Structure of IRF-3 Bound to the PRDIII-I Regulatory Element of the Human Interferon-β Enhancer

Molecular Cell

Article Structure of IRF-3 Bound to the PRDIII-I Regulatory Element of the Human Interferon-b Enhancer Carlos R. Escalante,1 Estanislao Nistal-Villa´n,2,3 Leyi Shen,1 Adolfo Garcı´a-Sastre,2,4 and Aneel K. Aggarwal1,* 1 Department of Structural and Chemical Biology, Mount Sinai School of Medicine, 1425 Madison Avenue, New York, NY 10029, USA 2 Department of Microbiology 3 Microbiology Training Area of the Graduate School in Biological Sciences 4 Emerging Pathogens Institute Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA *Correspondence: [email protected] DOI 10.1016/j.molcel.2007.04.022

SUMMARY

Interferon regulatory factor 3 (IRF-3) is a key transcription factor in the assembly of the mammalian interferon-b (IFN-b) enhanceosome. We present here the structure of IRF-3 DNA binding domain in complex with the complete PRDIII-I regulatory element of the human IFN-b enhancer. We show that four IRF-3 molecules bind in tandem to, variably spaced, consensus and nonconsensus IRF sites on the composite element. The ability of IRF-3 to bind these variable sites derives in part from two nonconserved arginines (Arg78 and Arg86) that partake in alternate protein-DNA contacts. We also show that the protein-DNA contacts are highly overlapped and that all four IRF sites are required for gene activation in vivo. In addition, we show that changing the nonconsensus IRF sites to consensus sites creates a more efficient enhancer in vivo. Together, the structure and accompanying biological data provide insights into the assembly of the IFN-b enhanceosome in mammals. INTRODUCTION The coordinated assembly of transcription factors on enhancers is central to the growth and development of mammals and to their cellular response to inflammatory and pathogenic signals (Maniatis et al., 1987; Tjian and Maniatis, 1994). The virus-inducible enhancer of the mammalian interferon-b (IFN-b) gene provides one of the best examples of how a set of transcription factors assemble on an enhancer to direct a specific gene expression program (Thanos and Maniatis, 1995; Wathelet et al., 1998). The IFN-b enhancer in humans, for example, is located between nucleotides 110 and 45 relative to the transcription start site, and it contains four overlapping

positive regulatory domains (PRDs) that are recognized by sequence-specific transcription factors to form a transcriptionally competent complex termed the ‘‘enhanceosome’’ (Fujita et al., 1985; Goodbourn and Maniatis, 1988; Kim and Maniatis, 1997; Merika and Thanos, 2001; Thanos and Maniatis, 1995). Interferon regulatory factor 3 (IRF-3) is one of the key transcription factors in the assembly of the mammalian IFN-b enhanceosome (Hiscott et al., 1999; Sato et al., 1998; Wathelet et al., 1998; Yoneyama et al., 1998). IRF-3 is expressed constitutively in all tissues in a latent state. Upon viral infection, IRF-3 is activated by the phosphorylation of a set of serine residues near the C terminus, leading to its dimerization and translocation from the cytoplasm to nucleus; whereupon, it activates the expression of type I IFNs (IFN-as and IFN-b) (Hiscott et al., 1999; Yoneyama et al., 2002). On the IFN-b enhancer, IRF-3 binds to both PRDI and PRDIII elements and it synergistically interacts with transcription factors NF-kB and ATF-2/ c-Jun bound to adjacent PRDII and PRDIV elements, respectively, and with the architectural HMG I(Y) protein that is also required for the assembly of the enhanceosome (Kim and Maniatis, 1997; Thanos and Maniatis, 1995). The enhanceosome in turn permits the ordered recruitment of chromatin modifying and general transcription factors to the promoter for the expression of the antiviral IFN-b gene (Agalioti et al., 2000; Merika et al., 1998; Munshi et al., 1998). IRF-3 belongs to the IRF family of transcription factors that has now expanded to include nine mammalian members (IRF-1, IRF-2, IRF-3, IRF-4/Pip, IRF-5, IRF-6, IRF-7, IRF-8/ICSBP, and IRF-9/ISGF3g) as well as viral members (vIRFs) that possess a homologous DNA binding domain (DBD) at the N terminus of 120 amino acids (Barnes et al., 2002; Hiscott et al., 1999; Taniguchi et al., 2001). The DBD is characterized by five conserved tryptophans, three of which are used to latch the DBD onto DNA (Escalante et al., 1998). The distinct role of each family member in biological processes ranging from antiviral response to hematopoietic development stems from different patterns of expression and pairings with coregulators, and slightly

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Molecular Cell Crystal Structure of IRF-3/PRDIII-I DNA Complex

Figure 1. Sequence of the Human IFN-b Enhancer and Protein Sequence Alignment (A) The 102 to 55 nucleotide region of the human IFN-b enhancer showing the traditional assignment of the four positive regulatory domains (PRDI–IV). The sequence of the 33 bp DNA oligomer used for cocrystallization is shown where the colored boxes indicate the sites recognized by the four IRF-3 molecules. Note the overlapping nature of the sites. (B) Sequence alignment of IRF-3 DNA binding domain (DBD) and other members of the IRF family from human. The secondary structure elements in IRF-3 DBD are shown on top of the alignment, and the key residues contacting DNA in the major and minor grooves are highlighted in red.

different DNA binding specificities within the broad IRF binding consensus sequence AANNGAAA (Barnes et al., 2002; Hiscott et al., 1999; Taniguchi et al., 2001). IRF-1 and IRF-2 were the first members of the family to be isolated and were early candidates for biochemical and structural studies examining their binding to the IFN-b enhancer (Fujita et al., 1988; Harada et al., 1989). However, more recent studies have shown IRF-3 and IRF-7 to be the key regulators of IFN-b expression, and this conclusion is supported by gene-knockout studies of IRF-3 and IRF-7 in mice that result in marked decrease in the production of IFN-b in response to viral infection (Honda et al., 2005; Sakaguchi et al., 2003; Sato et al., 2000). The structure of the IFN-b enhanceosome has been partially characterized via structures of various components (or related components). These include crystal structures of IRF-1 DBD bound to PRDI (Escalante et al., 1998), IRF-2 DBD bound to a PRDIII-like element (Fujii et al., 1999), NF-kB p50/p65 heterodimer bound to PRDII (Berkowitz et al., 2002; Escalante et al., 2002b), and ATF-2/c-Jun/ IRF-3 DBDs bound to a composite PRDIV-III element (Panne et al., 2004). In addition, the structure of the IRF-3 C-terminal domain has been determined (Qin et al., 2003; Takahasi et al., 2003). Collectively, the structures (and biochemical analyses) have helped to paint a picture of the enhanceosome; however, questions remain as to (1)

