Solution Structure of the SWIRM Domain of Human ... - Cell Press

Structure 14, 457–468, March 2006 ª2006 Elsevier Ltd All rights reserved

DOI 10.1016/j.str.2005.12.004

Solution Structure of the SWIRM Domain of Human Histone Demethylase LSD1 Naoya Tochio,1,6 Takashi Umehara,1,6 Seizo Koshiba,1 Makoto Inoue,1 Takashi Yabuki,1 Masaaki Aoki,1 Eiko Seki,1 Satoru Watanabe,1 Yasuko Tomo,1 Masaru Hanada,1 Masaomi Ikari,1 Miyuki Sato,1 Takaho Terada,1,2 Takahiro Nagase,3 Osamu Ohara,3,4 Mikako Shirouzu,1,2 Akiko Tanaka,1 Takanori Kigawa,1 and Shigeyuki Yokoyama1,2,5,* 1 RIKEN Genomic Sciences Center 1-7-22 Suehiro-cho Tsurumi Yokohama 230-0045 Japan 2 RIKEN SPring-8 center, Harima Institute Sayo Hyogo 679-5148 Japan 3 Department of Human Gene Research Kazusa DNA Research Institute 2-6-7 Kazusakamatari Kisarazu Chiba 292-0818 Japan 4 RIKEN Research Center for Allergy and Immunology 1-7-22 Suehiro-cho Tsurumi Yokohama 230-0045 Japan 5 Department of Biophysics and Biochemistry Graduate School of Science The University of Tokyo 7-3-1 Hongo Bunkyo-ku Tokyo 113-0033 Japan

Summary SWIRM is an evolutionarily conserved domain involved in several chromatin-modifying complexes. Recently, the LSD1 protein, which bears a SWIRM domain, was found to be a demethylase for Lys4-methylated histone H3. Here, we report a solution structure of the SWIRM domain of human LSD1. It forms a compact fold composed of 6 a helices, in which a 20 amino acid long helix (a4) is surrounded by 5 other short helices. The SWIRM domain structure could be divided into the N-terminal part (a1–a3) and the C-terminal part (a4–a6), which are connected to each other by a salt bridge. While the N-terminal part forms a SWIRMspecific structure, the C-terminal part adopts a helixturn-helix (HTH)-related fold. We discuss a model in which the SWIRM domain acts as an anchor site for a histone tail.

*Correspondence: [email protected] 6 These authors contributed equally to this work.

Introduction Eukaryotic genomes are highly condensed with histones and nonhistone proteins to form chromatin (reviewed in Kornberg and Lorch, 1999). Due to this condensed structure, eukaryotic DNA metabolisms are controlled by dynamic changes of the chromatin state, which are triggered by several histone-interactive protein complexes (reviewed in Jenuwein and Allis, 2001; Roth et al., 2001; Zhang and Reinberg, 2001; Bannister et al., 2002). For example, there are histone modification enzymes (Rice and Allis, 2001), ATP-dependent chromatin-remodeling complexes (Lusser and Kadonaga, 2003; Langst and Becker, 2004), and histone chaperones (Akey and Luger, 2003; Loyola and Almouzni, 2004). In many cases, these enzymes and chaperones form multisubunit complexes that have distinct or multiple chromatin-modulating activities. SWIRM (derived from Swi3p, Rsc8p, and Moira) is an evolutionarily conserved domain of about 85 amino acids that is often found in these chromatin-modulating complexes (Aravind and Iyer, 2002). From the viewpoints of the domain architectures and the amino acid sequence homology, the SWIRM domains can be classified into three main types (Figure 1A). The SWIRM domains of Swi3p and Rsc8p are representatives of the first type (Swi3p-type). Swi3p and Rsc8p are subunits of the SWI/SNF and the RSC complexes, respectively, which both facilitate the chromatin-remodeling process (Lusser and Kadonaga, 2003; Langst and Becker, 2004). At its C-terminal region, this type of SWIRM protein bears a SANT domain (derived from Swi3p, Ada2p, NcoR, and TFIIIB), an essential domain that is structurally related to the Myb DNA binding domain (Aasland et al., 1996; Boyer et al., 2002; Gru¨ne et al., 2003; de la Cruz et al., 2005) (Figure 1A). These SWIRM-containing subunits are thought to mediate protein-protein interactions in chromatin-remodeling complexes (Treich and Carlson, 1997; Treich et al., 1998). The second type (Ada2p-type) of SWIRM protein is represented by Ada2p, which is a transcription coactivator involved in several complexes (Candau et al., 1996; Eberharter et al., 1999; Sterner et al., 2002). The Ada2p-type SWIRM protein carries a SANT domain at its N-terminal region (Figure 1A). The intact Ada2p SANT domain is essential for the histone acetyltransferase activity of the Gcn5p-containing complex, and it is required for the Ada2p-dependent enhancement of histone tail binding (Boyer et al., 2002). The third type (LSD1-type) of SWIRM-containing protein bears the FAD-dependent aminooxidase-like domain (Figure 1A). LSD1 (Lysine-specific Demethylase 1) was initially identified as the KIAA0601 protein in several different transcription corepressor complexes, such as the NRD (Tong et al., 1998), Co-REST (You et al., 2001), CtBP (Shi et al., 2003), and HDAC complexes (Humphrey et al., 2001; Hakimi et al., 2002, 2003). Notably, LSD1 was identified as the first histone demethylase that specifically demethylates mono- or dimethylated histone H3 at Lys4 (Shi et al., 2004). The C-terminal

