and Phos3, respectively (Figure 3B and C). The side chains of Phe126 in R4 and Trp60 in R2 stack with Ade1 and Ade3, respectively. Furthermore, a quadruplex (Arg155–Phe126–Ade1–Ade2) and a triplex (Lys89–Trp60–Ade3) of stacking interactions are observed at R4 and R2, respectively. The side chains of Glu131 in R4 and Asn65 in R2 form hydrogen bonds with Ade1 and Ade3, respectively. It should be noted here that these 2-5A binding residues are completely conserved among human, mouse, and rat RNase Ls (Figure 2). Two insertions are found around the 2-5A binding region of ANK. One of these insertions is Glu57, which is inserted between R1 and R2. This insertion extends a finger between R1 and R2, which constitutes a wall of the third AMP binding site (Figure 1D). The other insertion is the insertion helix αI (residues 159–164), which is inserted between R4 and R5. This helix constitutes the bottom of the first AMP binding site (Figure 1D). The eight ankyrin repeats of ANK are kinked at the middle As shown in Figure 1C and D, the eight ankyrin repeats of ANK adopt an extremely curved structure. To obtain insight into the curved structure of ANK, we superposed repeats 1 and 2 of other ankyrin repeat proteins on those of ANK (Figure 4). In general, a longer ankyrin repeat protein has a more concave structure than a shorter ankyrin repeat protein does; for example, the structure of ankyrin (Michaely et al, 2002), which consists of 12 ankyrin repeats, is more concave than those of Bcl-3 (Michel et al, 2001) and gankyrin (Krzywda et al, 2004), which each consist of seven ankyrin repeats (Figure 4). However, it is of note that the degree of curvature across the eight ankyrin repeats of ANK is more remarkable than that of the 12 ankyrin repeats of ankyrin in two directions (Figure 4). Figure 4.The kinked structure of ANK. (A, B) Superposition of repeats 1 and 2 of human gankyrin (cyan, PDB ID: 1UOH), human Bcl-3 (yellow, 1K1A), and human ankyrin (green, 1N11) on those of ANK (magenta, 1WDY). The views in (A) and (B) are the same as those shown in Figure 1C and D, respectively. Download figure Download PowerPoint As shown in Figure 4, the eight ankyrin repeats of ANK are clearly kinked at R4 and R5 (15 and 11° in Figure 4A and B, respectively, as compared with ankyrin). A total of 10 residues (Thr157 to Lys166), including the insertion helix αI (residues 159–164), are inserted between R4 and R5 (Figure 2). This insertion of a longer polypeptide chain enables more bending. In addition, the more bulky side chains of Phe143 and Tyr145, instead of Leu residues, are found in the outer helix α2 of R4 (Figure 2). These arrangements appear to result in the wider separation of the outer helices α2 of R3, R4, and R5. The relationship between the higher degree of curvature of ANK and the 2-5A binding is still unclear, as we have not yet obtained the structure of ANK in the absence of 2-5A. However, the results of time-resolved fluorescence measurements and protease protection assays have indicated that the conformation of ANK does indeed change upon 2-5A binding (M Nakanishi et al, unpublished data). Therefore, we suggest here that the higher degree of curvature of ANK is due to 2-5A binding. Discussion 2-5A binding domain Extensive efforts employing various techniques have been directed toward functional analyses of RNase L. As summarized in Figure 5A, we can re-evaluate these results in terms of the newly established crystal structure of the ANK/2-5A complex. The results of the N-terminal truncations of RNase L (Dong and Silverman, 1997) are completely consistent with the present crystal structure analysis showing that the 2-5A molecule directly interacts with ankyrin repeats 2–4 (Figure 1C and D). Truncation of the 23 N-terminal amino acids preceding the first ankyrin repeat (R1), which are disordered in the present crystal structure and are not involved in interactions with 2-5A, does not result in the loss of 2-5A binding activity (NΔN(24–741)). Other N-terminal truncations, that is, mutants NΔR1(57–741), NΔR1-2(91–741), NΔR1-3(124–741), and NΔR1-6(238–741), lacking 1, 2, 3, and 6 ankyrin repeats, respectively, do show a loss of 2-5A binding activity. Although R1 is not directly involved in 2-5A binding, it constitutes a wall of the third AMP moiety of the 2-5A binding site (Figure 1D). Moreover, results showing the C-terminal truncation of RNase L (CΔC(1–335)) (Dong and Silverman, 1997) are also consistent with the present crystal structure analysis demonstrating that the N-terminal ankyrin repeat domain of RNase L is sufficient for 2-5A binding activity. Figure 5.Re-evaluation of previous structure–function relationship studies of