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how many and (2) how exactly the IRF-3 molecules are accommodated on the composite PRDIII-I element. We present here the structure of IRF-3 DBD in complex with the complete PRDIII-I regulatory element of the human IFN-b enhancer. We show that four IRF-3 molecules are bound to both consensus and nonconsensus sequences via surprising flexibility in both ‘‘spacing’’ and proteinDNA interactions. RESULTS Structure Determination We cocrystallized the human IRF-3 DBD (aa 1–113) with a DNA sequence that covers the PRDIII-I region (nucleotides 96 to 64) of the human IFN-b enhancer (Figure 1A). The complex crystallized in space group C2 with unit cell dimensions of a = 169.80, b = 47.42, c = 127.64 A˚, and b = 128.87 . The cocrystals were grown by vapor diffusion from solutions containing 12% PEG 400 and 200 mM NaCl. The structure was solved by molecular replacement and refined (at 2.31 A˚ resolution) to Rcryst of 19.2% and Rfree of 25.9% (Table 1). The final model contains four IRF-3 DBDs, namely IRF-3A (aa 5–43 and 52–110), IRF-3B (aa 7–112), IRF-3C (aa 4–110), and IRF-3D (aa 3–46 and 52–110), bound to 32 DNA base pairs (there is no electron density for the 50 A/T

Molecular Cell Crystal Structure of IRF-3/PRDIII-I DNA Complex

Table 1. Data Collection and Refinement Statistics Data Collection Resolution (A˚)

50–2.31

Number of measured

314059

Number of unique

30304 a

Data coverage (%)

91.7(74.8)

Rmerge (%)a,b

2.7(10.8)

a

34.8(5.5)

I/s

Refinement Statistics Resolution range Reflections c

Rcryst (%)

d

20–2.31 28759 19.2

Rfree (%)

25.9

Nonhydrogen atoms

4634

Protein

3205

DNA

1306

Water

123

Average B factors (A˚2)

49.8

Protein

54.7

DNA

43.8

Water

50.9

Rmsds Bonds (A˚)

0.007

Angles (A˚)

1.3

Ramachandran plot quality Most favored (%)

84.4

Additional allowed (%)

15.6

Generously allowed (%)

0

Disallowed (%)

0

a

Values for the outmost shells are given in parentheses. Rmerge = SjI  j / S I, where I is the integrated intensity of a given reflection. c Rcryst = S jjFoj  jFcjj / S jFoj. d For Rfree calculations, 5% of data was excluded from refinement. b

overhang), and 123 solvent molecules. The protein chains have excellent geometry with 85% of the residues in the most favored regions of the Ramachandran plot and no residues in the disallowed regions. Some of the missing residues in IRF-3A and IRF-3D correspond to a loop, which was omitted due to poor electron density.

Overall Arrangement and Architecture The IFN-b enhancer contains three consensus IRF sites (AANNGAAA), and a prevailing view has been that PRDI binds one IRF molecule and PRDIII binds two IRF mole-

cules. Instead, the structure reveals four IRF-3 DBD molecules bound to the composite PRDIII-I element (Figure 2A). The molecules bind in tandem in a head-totail orientation: two to the PRDIII element (IRF-3A and IRF-3B) and two to the PRDI element (IRF-3C and IRF-3D). Each PRD element is composed of a nonconsensus IRF binding site at the 50 end and a consensus site (AANNGAAA) at the 30 end. Thus, IRF3-A and IRF-3C DBDs bind to the nonconsensus sites (Boxes A and C, respectively), and IRF-3B and IRF-3D DBDs bind to the consensus sites (Boxes B and D, respectively). Because the sites are progressively separated by about half a turn of DNA, IRF-3A and IRF-3C occupy one face of the DNA and IRF-3B and IRF-3D are on the other (opposite) face of the DNA (Figure 2A). A closer look at this arrangement reveals variability in spacing between the binding sites. Thus, whereas IRF-3A and IRF-3B, and IRF-3B and IRF3C, bind relative to each other with a spacing of two nucleotides, IRF-3C and IRF-3D bind relative to each other with a spacing of three nucleotides. IRF-3 emerges from the structure as well adapted for alternate sets of proteinDNA contacts. As seen in previous IRF protein structures, the IRF-3 DBD consists of a trihelical bundle (a1, a2, a3) flanked on one side by a four-stranded antiparallel b sheet (Figure 2A). There are three long loops connecting different secondary structure elements. Loop L1 connects strand b2 with helix a2 and contains a conserved histidine residue (His40 in IRF-3) that interacts with the DNA minor groove (Figures 1B and 2A). Loop L2 is a long turn that links a2 with the recognition helix a3. The third loop, L3, connects b3 with b4. There are several features that differentiate the IRF-3 DBD from other members of the family and that are important for the discussion below: first, the presence of residue Leu42 on loop L1, which in other IRFs is either a glycine or an alanine (Figure 1B); second, the presence of two arginines (Arg78 and Arg86) at the N- and C termini of the recognition helix a3, which impart on IRF-3 the flexibility to recognize both consensus and nonconsensus DNA sites. These DNA sites are highly overlapped on PRDIII-I element, and DNA contacts made by one IRF-3 molecule extend into the binding site of the neighboring IRF-3 molecule. The PRDIII-I element (32 bps) weaves a sinusoidal path and has an overall curvature of 21 . At the same time, there is a reduction in the overall length by 4.5 A˚ (Figure 2B) and a lateral displacement of 8 A˚ with respect to the vertical axis of an idealized B-DNA. Each of the four IRF binding sites has a different local DNA curvature. Boxes A and B bend in opposite directions by 21 and 24 , respectively, and as a result, the PRDIII element has an overall curvature of 10 . Box C is the most deformed, with a local DNA curvature of 31 . Interestingly, compared to DNAs in other IRF complexes, IRF-3 DBD causes a widening of the minor groove by an extra 1–2 A˚ at the NN base steps (AANNGAAA) of the binding sites (Figure S1 in the Supplemental Data available with this article online).