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Figure 1. Tertiary Structure of Human LSD1SWIRM (A) Schematic representation of the SWIRM domain-containing proteins. Red, yellow, blue, and green indicate the SWIRM domain, the SANT domain, the zinc finger domain, and the demethylation domain, respectively. The amino acid length of each protein is shown to the right. (B) Ensemble of 20 structures. Residues 173– 268 are shown in a backbone wire presentation and in stereoview, with every tenth amino acid numbered and presented as a yellow sphere. The N and C termini are indicated as N and C, respectively. (C) Ribbon representation of the structure closest to the mean structure of the ensemble. The a helices are shown in red. (D) A salt bridge and hydrogen bonds. This view is rotated 180º around the z axis from that in (C), and the a helices are shown as pale-red cylinders. The side chains of the residues that participate in the formation of either a salt bridge or a hydrogen bond are indicated by ball-and-stick models. The salt bridge between Glu192 (magenta) and Arg214 (cyan) is shown by a red line. The threonine side chains are indicated in yellow, and the four hydrogen bonds are represented by blue lines. The position of the cis-proline (Pro241) is shown in dark gray.

aminooxidase-like domain is responsible for both the demethylase and transcriptional repression activities (Shi et al., 2004), suggesting that LSD1 contributes to repression of gene expression through demethylation of Lys4-methylated histone H3, which is a potent marker for active transcription (Litt et al., 2001; Noma et al., 2001; Santos-Rosa et al., 2002). The repression activity is also assumed to be exerted by other subunits of LSD1-containing complexes, such as the histone deacetylases HDAC1 and HDAC2 (Humphrey et al., 2001; You et al., 2001; Hakimi et al., 2002, 2003; Shi et al., 2003), and the histone methyltransferases G9a and EuHMT (Shi et al., 2003), which presumably methylate histone H3 at Lys9 and lead to transcriptional repression (Nielsen et al., 2001; Shi et al., 2003). However, the molecular mechanisms involving the deacetylase, methyltransferase, and demethylase activities for coordinating the histone modifications have yet to be revealed.

Although LSD1 is supposed to be localized in the nucleus by virtue of an N-terminal domain with putative nuclear localization signals (Aravind and Iyer, 2002), and to function as a histone demethylase through the C-terminal aminooxidase-like domain (Shi et al., 2004), little is known about the function of its SWIRM domain, which intervenes between these two domains. In addition, no SWIRM domain structure has been reported. To gain insight into the function of the SWIRM domain, we solved the tertiary structure of the LSD1 SWIRM domain (LSD1SWIRM). We found that LSD1-SWIRM forms a compact fold that is composed of N- and C-terminal parts, and that the C-terminal part adopts an HTH-related fold. Through the comparison with several different HTH folds, we discuss structural features of the SWIRM domain. Finally, we demonstrate a potential interaction between LSD1-SWIRM and a peptide of a histone H3 tail by surface plasmon resonance (SPR) experiments, and we

Solution Structure of the SWIRM Domain 459

Table 1. Structural Statistics, Based on 20 Structures, for Human LSD1-SWIRM NOE upper distance restraints Intraresidual (ji 2 jj = 0) Medium-range (1 % ji 2 jj % 4) Long-range (ji 2 jj > 4) Total Dihedral angle restraints (f and 4) CYANA target function value (A˚2) Number of violations Distance violations (>0.30 A˚) Dihedral angle violations (>5.0º) Rmsd from the averaged coordinates (A˚)a Backbone atoms Heavy atom Ramachandran plot (%)a Residues in most favored regions Residues in additional allowed regions Residues in generously allowed regions Residues in disallowed regions Rmsd from the ideal coordinates Bond length (A˚) Bond angle (º)

527 1221 600 2348 105 0.14 6 0.03 0 0 0.34 6 0.07 0.75 6 0.08 84.6 15.4 0.1 0.0 0.003 0.526

Data were analyzed by PROCHECK-NMR (Laskowski et al., 1996) and WHATCHECK (Hooft et al., 1996). a Residues 174–266.

discuss a possible role of the SWIRM domain for interaction with a histone. Results Structure Determination We determined the solution structure of human LSD1SWIRM (region from Glu169 to Gly279) by NMR spectroscopy. Statistics on experimental constraints, and structural quality as determined by PROCHECK (Laskowski et al., 1996) and WHATCHECK (Hooft et al., 1996), are summarized in Table 1. A total of 2348 NOE distance restraints and 105 backbone f and 4 constraints were used as the CYANA calculation input. A superimposed ensemble of the 20 lowest target function structures is shown in Figure 1B (Ca trace). The rootmean-square deviation (rmsd) from the mean structure was 0.34 6 0.07 A˚ for the backbone (N, Ca, and C0 ) atoms and 0.75 6 0.08 A˚ for all heavy (nonproton) atoms in the well-ordered region (residues 174–266) (Table 1). Based on the values of the target function, the rmsd, and the Ramachandran plot, we concluded that the structures are well defined. Solution Structure of the LSD1 SWIRM Domain The LSD1 SWIRM domain consists of six a helices (Figure 1C). A long central helix (a4: Gln204–Asp223) is surrounded by five other helices (a1: Val174–Phe179; a2: Ser190–Cys195; a3: Pro197–Ile200; a5: Phe231– Gln237; and a6: Thr246–Arg258) (Figure 1C). The a5 and a6 helices are packed against one side of a4, and the a1, a2, and a3 helices are located on the opposite side of a4. There are many hydrophobic contacts from a4 to a6 that are formed by 13 hydrophobic residues (Phe210, Phe212, Ile213, Leu218, Leu220, Leu229, Leu238, Tyr242, Leu248, Val249, Val252, Leu256, and Ile262). In contrast, the part composed of the N-terminal