ANK and 2-5As in terms of the present crystal structure analysis. (A) Biochemical studies of ANK truncation mutants (Dong and Silverman, 1997). (B) The two GKT motifs in repeats 7 and 8. The side chains of Lys240 and Lys274 are involved in an intrarepeat salt bridge (dashed lines) with the side chains of Glu248 and Glu282, respectively, rather than in 2-5A binding. (C) Schematic representation of the 2-5A binding mode. The side chains of ANK are colored as in Figure 1. Hydrogen bonds or salt bridges and a hydrogen bond network of water molecules are indicated by red and gray dashed lines, respectively, and distances (in Å) are given. Aromatic residues, Trp60 and Phe126, involved in the stacking interactions with 2-5A (seeFigure 3B and C) have been omitted for clarity. Water molecules are shown as 'Ow'. The [n] indicates important interactions for oligonucleotide [n] in (D). (D) Chemical studies of 2-5A and its analogs (Player and Torrence, 1998). The relative activities of 2-5A analogs, as compared with canonical 2-5A [4], for RNase L activation are shown as '+++' (1/2∼), '++' (1/20∼1/2), '+' (1/200∼1/20), and '−' (∼1/200). Download figure Download PowerPoint Before the present crystal structure analysis was conducted, two GKT motifs, Gly239–Lys240–Thr241 in R7 and Gly273–Lys274–Thr275 in R8 (Figure 2, boxed in gray), had been postulated as the 2-5A binding motifs (Zhou et al, 1993; Diaz-Guerra et al, 1999). Substitution of the two lysines with asparagines greatly reduced the affinity for 2-5A (Zhou et al, 1993). Similarly, one mutant (NΔN/CΔR7-9C(24–237)) (Dong and Silverman, 1997), which is truncated at both termini and lacks R7–R9, with which Lys240 and Lys274 are involved, was shown to lose 2-5A binding activity (Figure 5A). These observations, together with the lack of structural information about ANK at the time the observations were made, led to the assumption that the two GKT motifs were the 2-5A binding motifs. However, the present crystal structure has clearly revealed that the side chains of Lys240 and Lys274 are involved in an intrarepeat salt bridge with the side chains of Glu248 and Glu282, respectively (Figure 5B), rather than in 2-5A binding. Although the structural role of the two GKT motifs remains unclear without additional structural studies, one possible explanation for their role would be that the salt bridges in R7 and R8 contribute to maintaining the folding of the ankyrin repeat domain, and hence to its integrity. Consequently, structural perturbation at R7 and R8 greatly diminishes the 2-5A binding ability of RNase L, even though these regions are not directly involved in 2-5A binding. Structure–activity relationship of 2-5A and its analogs Several chemical approaches have produced a considerable amount of information about the atoms or groups of atoms in 2-5A that interact with RNase L. Similar to the re-evaluation of previous functional studies of RNase L described above, we can now re-evaluate previous studies of the structure–activity relationship of 2-5A and its analogs (Torrence et al, 1994; Player and Torrence, 1998) in terms of the present crystal structure (Figure 5C and D). The most intriguing feature of the 2-5A molecule is the presence of the 2′,5′-linked phosphodiester bonds (Figures 1B and 5C). Lesiak et al (1983) showed that replacing either of the 2′,5′-linkages in a 2-5A trimer, [1] and [2] in Figure 5D, decreased binding by 1 and 2 orders of magnitude; however, substituting both linkages [3] reduced binding drastically. The present structure thus accounts for the structural significance of the two 2′,5′-linkages, based on the assumption that the interactions between R4 and R2 of ANK and the first and third AMP, respectively, of 2-5A (Figures 3B, C, and 5C) must be maintained. Replacing the first 2′,5′-linkage with a 3′,5′-linkage [1] would drastically shift the adenosine moiety of the second AMP and the phosphate of the third AMP outwardly, and then the stacking interaction between Ade1 and Ade2 and the salt bridge between Phos3 and Lys89 would be weakened. Replacing the second 2′,5′-linkage [2] would shift the adenosine moiety of the second AMP inward (or drastically shift Phos3 outward), resulting in a weakening of Ade1–Ade2 stacking and possibly the steric hindrance of Ade2 at the protein surface (or the Phos3–Lys89 salt bridge might be weakened). Replacing both the 2′,5′-linkages [3] additively was found to weaken both the Ade1–Ade2 stacking and the Phos3–Lys89 salt bridge. Since the 2′,5′-linkages orient backbone-inwards toward the bases (Figure 1B), rather than away from the bases as in 3′,5′-linkages, 2-5A with both the 2′,5′-linkages enabled all of the essentia