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Molecular Cell Crystal Structure of IRF-3/PRDIII-I DNA Complex

Figure 2. Overall Structure of the IRF-3PRDIII-PRDI Complex (A) A ribbon and surface representation of the complex with the DNA oriented 50 to 30 as in Figure 1A. The four IRF-3 molecules are colored according to their binding sites in Figure 1A: IRF-3A in green, IRF-3B in red, IRF-3C in blue, and IRF-3D in orange. The core sequences are highlighted in yellow. All the secondary structure elements are labeled in IRF-3B and IRF-3C and only the helices and loops in IRF3A and IRF-3D. In the surface representation, the complex is rotated 90 along an axis down the paper from the view shown in the ribbon structure. (B) DNA curvature of the PRDIII-I element. The local helical axis is shown in red line.

Protein-DNA Interactions The IRF-3 DBDs latch onto DNA (stretches of 9 bps) through a series of phosphate contacts that position the recognition helix (a3) in the major groove and loop L1

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near the minor groove (Figure 2A). In particular, two of the five conserved tryptophans (Trp38 on loop L1 and Trp57 on helix a2) make phosphate contacts that serve as ‘‘anchors’’ for establishing the register of each IRF-3

Molecular Cell Crystal Structure of IRF-3/PRDIII-I DNA Complex

Figure 3. Protein-DNA Contacts The contacts are colored according to which IRF-3 DBD makes them: IRF-3A, green; IRF-3B, red; IRF-3C, blue; and IRF-3D, orange. Straight lines correspond to hydrogen bonds, and dotted lines represent van der Waals contacts. Black circles represent water molecules. In the IRF-3A, IRF-3B, IRF-3C, and IRF-3D insets, hydrogen bonds are represented by yellow dotted lines and water molecules are represented as red dots.

DBD on the PRDIII-I element. As such, IRF-3B and IRF-3D bind to the more consensus-like (AACTGAAA and AAGTGAAA) sequences, whereas IRF-3A and IRF-3C bind to nonconsensus (TAGGAAAA and AAGGGAGA) sequences. The recognition helix of each IRF-3 DBD is almost parallel to the sugar-phosphate backbone and carries residues (including Arg78 and Arg86 that are unique to IRF-3) that interact with the downstream base pairs (A/TANNG/AAG/AA) in the major groove, whereas loop L1 carries His40 and Leu42 that play a key role in

specifying the upstream base pairs (A/TANNG/AAG/AA) in the minor groove. IRF-3A IRF-3A binds the most upstream portion of PRDIII-I and is equivalent to IRF-3A in the ATF-2/c-Jun/IRF-3/PRDIV-III quaternary complex reported by Panne et al. (2004) (Figure 3). In this quaternary complex, IRF-3A binds to the nonconsensus TAGGAAAA sequence and it interacts with ATF-2/c-Jun bound to the adjacent PRDIV element.

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Molecular Cell Crystal Structure of IRF-3/PRDIII-I DNA Complex

An interesting question that arose from the quaternary complex was whether IRF-3 in the absence of ATF-2/ c-Jun would bind to the same nonconsensus sequence or ‘‘move’’ over by a nucleotide to the more consensuslike ATAGGAAA sequence. In fact, IRF-3 turns out in our structure to bind the same nonconsensus sequence as in the quaternary complex, though some of the contacts to bases in the major groove are different. In particular, the nonconserved arginines Arg78 and Arg86 are positioned opposite different bases in the two structures. In the quaternary complex, for example, Arg78 and Arg86 specify the second A:T base pair of the core of base pairs recognized in the major groove (AAAA), wherein Arg86 donates a hydrogen bond to the N7 of the adenine and Arg78 makes van der Waals contacts with the methyl group of the paired thymine (Panne et al., 2004). In the present structure, Arg78 takes on a different conformation, whereby its terminal guanidinium group is shifted by as much as 5.5 A˚ from the position in the quaternary complex and it makes contacts with the two guanines upstream of the AAAA core (GGAAAA). The N7 of the first guanine (GGAAAA) receives a hydrogen bond from Nh1 of Arg78, whereas the N7 of the second guanine (GGAAAA) receives a hydrogen bond from a water molecule linked to N3 of Arg78 (Figure 3). The Arg86 guanidinium group shifts in the opposite direction and donates a hydrogen bond to the O6 of guanine of a G:C base pair on the 30 side of the core sequence (AAAAC). Thus, both Arg78 and Arg86 specify base pairs outside of the core sequence (GGAAAAC). From the structure, IRF-3 appears to be the only IRF able to interact with the first guanine on the 50 side of the core sequence (GGAAAAC), as the other IRFs contain either alanine or threonine in place of Arg78 that is too short to reach the purine base. However, in addition to IRF-3, several other IRFs (IRF-4, IRF-5, IRF-6, IRF-8, and IRF-9) appear able to recognize a G:C base pair on the 30 side of the core sequence (GGAAAAC), as they contain a lysine in place of Arg86. Indeed, IRF-4 has been observed making bidentate hydrogen bonds with a 30 G:C base pair in the structure of IRF-4/PU.1/DNA ternary complex (Escalante et al., 2002a). IRF-3A specifies the core A:T base pairs (AAAA) primarily by van der Waals interactions: Arg81 with the adenine of the first A:T base pair (AAAA), Ser82 with the methyl group of thymine of the third A:T base pair (AAAA), and Ala83 and Arg86 with the methyl group of the fourth A:T base pair (AAAA). Arg81, Ser82, and Ala83 are positioned almost identically with respect to the DNA as in the quaternary complex (Panne et al., 2004). These interactions are different from those observed with IRF-1, IRF-2, and IRF-4, wherein the arginine equivalent to Arg81 makes a direct hydrogen bond with the guanine of the first core base pair (GAAA) and the cysteine equivalent to Ser82 makes hydrogen bonds with the adenine and thymine of the second and third base pairs (GAAA), respectively. Altogether, the IRF-3A recognition helix appears to be positioned slightly further away from the core base pairs