three helices has fewer hydrophobic contacts. Instead, Glu192 at a2 forms a salt bridge with Arg214 at a4, which is located in the core region of the protein (Figure 1D). Furthermore, in most of the conformers, hydrogen bonds involving threonine side chains were observed for Thr189 (Og1) with Glu192 (HN) in a2, Thr217 (Hg1) with Ile213 (O) in a4, Thr230 (Og1) with Ala233 (HN) in the a4-a5 loop, and Thr234 (Hg1) with Thr230 (O) in a5 (Figure 1D). These results are consistent with the observation of labile side chain protons such as the Hg1 of threonine and the H3/Hh of arginine. Overall Structural Features of the SWIRM Domain To clarify the structural features of the SWIRM domain, we aligned all of the nonredundant SWIRM domain sequences (87 sequences in total), defined as SWIRM in the Pfam database, by using CLUSTAL W (Higgins et al., 1992). The sequence alignment of 15 representative SWIRM domains is shown in Figure 2. Subsequently, we calculated the degree of conservation among the 87 SWIRM sequences at each amino acid position, by using the program ConSurf (Armon et al., 2001; Glaser et al., 2003), and mapped this result onto the structure of LSD1-SWIRM (Figure 3A). We found that Asp198 and Asp245 are evolutionarily highly conserved surface residues (Figure 3A). In addition, Arg251 (shown by a closed circle in Figure 2) is also a conserved charged residue on the molecular surface. Other conserved residues include the internal hydrophobic residues, such as Phe210, Val252, Tyr255, Leu256, and Ile262, as well as Glu192 and Arg214, which are responsible for salt bridge formation (Figure 2). Next, we investigated the hydrophobicity of the LSD1SWIRM surface by using the program Pymol (Figure 3B). This analysis revealed the presence of a hydrophobic cleft, composed of Phe196, Pro197, Ile199, and Leu248 (open circles in Figure 2), within the area surrounded by the conserved charged residues, Asp198, Asp245, and Arg251 (Figure 3B, indicated by an oval). The residues composing this hydrophobic cleft are highly conserved in the mammalian SWIRM domains (data not shown). Structural Homology to Other Proteins To understand the potential function of the SWIRM domain, we searched for structural homologs of LSD1SWIRM by using the Dali server (Holm and Sander, 1996). This Dali search identified several different structures as candidates of LSD1-SWIRM homologs. These include the N-terminal domain of the DNA topoisomerase VI A subunit (1d3y, Z score: 4.6, rmsd56CA: 2.2 A˚; [Nichols et al., 1999]), the Z-DNA binding domain (Za domain) of double-stranded RNA adenosine deaminase (1qbj, Z score: 4.5, rmsd52CA: 2.0 A˚; [Schwartz et al., 1999]), and the DNA binding region of the archaeal transcription initiation factor IIB (TFIIB) homolog (1ais, Z score: 3.7, rmsd68CA: 3.2 A˚; [Kosa et al., 1997]). Interestingly, the structural similarity was detected between an HTH motif involved in each of these structures and the part of LSD1-SWIRM composed of helices a4–a6 (colored green in Figure 3C). We considered the LSD1-SWIRM structure to be divisible into two parts—the N-terminal part comprising helices a1–a3 (residues 174–203; colored red in Figure 3C) and the C-terminal part comprising helices a4–a6

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Figure 2. Sequence Alignment of the SWIRM Domains The alignment includes representative SWIRM sequences of LSD1-, Swi3p-, and Ada2p-type subfamily members. Protein names and corresponding species (in parentheses) are indicated on the left. The secondary structure and the residue number of LSD1-SWIRM are shown at the bottom and the top of the alignment, respectively. The conserved hydrophobic residue composing the cleft is indicated by an open circle. The conserved acidic residue is indicated by a filled circle. Two conserved ionic residues involved in the salt bridge formation are indicated by ‘‘b.’’ The conserved basic residues among the LSD1-type SWIRM domains are indicated by filled triangles. The sequence alignment was produced with ClustalX (Thompson et al., 1997).