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than IRF-1, IRF-2, and IRF-4, perhaps to create room for the nonconserved and lengthy Arg78 and Arg86 within the major groove. Interactions with A:T base pairs upstream of the core (TANNAAAA) occur in the minor groove, wherein His40 on loop L1 partially penetrates the groove. That is, a water molecule bonded to N32 of His40 makes two hydrogen bonds in the minor groove: the first with O2 of thymine (TANNAAAA) and the second with N3 of adenine (TANNAAAA) (Figure 4A). The configuration of the loop of L1 (and the precise juxtaposition of His40) is stabilized by residues that flank His40, including Trp38 and Gly41 that make contacts with the DNA backbone. Interestingly, in IRF-3B, IRF-3C, and IRF-3D, Leu42 on loop L1 also enters the minor groove (described below) but there is no clear density for the side chain in IRF-3A. IRF-3B IRF-3B binds a more consensus-like sequence (AANN GAAA) at the 30 end of PRDIII (Figure 2). The consensus guanine (GAAA) is specified by a pair of hydrogen bonds with Arg81, one of which is mediated by a water molecule (Figure 3). The A:T base pairs within the core (GAAA) are specified by van der Waals contacts between the methyl groups of the thymines and the long Arg78 and Arg86 side chains. Thus, whereas Arg78 and Arg86 in IRF-3A specify base pairs outside of the consensus core, in IRF3B, these two nonconserved arginines are closer to the conformation in the quaternary complex and interact with bases within the consensus core. Interactions with A:T base pairs upstream of the core (AANNGAAA) occur in the minor groove via His40, in the manner described above for IRF-3A. In addition, Leu42 on loop L1 penetrates the minor grove (deeper than His40) and makes van der Waals contacts with the sugar and the minor groove edge of the first adenine (AANN GAAA) (Figure 4A). These contacts are specific to IRF-3 as all of the other IRFs contain glycine or alanine in place of Leu42. Importantly, the A:T base pairs specified in the minor groove by IRF-3B are the same A:T base pairs recognized in the major groove by IRF-3A. This mutual dependence between IRF-3B and IRF-3A in recognizing ‘‘middle’’ A:T base pairs in PRDIII (TAGGAAAACTGAAA) extends to interactions with DNA backbone; wherein, IRF-3B makes several DNA backbone contacts that extend into the IRF-3A recognition sequence, and conversely, IRF-3A makes DNA backbone contacts that continue into the IRF-3B recognition sequence (Figure 3). For example, the main-chain amide of Gln44 on loop L1 of IRF-3B donates a hydrogen bond to the 50 phosphate of cytosine of a G:C base pair upstream of the IRF-3A consensus core (TAGGAAAACTGAAA), and the main-chain amide of Ile8 on an N-terminal extension of IRF-3A makes a hydrogen bond with the 50 phosphate of guanine of a C:G base pair well within the IRF-3B recognition sequence (TAGGAAAACTGAAA). Taken together, the IRF-3A and IRF-3B binding sites are highly overlapped on PRDIII, and as in the quaternary

Molecular Cell Crystal Structure of IRF-3/PRDIII-I DNA Complex

Figure 4. Conformational Variability and Minor Groove Contacts (A) Minor groove interactions mediated by His40 and Leu42 in IRF-3B. His40 binds to a water molecule (red sphere) that in turn makes hydrogen bonds (yellow dotted lines) with bases. The other IRF-3 DBDs make similar minor groove interactions. (B) A superposition of the IRF-3A (green), IRF-3B (red), IRF-3C (blue), and IRF-3D (orange) recognition helices to show conformational variability of Arg78, Arg81, and Arg86. Note that arginines that interact with consensus sites (IRF-3B, red; IRF-3D, orange) are closer in conformation than those that interact with the nonconsensus sites (IRF-3A, green; IRF-3C, blue). (C) Sedimentation velocity analysis showing the g(s*) distribution of IRF-3 DBDWT/PRDIII-I complex (black) and the mutant IRF3-2Rmut/ PRDIII-I complex (red).

complex, the two consensus cores are separated by two nucleotides (TANNAAAANNGAAA). There are no proteinprotein interactions between the two DBDs. The closest approach occurs across the minor groove over the middle A:T base pairs (TAGGAAAACTGAAA), wherein Ca of Ala63 on loop L2 (between helices a2 and a3) of IRF-3A is 18.4 A˚ from Ca of Asp95 on strand b3 (leading to loop L3) of IRF-3B.

IRF-3C IRF-3C marks the first time that an IRF molecule has been observed bound to this nonconsensus site (AANNGAGA) (Figure 2), which is the most locally curved (31 ) segment of PRDIII-I element (Figure S1). Compared to the other IRF-3 molecules, the C-terminal end of the IRF-3C recognition helix is slightly more angled away from the major groove (based on superposition of the complexes). This may be one reason why the Arg86 side chain in IRF-3C is disordered (and is not visible in our electron density map). Otherwise, interactions with bases are more akin to IRF-3A (than IRF-3B and IRF-3D), which also binds to a nonconsensus sequence. In particular, Arg78 extends ‘‘outward’’ and is in a position to form hydrogen bonds with guanines on the 50 side of the core (AAGGGAGA) (Figures 3 and 4B). However, the hydrogen bonds in this case are donated to the O6 atoms of the guanines rather than to the N7 atoms. Another difference is that whereas Ser82 in IRF-3A makes van der Waals contacts with a thymine of an A:T base pair within the consensus core (AAAA), Ser82 in IRF-3C forms a direct hydrogen bond with the equivalent cytosine (GAGA). As with IRF-3B and IRF-3A, the IRF-3C and IRF-3B binding sites overlap and the spacing between the consensus cores is two nucleotides (AACTGAAAGGGAGA). Thus, the conserved adenines at the 30 end of PRDIII (TAGGAAAACTGAAA) are recognized in the major groove by IRF-3B (via Arg78 and Arg86 on the recognition helix a3) and are specified in the minor groove by IRF-3C (via His40 and Leu42 on loop L1). Interestingly, the only significant protein-protein interactions in our structure occur between IRF-3C and IRF-3A. Because of the juxtaposition of the IRF-3C and IRF-3A binding sites on the same side of the DNA (boxes C and A, separated by two nucleotides), loop L1 of IRF-3C lays in close proximity to the N terminus of IRF-3A. Specifically, Gln44 in IRF-3C makes a series of van der Waals contacts with Arg7 and Pro10 in IRF-3A (Figure 2). IRF-3D IRF-3D binds to an IRF consensus sequence (AANN GAAA) at the 30 end of PRDI (Figure 2). Intriguingly, the spacing between IRF-3D and IRF-3C consensus cores is three nucleotides, as opposed to two nucleotides between the other adjacent IRF pairs. And, because of the 36 rotation introduced by the ‘‘extra’’ nucleotide, IRF3D and IRF-3C are spatially closer than other adjacent pairs such as IRF-3C and IRF-3B or IRF-3B and IRF-3A. Thus, whereas the distance across the minor groove between Ala63 Ca of IRF-3A and Asp95 Ca of IRF-3B is 18.4 A˚, the corresponding distance between IRF-3C and IRF-3D is only 10.1 A˚. However, even at this closer apposition, there are no meaningful protein-protein interactions between IRF-3C and IRF-3D. There are no significant interactions between IRF-3D and IRF-3B, even though, like IRF-3C and IRF-3A, they are on the same face of the DNA. In this case, the extra-spacing nucleotide has the effect of increasing the spatial separation between the two IRFs.