(residues 204–266; colored green in Figure 3C)—for the following three reasons: (1) there is a gap between helices a3 and a4 in the sequence alignment (Figure 2); (2) the salt bridge is formed between helix a2 at Glu192 and helix a4 at Arg214 (Figure 1C); and (3) while the part composed of helices a4–a6 showed structural similarity to HTH motifs, the part composed of helices a1–a3 cannot superimpose with any structures, as indicated by Dali homology search analyses. Structural Features of the N-Terminal Part Among all of the SWIRM family members, the conservation of the surface residues in the N-terminal part (Figure 3C, colored red) is relatively low, as compared with that in the C-terminal part (Figure 3C, colored green). However, when we examined the conserved residues among the members of the aminooxidase-like domain-containing SWIRM subfamily (LSD1-type subfamily), we found that the conserved surface residues were more prevalent within the N-terminal part (Figure 4A, left) than the C-terminal part (Figure 4A, right). In particular, we found that Arg182 and Arg187 (shown by closed triangles in Figure 2) are highly and specifically conserved among the LSD1-type subfamily members (Figure 4A). In LSD1-SWIRM, these residues are located on the surface with protruding, positively charged side chains (Figure 4B). Comparison of the C-Terminal Part to DNA Binding HTH Motifs As described, we detected the structural similarities between the C-terminal part (a4–a6) of LSD1-SWIRM and different variations of HTH motifs (Figure 5A). All of these

types of HTH motifs are involved in DNA binding and are commonly composed of a right-handed helical bundlelike triangle (Figure 5A). We also compared the C-terminal SWIRM part with the Myb DNA binding domain of Avian myeloblastosis virus Myb transforming protein (1h8a; Tahirov et al., 2002), which is a canonical threehelical bundle HTH motif (Figure 5A). Importantly, the relative orientation of a6 in SWIRM is different from that of the corresponding helix III in each of the HTHs (Figure 5A), and the angle made by a4 and a6 of LSD1SWIRM is sharper than the angles made by helices I and III of HTHs (Figure 5A). In addition, SWIRM has 8 residues of a helical loop between a5 and a6, while HTHs have less than 5 residues of a loop at the corresponding positions (between helices II and III in Figure 5A). Furthermore, helix a4 of LSD1-SWIRM (20 residues) is much longer than that of the corresponding helix (helix I) of HTHs (14–16 residues). The structure-based sequence alignment with DaliLite indicates that the amino acid sequences of the C-terminal SWIRM part and the HTH motifs are almost different (Figure 5B). We detected 14% sequence identity to the HTH of the topoisomerase VI A subunit, 6% to Myb, 13% to TFIIB, and 15% to Za (Figure 5B). The hydrophobic residues are rather highly conserved in the corresponding region; however, the residues involved in DNA binding (highlighted in Figure 5B) are not conserved between SWIRM and these HTH motifs (Figure 5B). Comparison of the C-Terminal Part to Non-DNA Binding HTH Motifs The aforementioned features imply that the C-terminal part of SWIRM might not be a DNA binding motif (see


Figure 4. Structural Fold of the N-Terminal SWIRM Part (A) Conserved residues among the LSD1-type subfamily members. The residues with a ConSurf score greater than 8 are shown in red. The orientation is the same as in Figure 3A. The view on the right is rotated 180º around the z axis from that on the left. (B) Electrostatic surface. The molecular surface is represented, and it is contoured from negative (red) to positive (blue) potentials. The orientation is the same as the left view in (A).

Figure 3. Overall Structural Features of LSD1-SWIRM (A) Conserved residues. The positions of the residues with a ConSurf score greater than 8 are highlighted in red on the gray overall structure. The view on the right is rotated 180º around the z axis from that on the left. (B) Surface properties. The positions of the hydrophobic residues located on the molecular surface are shown in yellow. The conserved hydrophobic residues (Phe196, Pro197, Ile199, and Leu248) are highlighted in green. The conserved Asp198 and Asp245 are shown in red, and the conserved Arg251 is shown in blue. The cleft formed by these residues is shown by a black oval. The orientation is the same as in (A). The view on the right is rotated 180º around the z axis from that on the left. (C) The N-terminal and C-terminal parts. Helices a1–a3 are shown in red in a ribbon representation, and a4–a6 are shown in green. The orientation is the same as in (A). The view on the right is rotated 180º degrees around the z axis from that on the left.

also Discussion). Since the C-terminal part of SWIRM showed the highest similarity to a three-helical bundletype HTH motif, we next compared it to non-DNA binding three-helical bundle-type HTH motifs, such as the deacetylase activation domain (DAD) and SANT domains. The DAD domain of the silencing mediator of retinoid acid and thyroid hormone receptor (SMRT) interacts with histone deacetylase (HDAC3) (Codina et al., 2005). The SANT domain seems not to bind to DNA (Gru¨ne et al., 2003), but it is considered to be a histone-interaction module (Boyer et al., 2002). Though we could not detect structural similarity between SWIRM and DAD or SANT domains by the DaliLite program, we superimposed the LSD1-SWIRM structure (red in Figure 6A)