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Molecular Cell Crystal Structure of IRF-3/PRDIII-I DNA Complex

IRF-3D recognizes the consensus sequence (at the 50 end of PRDI) in much the same way as IRF-3B (at the 30 end of PRDIII). Accordingly, Arg78 is positioned near the first two A:T base pairs within the consensus core (GAAA), making van der Waals contacts with the methyl groups of the thymines (Figure 3). Arg81 specifies the consensus guanine (GAAA) via a hydrogen bond, and Arg86 (in two possible conformations) makes van der Waals contacts with the third and fourth adenines (GAAA) (Figure 3). Minor groove contacts to upstream adenines (AANN GAAA) do not extend as far into the IRF-3C binding site (as compared to the other IRF pairs) because of the extra spacing between the IRF-3D and IRF-3C consensus cores. Mutational Analysis To assess the role of two nonconserved arginines (Arg78 and Arg86) in binding to the PRDIII-I element in vitro, we used analytical ultracentrifugation (AU). We mutated these arginines to alanines (IRF3-2Rmut) and used sedimentation velocity to follow the formation of a complex with the 33-mer used in cocrystallization. Figure 4C compares the g(s*) profiles of the complexes obtained with IRF-3 DBDWT and IRF3-2Rmut. IRF-3 DBDWT forms a complex that sediments at 5.12 S (or at corrected S0w,20=5.21), which corresponds to the expected molecular weight (72 kDa) for a complex containing four IRF-3 DBDs. IRF3-2Rmut, on the other hand, forms a complex that has a much lower sedimentation coefficient of 3.79 S, corresponding to the binding of only two mutant IRF-3 DBDs. Together, these data show the importance of Arg78 and Arg86 in PRD III-I binding and suggest that some IRF sites are more sensitive than others to mutations in these nonconserved arginines. Functional Requirement for IRF-3A, IRF-3B, IRF-3C, and IRF-3D In Vivo and In Vitro From the structure, the four IRF-3 molecules are accommodated in close proximity to each other on the IFN-b enhancer. DNA contacts made by one IRF-3 molecule extend into the binding site of the neighboring IRF-3 molecule (Figures 2 and 3). To test the importance of these consensus and nonconsensus binding sites (Boxes A–D) and the requirement for four IRF-3 molecules, we mutated each box on the IFN-b enhancer and then assayed the ability of the mutated enhancers to drive the expression of a reporter gene in vivo—in response to infection by Sendai virus or to overexpression of a constitutively activated form of IRF-3 (IRF-3 5D). Because of the overlapping nature of the sites, we changed only those bases that are contacted uniquely by each IRF-3 molecule (Figure 5A), namely the first two bases of the consensus or nonconsensus cores (A/TANNG/AAG/AA) in sites A and B, AANN GAGA in site C, and AANNGAAA in site D. (The other bases are contacted in the major groove by one IRF-3 and in the minor groove by the adjacently bound IRF-3.) This allowed us to assess the effect of each IRF-3 binding site on transcription without impinging on a neighboring

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site. Figure 5 shows the results obtained when we cotransfected 293T cells with an IFN-b promoter reporter vector (pIFNb-RFP-CAT) or with plasmids with the individually mutated boxes. As a control, we also transfected the Renilla luciferase reporter vector pRL-TK (Promega). By directly observing the red fluorescence protein (RFP) (Figure 5B), and by measuring the activity of chloramphenicol acetyltransferase (CAT) (Figure 5C), after normalization using the luciferase values, we could assess the effect of mutating each box on gene activation. From these experiments, the ability of the IFN-b enhancer to activate the expression of reporter genes was found to be severely compromised by mutations in the individual IRF binding sites. These results were confirmed as specific for IRF-3 by comparing the ability of a constitutively active mutant of IRF-3, namely IRF-3 5D, to activate the expression of reporter genes from WT and mutant IFN-b enhancers. In the IRF-3 5D mutant, five of the serines that are believed to be phosphorylated in order to activate IRF-3 are changed to phospho-mimetic aspartates, and this mutant is constitutively active for IFN-b expression in the absence of viral infection. As shown in Figure 5D, the ability of the IRF-3 5D mutant to activate the expression from reporter genes is severely impaired by mutations in each of the four IRF binding sites. Taken together, these results in vivo show not only a requirement of four IRF-3 binding boxes on the IFN-b enhancer but also a level of cooperativity where mutations in any individual IRF binding site compromise the ability to activate transcription. To assess the effects of mutations in sites A–D on IRF-3 binding in vitro, we used sedimentation velocity to follow the formation of the IRF-3 DBD/DNA complex with the WT PRDIII-I element (namely, the 33-mer used in cocrystallization) and elements carrying the mutations described above in Boxes A–D. As shown in Table S1, whereas the WT element forms a complex that sediments at 5.21 S, corresponding to the binding of four IRF-3 DBDs, elements with mutations in Box A, B, C, or D give rise to complexes with lower sedimentation coefficients (4.4–4.7 S), corresponding to the binding of three IRF-3 DBDs. Together, these data are consistent with the binding of one IRF-3 molecule to each of the four IRF sites on the PRD III-I element. Changing Nonconsensus Sites to Consensus Sites Creates a More Efficient IFN-b Enhancer The finding that four IRF-3 molecules bind to variably spaced, consensus, and nonconsensus IRF sites on the PRDIII-I element raises the intriguing question of whether one could make the IFN-b enhancer more optimal by (1) changing the nonconsensus sites to consensus sites and (2) changing the 3 bp spacing between Boxes C and D to the more optimal 2 bp spacing. To test this, we generated four mutant enhancers: C+, in which we changed Box C from a nonconsensus AANNGAGA to a consensus AANNGAAA sequence; AC+, in which we also changed Box A from a nonconsensus TANNAAAA to a consensus AANNGAAA sequence; CD+, in which we changed the

Molecular Cell Crystal Structure of IRF-3/PRDIII-I DNA Complex

Figure 5. Mutations of Individual IRF Binding Sites, Boxes A–D, Inhibit the Induction of the Interferon-b Enhancer (A) Alignment of the sequences of the different IFN b box mutants constructs. The different mutations on top are highlighted in red, and wild-type murine IFN-b core PRD enhancer is at the bottom. (B) Visualization of 293T cells transfected with 1 mg of the indicated plasmids and infected 12 hr after transfection with Sendai Cantell virus at high multiplicity of infection. The images were taken 24 hr postinfection. Red fluorescence is indicative of the enhanceosome interferon reporter activation by Sendai virus infection. (C) Quantification of the levels of IFN-b promoter induction visualized in (B) by the CAT reporter protein. The experiment was done in triplicate, and the error bars represent the standard deviation between experiments. (D) Quantification of the levels of IFN-b promoter induction driven by the expression of the constitutively activated IRF3 5D protein. 293T cells were transfected with 1 mg of the indicated wild-type or the corresponding box mutant of the IFNb-RFP-CAT enhancer reporter plasmid together with 1 mg of the plasmid expressing IRF3 5D or control empty plasmid and 0.5 mg of the pRL-TK plasmid in order to normalize the CAT values. The experiment was done again in triplicate, and the error bars represent the standard deviation between experiments.

3 bp spacing between Box C+ and Box D to the more optimal 2 bp spacing; and ACD+, which encompassed all of the above changes and thus specified an IFN-b enhancer that is consensus in sequence and spacing across all four IRF sites, namely AANNGAAANNGAAANNGAAANGAAA. Figure 6A shows the results obtained when we cotransfected 293T cells with an IFN-b reporter vector (pIFNbRFP-CAT) or with plasmids carrying the C+, AC+, CD+, and ACD+ mutations and then measured CAT activity in response to Sendai virus infection (Figure 6C) or to the overexpression of constitutively active IRF-3 5D (Figure 6D). The ability of the IFN-b enhancer to activate the expression of the reporter gene is found to increase substantially via these ‘‘consensus-like’’ mutations and deletions. The ACD+ enhancer, which embodies a con-

sensus sequence across all four IRF sites, is the most efficient, showing almost 300% enhancement in CAT activity in response to viral infection, and even greater enhancement in response to the overexpression of IRF-3 5D. Together, these results show that the IFN-b enhancer can indeed be made more efficient by changing the nonconsensus IRF sites to consensus sites. We next asked whether with the more optimal ACD+ IFN-b enhancer one can obviate a requirement for ATF-2/c-Jun and NF-kB in gene activation. To address this, we mutated the PRDIV and PRDII elements in the context of both WT (PRDIVmut and PRDIImut) and ACD+ (PRDIVmutACD+ and PRDIImuACD+) IFN-b enhancers. These mutations have previously been shown by Maniatis and coworkers to reduce expression from reporter plasmids carrying the

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Molecular Cell Crystal Structure of IRF-3/PRDIII-I DNA Complex

Figure 6. Changing the IRF Nonconsensus Sites to Consensus Sites Enhances the Ability of the IFN-b Enhancer to Induce Transcription (A) Alignment of the sequences of the different IFN-b mutant constructs. The wild-type murine IFN-b core PRD enhancer is on top followed by the sequences of the different mutants. (B) Visualization of 293T cells transfected with WT IFNb-RFP-CAT and mutant ACD+ IFNb-RFP-CAT reporter vectors. (C and D) Quantification of the levels of reporter CAT activity produced by WT and different mutant IFNb-RFP-CAT reporter vectors in response to Sendai virus (C) or to constitutively active GFP-IRF3 5D (D). The experiment was done in triplicate, and the error bars represent the standard deviation between experiments. (E) Visualization of 293T cells transfected with IFNb-RFP-CAT and ACD+ IFNb-RFP-CAT reporter vectors carrying mutations in PRDIV and PRDII in response to constitutively active RIG-I CARD (RIG-I residues 1–210).

IFN-b enhancer (or portions of it) (Du and Maniatis, 1992; Goodbourn and Maniatis, 1988). Indeed, as expected, PRDIVmut and PRDIImut strongly inhibit the activation of the reporter gene in our assay. In this case, we used the constitutively active CARD RIG-I (RIG-I aa 1–210) to induce the expression of the reporter plasmids. RIG-I is an upstream inducer of the three routes of IFN-b pathway, namely IRF-3, ATF-2/c-Jun, and NF-kB, and its CARD domain has been shown to constitutively activate IFN-b expression in the absence of viral infection (Saito et al., 2007). As shown in Figure 6E, in contrast to PRDIVmut and PRDIImut, PRDIVmutACD+ and PRDIImuACD+ have only a marginal effect on the expression of the reporter gene in vivo.

DISCUSSION In the past decade, much has been learned about the role of IRF-3 in triggering IFN-b expression in mammals, in response to viral infection. The structure we present here provides molecular details on how four IRF-3 DBDs (IRF-3A, IRF-3B, IRF-3C, and IRF-3D) come together on the PRDIII-I element of the IFN-b enhancer. The structure reveals mutually dependent DNA binding. IRF-3C, for example, makes DNA contacts that extend into PRDIII, and conversely, IRF-3B makes contacts that continue into PRDI. Moreover, IRF-3C bears the same spatial relationship to IRF-3B as IRF-3B bears to IRF-3A. Altogether, the structure resists the traditional delineation of the IFNb enhancer into PRDI and PRDIII elements and shows that

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the functional regulatory unit (in terms of IRF-3 binding) is the composite PRDIII-I element. The close proximity of IRF-3 DBDs and the overlapping nature of their interactions with PRDIII-I will contribute to the cooperative assembly of IRF-3 on the IFN-b enhancer. Indeed, IRF-3A and IRF-3B DBDs have been shown to bind cooperatively to PRDIII (Falvo et al., 2000; Panne et al., 2004), and on a composite PRDIII-I element, we observe only a single complex, which by AU corresponds to a species with four bound IRF-3 DBDs. Similar cooperativity from adjacent binding has been seen in a number of cases, where the sites overlap, including the transcription factor Oct-1 (in which the linker was cleaved between its homeodomain and its POU-specific domain) (Klemm and Pabo, 1996), the homeodomains of homeotic Ultrabithorax (Ubx) and its partner Extradenticle (Exd) (Passner et al., 1999), IRF-4 DBD and its partner PU.1 (Escalante et al., 2002a), and adjacently bound IRF-3A and ATF-2 DBDs on the PRDIV-III element (Panne et al., 2004). Collectively, these and other examples highlight an emerging theme in combinatorial transcription, wherein the binding of one transcription factor can aid the binding of another (next to it) by helping to configure the DNA around it. The IRF-3 DBD emerges from our structure as remarkably versatile in its ability to recognize both consensus and nonconsensus IRF sites on the PRDIII-I element. Much of this versatility derives from a pair of arginines, at the N- (Arg78) and C termini (Arg86) of the recognition helix, which are unique to IRF-3. IRF-3A, for example, binds a nonconsensus site with Arg78 and Arg86 splayed ‘‘outward’’ to access bases outside of the consensus