with the DAD domain of SMRT (1xc5: blue) and the SANT domain of ISWI (1ofc: yellow) by using the HTH of the the topoisomerase VI A subunit as a mediating model structure. Structural features of these domains are individually shown in Figure 6B. We found that SWIRM also differs from both DAD and SANT (Figure 6A), based on the same reasons as those observed in comparison with the DNA binding types of HTH motifs (Figure 5A). Accordingly, LSD1-SWIRM forms a narrower concave between helices a4 and a6 (shown by a triangle in Figure 6B) than those formed by helices I and III (shown by squares in Figure 6B) of the DAD and SANT domains. The comparison of electrostatic potential surfaces indicates that the SANT domain reveals a considerably acidic surface, while SWIRM and DAD domains are obscure in electrostatic character (Figure 6C). Furthermore, the comparison of hydrophobic surfaces (Figure 6D) indicates that the concave surface formed by helices I and III are highly hydrophobic in both DAD and SANT domains (circled regions in Figure 6D), while the corresponding SWIRM region (a dotted circle in Figure 6D) is not hydrophobic. In SWIRM, hydrophobic residues are rather prominent at the cleft, composed of residues at helix a6 and the loop between a2 and a3, which specifically exists in the SWIRM domain structure (shown by an oval in Figure 6D; see also the right side of Figure 3B). Interaction with a Histone H3 Tail The presence of the hydrophobic cleft (shown in an oval on the right side of Figure 3B), with a hydrophobic surface composed of Phe196, Pro197, Ile199, and Leu248, and a potentially charged surface comprising

Structure 462

Figure 5. Comparison of the C-Terminal SWIRM Part and DNA Binding HTH Motifs (A) Superimposition of human LSD1-SWIRM (red) and HTH motifs of the topoisomerase VI A subunit (green; [Nichols et al., 1999]), v-Myb (cyan; [Tahirov et al., 2002]), archaeal TFIIB homolog (yellow; [Kosa et al., 1997]), and Za of human ADAR1 (blue; [Schwartz et al., 1999]). The orientation of LSD1-SWIRM is the same as the right views of Figure 3. All of these types of HTH motifs are involved in DNA binding, and they are commonly composed of a right-handed helical bundle-like triangle. (B) Structure-based sequence alignment of LSD1-SWIRM and HTH motifs. The residues of the HTH motifs involved in DNA interaction are highlighted. The helical regions are indicated by boxes. The residue numbers of LSD1 are shown at the top.

Asp198, Asp245, and Arg251, led us to postulate that this cleft might be a binding pocket for chromosomal molecules. The SWIRM domains exist in several histone-interactive proteins, such as a histone demethylase and some ATP-dependent chromatin-remodeling subunits. In addition, the LSD1-type subfamily members lack the SANT domains, which may bind histone tails (Boyer et al., 2004; de la Cruz et al., 2005). Furthermore, LSD1 specifically demethylates mono- or dimethylated H3 at Lys4 (Shi et al., 2004). We thus hypothesized that this cleft could be a potential binding pocket for histone H3. To investigate the interaction between LSD1-SWIRM and a histone H3 tail, we performed an in vitro binding assay by the surface plasmon resonance (SPR) technique, by using synthetic peptides of core histone tails. Notably, we detected that LSD1-SWIRM interacted with an N-terminal tail (residues 1–20) of human histone H3 (Figure 7A and Table 2), while it did not bind to a C-terminal tail of H3 (Figure 7E) or an N-terminal tail of H4 (Figure 7F). LSD1-SWIRM also bound to the Lys4-dimethylated and the Lys9-dimethylated H3 tails with almost the same affinity (Figures 7B and 7C). Since we could build a docking model of the cleft of LSD1-SWIRM complexed with the N-terminal tail (residues Lys4–Gly13) of histone H3 (data not shown), we next examined the

LSD1-SWIRM interaction with the H3 peptide bearing mutations of the Arg8 and Thr11 residues, which are predicted to interact with LSD1-SWIRM from our structural model. We changed the H3 sequence at position 8 from Arg to Glu, and that at position 11 from Thr to Lys, to disrupt possible interactions with Asp198, and Phe196 and Asp198, of LSD1-SWIRM, respectively. As expected, we found that the interaction between LSD1SWIRM and this mutant H3 peptide (H3[1–20]-R8E/ T11K) was weaker (Figure 7D; also see Table 2). Taken together, these results indicate that LSD1-SWIRM could interact with an N-terminal tail sequence of histone H3. Discussion The SWIRM Domain Structure In this study, we have described the first, to our knowledge, tertiary structure of a SWIRM domain, which exists in several different chromosomal proteins. The SWIRM domain of human histone demethylase LSD1 forms a compact domain architecture consisting of 6 a helices, where the 20 amino acid long helix (helix 4) is surrounded by 5 other helices (Figure 1C). We found that SWIRM is composed of two different parts: an Nterminal fold part (colored red in Figure 3C) and a C-terminal HTH-related fold part (colored green in Figure 3C).


Figure 6. Comparison of the C-Terminal SWIRM Part and Non-DNA Binding HTH Motifs (A) Superimposition of LSD1-SWIRM (red) and HTH motifs of the DAD domain of SMRT (blue; [Codina et al., 2005]) and SANT domain of ISWI (yellow; [Gru¨ne et al., 2003]). The orientation of LSD1-SWIRM is the same as the right views of Figure 3. (B) Ribbon representation of the SWIRM domain (red; middle), the DAD domain (blue; left), and the SANT domain (yellow; right). The space between helices I and III is schematically indicated by a filled triangle or square. (C) Electrostatic potential surfaces of SWIRM (middle), DAD (left), and SANT (right). Positive, negative, and neutral electrostatic potentials are shown in blue, red, and white, respectively. (D) Distribution of hydrophobicity on the surfaces of SWIRM (middle), DAD (left), and SANT (right). The hydrophobic residues on the molecular surface are colored in yellow. In DAD and SANT, possible binding sites are indicated by circles. In SWIRM, the region corresponding to the possible binding site of DAD or SANT is indicated by a dotted oval, and the SWIRM-specific hydrophobic cleft is indicated by an oval.

The conserved residues among the SWIRM family members are located in the core hydrophobic domain and at the positions that form a salt bridge (Glu192 and Arg214; Figure 1C) to connect these two parts. We thus propose that the SWIRM domains generally form a similar backbone structure to this LSD1-SWIRM structure. From the viewpoints of sequence conservation (Figure 2) and domain architecture, the SWIRM domains have been classified into three types (described in the Introduction, see also Figure 1A). The Swi3p-type and Ada2p-type proteins both bear a SANT domain, although their domain compositions differ. The LSD1-type proteins, including LSD1 histone demethylase, carry an aminooxidase-like domain at their C termini. Several surface residues, such as Asp198, Asp245, and Arg251 (Figure 3B, right), are highly conserved among these three SWIRM types, indicating that they are commonly utilized for the structure and/or function of the SWIRM domain. On the other hand, Arg182 and Arg187, located between a1 and a2, are exclusively conserved in the LSD1-type subfamily (Figures 2 and 4A). These conserved residues extend beyond the compact SWIRM do-

main (Figure 4B). The sequence conservation (40% similarity) between the LSD1 demethylase domain and the polyamine oxidase domain suggests that the LSD1 demethylase domain shares a similar fold with that of Zea mays polyamine oxidase (1b5q), which has a 30 A˚ long, U-shaped catalytic tunnel (Binda et al., 1999). The polyamine oxidase has several anionic surfaces, including the two tunnel openings (Binda et al., 1999). Considering that the N-terminal parts of the LSD1-type SWIRM domains specifically conserve the arginine-extended surface, the SWIRM domain belonging to the LSD1-type subfamily might form an intramolecular ionic interaction with the aminooxidase-like domain. We obtained a good, complementary fit of this N-terminal SWIRM structure with the anionic concave region of the polyamine oxidase (data not shown). In addition to the aforementioned features, LSD1 is involved in the huge CtBP protein complex, which includes several enzymatic activities (Shi et al., 2003), and LSD1SWIRM might be a potential domain for tethering the LSD1 histone demethylase to the CtBP complex. The N-terminal LSD1-SWIRM part might also function in

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Figure 7. Interaction between LSD1-SWIRM and the Peptides of the Histone Sequences (A–F) Biotinylated peptides were immobilized onto the streptavidin chips, and 64 mM (blue), 32 mM (green), 16 mM (cyan), or 8 mM (magenta) LSD1SWIRM protein solution was passed over the chip at a 20 ml/min flow rate for 2 min. Biotinylated peptides used are (A) H3(1–20), (B) H3(1–20)K4diMe, (C) H3(1–20)-K9diMe, (D) H3(1–20)-R8E/T11K, (E) H3(122–135), and (F) H4(1–15). The x axis indicates the time course. Each injection starts at 0 s and ends at 120 s. The y axis indicates the difference in the resonance units between the peptide bound and peptide unbound flow cells.

association with a subunit(s) of the CtBP multienzymatic complex. The HTH-Related Fold of the SWIRM Domain The SWIRM structure has a HTH-related fold at its C terminus. From the point of structural features, the HTH folds can be classified into roughly two major types (reviewed in Aravind et al., 2005). One type is the HTH domain with a simple three-helical bundle and its extensions, and the other type is the winged HTH domain (Aravind et al., 2005). We compared the LSD1-SWIRM structure with both types (three- or multihelical bundle types, and winged type) of DNA binding HTHs, and the three-helical bundle type of non-DNA binding HTHs (Figures 5A and 6), and we found three major difference in their structures. We postulated that the DNA binding mode for canonical DNA binding HTH motifs (Myb, TFIIB, and Za) is unlikely for the C-terminal part of SWIRM. Helix a6 of SWIRM, which is corresponding to DNA binding helix III in HTH, is oriented differently from those of HTHs. This atypical orientation of helix a6 is supposed to be strained by the longer helical loop between helices a5

and a6 of SWIRM. Furthermore, the HTH residues involved in DNA binding are not conserved at corresponding SWIRM residues in a structure-based alignment (Figure 5B). It is also difficult to consider that the SWIRM domain exhibits a similar protein binding mode of canonical protein binding HTHs, because (1) SWIRM has a narrower concave than the corresponding protein binding concaves of HTHs (Figure 6B) and (2) the concave surface of SWIRM is neither conserved nor hydrophobic (Figure 6D). On the other hand, SWIRM has a conserved hydrophobic cleft between a6 and the loop between a2 and a3, including highly conserved acidic residues Asp198 and Asp245 (highlighted by an oval on the right side of Figure 3B; also shown in Figure 6D). The extension of helix a4 seems to be a key determinant of this cleft as well as the overall SWIRM conformation, by forcing N- and C-terminal parts to maintain a stable distance. Implications for the Interaction with Histone Tails We hypothesized that this SWIRM-specific cleft could be a binding pocket for a histone. By using the SPR technique, we detected an interaction between


Table 2. LSD1-SWIRM Binding to the Peptides of the Histone Sequences Peptide

Sequence

Binding

H3(1–20) H3(1–20)-K4diMe H3(1–20)-K9diMe H3(1–20)-R8E/T11K H3(122–135) H4(1–15)

ARTKQTARKSTGGKAPRKQLka-biotin ARTKbQTARKSTGGKAPRKQLka-biotin ARTKQTARKbSTGGKAPRKQLka-biotin ARTKQTAEKSKGGKAPRKQLka-biotin biotin-KDIQLARRIRGERA SGRGKGGKGLGKGGAgsgska-biotin

++ ++ ++ +/2 — —

a b

An amino acid in lower-case indicates a spacer sequence. Dimethylated lysine.