Molecular Cell Crystal Structure of IRF-3/PRDIII-I DNA Complex

core. IRF-3B and IRF-3D, on the other hand, bind consensus sites with Arg78 and Arg86 directed more inward to contact bases within the consensus core (Figures 3 and 4B). Overall, the IRF-3 DBD appears to be well adapted for IFN-b enhancer recognition. The length of the nonconserved arginine side chains and their ability to mediate hydrophobic and polar contacts confer on IRF-3 (among other IRFs) the flexibility to bind both consensus and nonconsensus IRF sites on the IFN-b enhancer. Curiously, when we mutate Arg78 and Arg86 to alanines, we observe by AU a DNA complex that contains two bound IRF-3 DBDs. The two nonconserved sites (A and C) appear from the structure to be perhaps more dependent on interactions with Arg78 and Arg86 (making direct hydrogen bonds with outside of the consensus core), and it will be interesting to know if these are the sites vacated in the binding experiment. A 3 bp spacing between IRF-3C and IRF-3D is unusual. In previous structures with adjacently bound two IRFs, the DBDs bind with a 2 bp spacing between the consensus cores (AANNG/AAAANNGAAA). As such, the middle A:T base pairs (AANNG/AAAANNGAAA) are recognized in the minor groove by the downstream IRF and in the major groove by the upstream IRF. The DNA is configured optimally for such dual recognition, whereby, over the middle A:T base pairs the major groove is relatively wide (for accommodating the recognition helix a3 of upstream IRF) and the minor groove is relatively narrow (for accommodating His40 of downstream IRF). Thus, a combination of maximal overlapping DNA contacts and the conformability of DNA appears to favor a 2 bp spacing between adjacently bound IRFs (Panne et al., 2004). Indeed, IRF3A-IRF-3B and IRF-3B-IRF-3C bind with a 2 bp spacing in our structure. Why a 3 bp spacing between IRF-3C and IRF-3D then? A closer look at the IFN-b enhancer sequence suggests that it may be the more favorable choice on the WT enhancer. A 2 bp spacing would position IRF-3D over a sequence, GANNTGAA, that is suboptimal for contacts in the minor groove (GAAGTGAA) and in the major groove (GAAGTGAA). By moving over by a nucleotide, IRF-3D loses some overlapping contacts but gains a fully consensus IRF binding sequence (AANNGAAA). When we delete a nucleotide between Boxes C and D, the mutant enhancer is significantly more efficient in driving the expression of a reporter gene in vivo. The resulting 2 bp spacing allows IRF-3C and IRF-3D to regain some of the overlapping DNA contacts. In addition to IRF-3, knockout of the IRF-7 gene in mice results in a decrease in IFN-b production in response to viral infection (Honda et al., 2005). However, unlike IRF-3, IRF-7 is not expressed constitutively in most cells but is expressed in a second wave of signaling. That is, IFNs that are transcriptionally activated by IRF-3 (in the early phase) induce the expression of IRF-7, which then cooperates with IRF-3 for subsequent rounds of IFN-b production (in the late phase) (Hiscott et al., 1999; Taniguchi et al., 2001). Does the structure offer any insights into how IRF-7 may be accommodated alongside IRF-3 on the IFN-b

enhancer (in the late phase)? In answer to this question, we note an intriguing feature of IRF-7 DBD, namely an extra-long loop L2 between helices a2 and a3. IRF-7 is the only IRF family member to carry a nine amino acid insertion (rich in glutamates and arginines) in an otherwise conserved loop L2 (Figure 1B). We believe this to be relevant because the closest approach-adjacent IRF-3s in our structure occur between loop L2 of the upstream IRF-3 and loop L3 of the downstream IRF-3. However, the distance between these elements is long (>18 A˚ for IRF-3AIRF-3B and IRF-3B-IRF-3C, and >10 A˚ for IRF-3C-IRF3D) and cooperativity between neighboring IRF-3 DBDs is determined mainly by overlapping DNA contacts (as discussed above). IRF-7 DBD, on the other hand, could partake in protein-protein interactions. That is, when IRF-7 DBDs are bound at upstream sites (as IRF-7A and IRF-7C), their extra-long loop L2 can reach across the minor groove and contact loop L3 of the downstream IRF-3 DBDs (IRF-3B and IRF-7D). Whether IRF-7 DBDs do indeed bind to upstream sites (Boxes A and C) and mediate protein-protein interactions via their loop L2 may become clearer with the structure of IRF-3/IRF-7/DNA ternary complex. Overall, the structure we present here represents in many ways the missing piece in the jigsaw of structures depicting the binding of various sequence-specific transcription factors to the IFN-b enhancer. The structure permits a model of the IFN-b enhanceosome (Figure 7) with four IRF-3 molecules (on PRDIII-I) bracketed by ATF-2/ c-Jun on the 50 side (on PRDIV) and NF-kB on the 30 side (on PRDII). Upon viral infection, IRF-3 is activated by the phosphorylation of serine residues near the C terminus, leading to its dimerization and translocation to nucleus (Hiscott et al., 1999; Yoneyama et al., 1998). The structure is consistent with the recruitment of two (activated) full-length IRF-3 dimers on PRDIII-I, and we also show by transient cotransfection assays a requirement for all four IRF boxes in the activation of reporter genes in vivo (in response to Sendai virus infection and to constitutively active IRF3-5D). We also show by analytical ultracentrifugation that mutations in any one of the four IRF boxes results in the destabilization of the complex and the dismissal of an IRF-3 molecule. From the model in Figure 7, although the DNA deviates locally, the overall path of the DNA through the enhanceosome is relatively straight. IRF-3A and ATF-2 DBDs make only a few protein-protein contacts and bind cooperatively (on PRDIVIII) mainly as the result of overlapping DNA contacts. The same mechanism appears to apply to neighboring IRF-3 DBDs (on PRDIII-I), and between IRF-3D and NF-kB (p50 and p65 DBDs) (on PRDII-I), which barely touch each other in the model (Figure 7). Overall, the IFN-b enhancer is prototypic of a class of mammalian enhancers that are marked by overlapping and repeated DNA sites with strong and weak consensus sequences (Maniatis et al., 1987; Tjian and Maniatis, 1994). Strikingly, when we change the nonconsensus IRF sites to consensus sites in PRDIII-I, it increases the ability of the IFN-b enhancer to