LSD1-SWIRM and the N-terminal tail (residues 1–20) of histone H3 (Figure 7A). Since LSD1 is responsible for the demethylation of mono- or dimethylated Lys4 of histone H3 (Shi et al., 2004), we tested LSD1-SWIRM binding to dimethylated H3 peptides. However, LSD1SWIRM binding to histone H3 was independent of the dimethylation of Lys4 or Lys9 (Figures 7B and 7C), suggesting that LSD1-SWIRM binds to the N-terminal tail of histone H3 without recognizing the side chain methylation of lysine residues. On the other hand, the mutations in the histone H3 sequence affected the interaction with LSD1-SWIRM (Figure 7D), suggesting specific recognition of the histone H3 tail sequence by LSD1-SWIRM. Such an interaction between LSD1-SWIRM and the histone tail would help the C-terminal enzymatic domain of LSD1 demethylate the Lys4-methylated histone H3. On the other hand, we could not detect the interaction between LSD1-SWIRM and the peptide of the N-terminal H3 sequence by our NMR chemical shift perturbation analysis (data not shown). In our SPR analysis, the peptide of the N-terminal H3 sequence (residues 1–20), or its relative peptide, was immobilized at its C terminus onto the chip, to mimic the chromosomal architecture that is assumed to be abundant in the flanking N-terminal tails of core histones. Our results indicate that LSD1-SWIRM might need such an architecture for interaction with the N terminus of histone H3, and might not, or quite scarcely, bind to the free form of the peptide in solution. Additional biochemical studies are required to understand this interaction mechanism. The Swi3p-type and Ada2p-type SWIRM proteins both carry the SANT domains (Aasland et al., 1996). Although the SANT domain is structurally related to the DNA binding domain of Myb (Gru¨ne et al., 2003), the intact Ada2p SANT domain may be required for an interaction with unmodified histone H3 tails (Boyer et al., 2002), supporting the view that the SANT domain is a histone binding motif (Boyer et al., 2004; de la Cruz et al., 2005). Our results also raised the possibility that SWIRM is a novel histone tail binding domain. It would be important to investigate the abilities of the LSD1-type SWIRM domains, as well as the Swi3p-type and Ada2p-type SWIRM domains, to interact with intact histones in vitro and in vivo. Recently, LSD1 was found to interact with androgen receptor (AR) through the SWIRM domain (Metzger et al., 2005). The LSD1 SWIRM domain was reported to interact with several different domains of AR, though it showed the strongest interaction with the DNA binding domain of AR (Metzger et al., 2005). Thus, the SWIRM domain might interact with several different components involved in chromatin-related reactions.

Additional structural studies of the SWIRM-containing proteins and functional identification of SWIRM-interactive proteins will be essential to clarify the regulatory mechanisms of the histone demethylation reaction and the chromatin-remodeling process.

Experimental Procedures Protein Expression The cDNA fragment encoding the SWIRM domain (the amino acid sequence from Glu203 to Gly313) of human LSD1/KIAA0601 (AB011173.1) was amplified by PCR. Regarding Met35 of KIAA0601 as the putative first methionine (Met1), we defined the position of the LSD1 SWIRM domain as Glu169–Gly279. This sequence position is consistent with that of the translated amino acid sequence of the human AOF2 transcript variant 2 (NM_015013). The amplified cDNA fragment was subcloned into the expression vector pCR2.1 (Invitrogen) as a fusion with an N-terminal histidine affinity tag and a TEV protease cleavage site. The 13C/15N-labeled fusion protein was synthesized by a cell-free protein expression system (Kigawa et al., 1999, 2004).

Protein Purification The cell-free reaction solution was first adsorbed to a HiTrap Chelating column (Amersham Biosciences), which was washed with buffer A (50 mM sodium phosphate buffer [pH 8.0] containing 500 mM sodium chloride and 20 mM imidazole) and was eluted with buffer B (50 mM sodium phosphate buffer [pH 8.0] containing 500 mM sodium chloride and 500 mM imidazole). To remove the histidine tag, the eluted protein was incubated at 30ºC for 1 hr with the TEV protease. After dialysis against buffer A without imidazole, the dialysate was mixed with imidazole (20 mM final concentration) and then applied to a HiTrap Chelating column, which was washed with buffer A. The flow-through fraction was dialyzed against buffer C (20 mM sodium phosphate buffer [pH 7.2] containing 1 mM PMSF). The dialysate was fractionated on a HiTrap SP column by a concentration gradient of buffer C and buffer D (20 mM sodium phosphate buffer [pH 7.2] containing 1 M sodium chloride and 1 mM PMSF), and the SWIRM-containing fractions were collected. For NMR measurements, the purified protein was concentrated to w1 mM in 1 H2O/2H2O (9:1) 20 mM d-Tris buffer (pH 7.0), containing 100 mM NaCl, 1 mM 1,4-DL-dithiothreitol-d10 (d-DTT), and 0.02% NaN3.