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Molecular Cell Crystal Structure of IRF-3/PRDIII-I DNA Complex

Figure 7. A Model of IFN-b Enhanceosome The model is based on the structure of c-Jun-ATF-2-IRF-3 bound to PRDIV (PDB: 1T2K), the present structure with four IRF-3 DBDs bound to PRDIII-I, and the structure of NF-kB p65-p50 heterodimer bound to PRDII (PDB: 2I9T). c-Jun is shown in violet, ATF-2 in magenta, p50 in smudge, and p65 in wheat. The DNA is aligned as in Figure 1A, from 50 (PRDIV) to 30 (PRDII).

activate a reporter gene in vivo. Moreover, this more efficient IFN-b enhancer averts the requirement for ATF-2/ c-Jun and NF-kB to bind PRDIV and PRDII, respectively, suggesting that nonconsensus IRF sites may help to ‘‘curb’’ IFN-b expression (via IRF-3), so as to allow the coincidence of the IRF-3, ATF-2/c-Jun, and NF-kB signaling pathways in the cellular response to viral infection. Taken together, the structural and biological data we present provide insights into the determinants of enhanceosome assembly in eukaryotes. EXPERIMENTAL PROCEDURES Protein and DNA Purification DNA encoding the human IRF-3 DBD (aa 1–113) was subcloned in vector PET-15b and expressed in E. coli BL21pLysS(DE3) cells. Purification is described in detail in the Supplemental Data. Singlestranded DNA oligonucleotides were chemically synthesized, purified, and annealed according to the procedure described previously (Aggarwal, 1990). Plasmids The plasmids and the sequence of the primers used to mutate the IRF-3 binding sites (Boxes A–D) are described in the Supplemental Data. Tissue Culture and Viruses 293T cells were maintained in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum and penicillin/streptomycin. Sendai virus (SeV) strain Cantell was grown in 10-day-old embryonated chicken eggs at 37 C for 48 hr. Reporter Gene Assays The reporter gene assays are described in detail in the Supplemental Data.

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Analytical Ultracentrifugation Sedimentation velocity experiments, performed with a Beckman XL-I Analytical Ultracentrifuge, are described in detail in the Supplemental Data. Crystallization and Data Collection IRF-3 DBD was mixed with DNA in a 4:1 ratio and cocrystals measuring 0.4 3 0.4 3 0.3 mm obtained from solutions containing 12%–14% PEG 400 and 200 mM NaCl. The cocrystals belong to space group C2 with unit cell dimensions of a = 169.80, b = 47.42, c = 127.64 A˚, and b = 128.87 . Data (extending to 2.31 A˚ resolution) were measured from a single frozen crystal at the Advance Photon Source (APS, beamline 17ID) of Argonne National Laboratory at l = 1A˚, using an ADSC Quantum 210 CCD detector. Images were processed with the HKL2000 package (Otwinowski and Minor, 1997). Structure Determination The structure was solved by molecular replacement (MR) using the program PHASER (McCoy et al., 2005). The initial search model comprised of two IRF-3 DBDs (IRF-3B and IRF-3C) bound to a 31 bp long DNA, and it was partially based on the ATF-2/c-Jun/IRF-3/PRDIV-III quaternary complex (PDB id 1T2K). After rigid body refinement and solvent flattening with the program CNS (Brunger et al., 1998), the electron density map showed clear densities for molecules IRF-3A and IRF-3D. Rigid body refinement of all four IRF DBDs and the DNA led to an Rfree of 37%. Further rounds of refinement were again carried out with CNS, using the maximum likelihood refinement target function and a bulk solvent correction, and were interspersed with rebuilding with program O (Jones et al., 1991). The final rounds of refinement and rebuilding were performed with programs REFMAC (Murshudov et al., 1997) and Coot (Emsley and Cowtan, 2004), respectively. Water molecules were selected by distance criteria and optimal hydrogen bonding geometry. The final model has excellent stereochemistry with no protein residues in the disallowed regions of the Ramachandran plot. Analysis of DNA geometric parameters was carried out with the programs 3DNA and Curves (Lavery and Sklenar, 1988). The

Molecular Cell Crystal Structure of IRF-3/PRDIII-I DNA Complex

final refinement statistics are shown in Table 1. All figures were made with the program PyMOL (DeLano, 2002). Supplemental Data Supplemental Data include Supplemental Experimental Procedures, Supplemental References, one figure, and one table and can be found with this article online at http://www.molecule.org/cgi/content/full/26/ 5/703/DC1/. ACKNOWLEDGMENTS

Fujii, Y., Shimizu, T., Kusumoto, M., Kyogoku, Y., Taniguchi, T., and Hakoshima, T. (1999). Crystal structure of an IRF-DNA complex reveals novel DNA recognition and cooperative binding to a tandem repeat of core sequences. EMBO J. 18, 5028–5041. Fujita, T., Ohno, S., Yasumitsu, H., and Taniguchi, T. (1985). Delimitation and properties of DNA sequences required for the regulated expression of human interferon-beta gene. Cell 41, 489–496. Fujita, T., Sakakibara, J., Sudo, Y., Miyamoto, M., Kimura, Y., and Taniguchi, T. (1988). Evidence for a nuclear factor(s), IRF-1, mediating induction and silencing properties to human IFN-beta gene regulatory elements. EMBO J. 7, 3397–3405.

We thank the staff at Advanced Photon Source (beamline 17ID) for facilitating X-ray data collection, Richard Cadagan for technical assistance, Luis Martinez-Sobrido for suggestions and plasmids, and Dimitris Thanos for discussions during the early stages of the project. This work was supported by grant AI41706 from the National Institutes of Health (NIH) (A.K.A) and by CIVIA, a human immunology center supported by NIH/NIAID U19 grant AI62623 (A.G.-S.).

Goodbourn, S., and Maniatis, T. (1988). Overlapping positive and negative regulatory domains of the human beta-interferon gene. Proc. Natl. Acad. Sci. USA 85, 1447–1451.

Received: November 17, 2006 Revised: March 14, 2007 Accepted: April 25, 2007 Published: June 7, 2007

Hiscott, J., Pitha, P., Genin, P., Nguyen, H., Heylbroeck, C., Mamane, Y., Algarte, M., and Lin, R. (1999). Triggering the interferon response: the role of IRF-3 transcription factor. J. Interferon Cytokine Res. 19, 1–13.

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Accession Numbers The coordinates have been deposited in the RCSB Protein Data Bank under accession codes RCSB042395 and 2PI0.