NMR Spectroscopy All NMR data were collected on a Bruker AVANCE600 spectrometer equipped with a triple-resonance CryoProbe, while the NOESY spectra were obtained on a Bruker AVANCE800 spectrometer at 298 K. Sequence-specific backbone chemical shift assignments were achieved with the 13C/15N -labeled sample, by using standard triple-resonance experiments (Cavanagh et al., 1996). Assignments of side chains were obtained from HBHA(CBCA)(CO)NH, C(CO)NH, HC(C)H-TOCSY, and HC(C)H-COSY spectra (Ikura et al., 1990; Bax et al., 1994; Kay, 1997; Sattler et al., 1999). 15N-edited NOESY and 13 C-edited NOESY spectra with 80 ms mixing times were used to determine the distance restraints.

Structure 466

Resonance Assignments Protein samples of the 13C/15N-labeled LSD1 SWIRM domain, composed of 124 amino acid residues (region from Glu169 to Gly279), had nonnative expression tag-derived sequences at the N terminus (GSSGSSG) and the C terminus (SGPSSG). The backbone resonance assignments were almost complete, except for Ser190, Gln204, Lys277, and 9 residues in the nonnative expression tag-derived sequence. The side chain assignments were complete, with the exception of Hz of Phe179, H31 of His185, Hz of Phe231, H31 of His185, Hg of Pro272, and Ha/Hb/Hg/Hd/H3 of Lys276. Among the labile side chain protons, the amide groups of all of the asparagine and glutamine residues, the 3-proton resonances of Arg214 and Arg216, and the h-proton resonance of Arg214 were assigned by using NOEs. The hydroxyl proton resonances of Thr189, Thr217, Thr230, and Thr234 were observed and assigned. For residue Pro241, the cis conformation was confirmed independently by the intensity of the sequential NOESY crosspeaks (Wu¨thrich, 1986) and by the Cb and Cg chemical shift differences (Schubert et al., 2002).

Structure Determination and Analysis The spectra were processed with the program NMRPipe (Delaglio et al., 1995). The programs KUJIRA (N. Kobayashi, personal communication) and NMRView (Johnson and Blevins, 1994; Johnson, 2004) were employed for visualization of the NMR spectra and chemical shift assignments. Automated NOE assignments and structure calculations with torsion angle dynamics were performed by using the software package CYANA 2.0.17 (Gu¨ntert et al., 1997; Herrmann et al., 2002; Jee and Gu¨ntert, 2003; Gu¨ntert, 2004). Dihedral angle restraints were derived by using the program TALOS (Cornilescu et al., 1999). A total of 100 structures were independently calculated. The 20 conformers with the lowest target-function values were finally selected. Structural quality was evaluated with PROCHECK-NMR (Laskowski et al., 1996). The programs MOLMOL (Koradi et al., 1996) and PyMol (DeLano, 2002) were used to analyze the calculated structures and to prepare drawings of the structures.

Binding Assay Surface plasmon resonance (SPR) was carried out on a Biacore 3000 instrument, by using streptavidin chips from the manufacturer (Biacore). Approximately 1000 resonance units of biotinylated peptides were immobilized to the chip with the HBS-EP running buffer, containing 10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, and 0.005% Tween-20. Purified LSD1-SWIRM protein (concentrations: 64–8 mM) was loaded for 2 min with a 20 ml/min flow rate onto the chip, and the flow continued for 2 min to detect the dissociation of LSD1-SWIRM from the immobilized peptides. After each binding experiment, the immobilized peptide was regenerated by stripping the protein from the surface with 20 or 40 ml 10 mM glycine-HCl (pH 1.5 or 2.0) for 1 or 2 min. Control surfaces with no peptide attached were used to correct for refractive index differences between the samples and the running buffer. The results were analyzed and visualized with BIAevaluation 4.1 software.

Acknowledgments We thank T. Matsuda, Y. Fujikura, N. Matsuda, Y. Motoda, Y. Miyata, A. Kobayashi, F. Hiroyasu, Y. Nishimura, M. Watanabe, T. Harada, S. Yasuda, Y. Tomabechi, C. Ogasa, A. Kikuchi, and Y. Yamaguchi for kind assistance; B. Padmanabhan and Y. Nakamura for unpublished results; H. Hirota and K. Yasumuro for discussion; and N. Nameki for critically reading the manuscript. This work was supported by the RIKEN Structural Genomics/Proteomics Initiative (RSGI) and by the National Project on Protein Structural and Functional Analyses, Ministry of Education, Culture, Sports, Science and Technology of Japan. Received: July 22, 2005 Revised: November 6, 2005 Accepted: December 15, 2005 Published online: March 14, 2006

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Accession Numbers The structure coordinates of the human LSD1 SWIRM domain have been deposited in the Protein Data Bank under the accession code 2com. The assignments have also been deposited in the Wisconsin chemical database under the accession code 10011.