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Molecular Mimicry and Ligand Recognition in Binding and Catalysis by the Histone Demethylase LSD1-CoREST Complex

Riccardo Baron,1,* Claudia Binda,2 Marcello Tortorici,2 J. Andrew McCammon,1 and Andrea Mattevi2,* 1Department of Chemistry and Biochemistry, Center for Theoretical Biological Physics, and Department of Pharmacology, Howard Hughes Medical Institute, University of California at San Diego, La Jolla, CA 92093-0365, USA 2Department of Genetics and Microbiology, University of Pavia, Via Ferrata 1, 27100, Pavia, Italy *Correspondence: [email protected] (R.B.), [email protected] (A.M.) DOI 10.1016/j.str.2011.01.001

SUMMARY (LSD1 or KDM1A, according to the newly adopted nomencla- ture) (Shi et al., 2004; Forneris et al., 2005). This enzyme acts Histone demethylases LSD1 and LSD2 (KDM1A/B) on mono- and dimethylated Lys4 of histone H3 through a catalyze the oxidative demethylation of Lys4 of FAD-dependent oxidative process (Figure 1A). LSD1 is often histone H3. We used molecular dynamics simula- associated to the histone deacetylases (HDAC) 1 and 2 and to tions to probe the diffusion of the oxygen substrate. the corepressor protein CoREST, which tightly binds to LSD1 Oxygen can reach the catalytic center independently enhancing both its stability and enzymatic activity. A number of from the presence of a bound histone peptide, studies have indicated that LSD1-CoREST interacts with various protein complexes involved in gene regulation and chromatin implying that LSD1 can complete subsequent deme- modification (Forneris et al., 2008; Mosammaparast and Shi, thylation cycles without detaching from the nucleo- 2010). Especially relevant for our investigations is the finding somal particle. The simulations highlight the role of that LSD1 is recruited to target gene promoters by interacting a strictly conserved active-site Lys residue providing with the N-terminal SNAG domain of SNAIL1, a master regulator general insight into the enzymatic mechanism of of the epithelial-mesenchymal transition, which is at the heart of oxygen-reacting flavoenzymes. The crystal structure many morphogenetic events including the establishment of of LSD1-CoREST bound to a peptide of the transcrip- tumor invasiveness (Lin et al., 2010)(Figure 1B). A further addition factor SNAIL1 unravels a fascinating example of tion to this biological complexity has been the discovery of molecular mimicry. The SNAIL1 N-terminal residues LSD2 (or KDM1B), a mammalian homolog of LSD1 (Karytinos bind to the enzyme active-site cleft, effectively et al., 2009). LSD2 exhibits the same H3-Lys4 demethylase mimicking the H3 tail. This finding predicts that other activity as LSD1 but it functions in distinct transcriptional complexes with specific biological functions (Ciccone et al., members of the SNAIL/Scratch transcription factor 2009; Fang et al., 2010; Yang et al., 2010). family might associate to LSD1/2. The combination LSD1 and LSD2 are distinguished from the histone demethy- of selective histone-modifying activity with the lases of JmjC class that have been identified in the last years. distinct recognition mechanisms underlies the bio- The JmjC enzymes display wider substrate specificity by acting logical complexity of LSD1/2. on mono-, di-, and/or trimethylated Lys residues and function through an iron-dependent catalytic mechanism that produces formaldehyde as side product (Horton et al., 2010; Tsukada INTRODUCTION et al., 2006). Conversely, LSD1/2 are flavoenzymes that use FAD to oxidatively demethylate their substrate. The reduced Large chromatin complexes finely regulate eukaryotic gene flavin, generated on Lys demethylation, is reoxidized by molec-

expression and are selectively recruited to DNA sequences by ular oxygen (O2) with the production of hydrogen peroxide in specific transcription factors. The post-translational modifica- addition to formaldehyde (Figures 1A and 2)(Forneris et al., tions on the histone N-terminal tails protruding from the nucleo- 2005; Shi et al., 2004). This peculiar hydrogen-peroxide gener- somal particle play fundamental roles in gene expression by ating activity of LSD1/2 represents an important aspect because dictating an epigenetic code that flags the activation or repres- the reaction product and its reactive oxygen species are poten- sion status of a gene (Jenuwein and Allis, 2001). These histone tially dangerous in the context of the chromatin environment. An marks are recognized by transcription factors and are dynami- intriguing hypothesis is that they might have a signaling role in cally regulated by specific histone-modifying enzymes (Ruthen- cellular processes, further expanding the biological roles and burg et al., 2007). functions of LSD1/2 (Amente et al., 2010; Forneris et al., 2008; Methylation of histone Lys residues is catalyzed by histone Winterbourn, 2008). methyl-transferases, a process thought to be irreversible for Here, we study the mechanisms and processes of molecular decades. This view was challenged by the discovery of the first recognition in LSD1 with a focus on the recognition of substrates histone lysine demethylase, lysine-specific demethylase 1 and protein ligands. How does oxygen diffuse into the catalytic

anisms underlying the ability of LSD1/2 to interact and recruit many distinct protein partners and does binding to these partners affect substrate recognition (Forneris et al., 2008; Mosammaparast and Shi, 2010)? To advance our understanding about these points, we have investigated the crystal structure of LSD1-CoREST in complex with an N-terminal peptide of human SNAIL1 (Figure 1B). Furthermore, we have probed the mecha-

nisms of O2 diffusion in LSD1-CoREST using molecular dynamics (MD) simulations. Our studies provide a molecular framework for the processivity of LSD1, which is able to catalyze the subsequent removal of two methyl groups from dimethylated H3-Lys4 residue (Figure 1A). They also highlight the factors that determine peptide recognition and predict that a whole class of transcription factors is likely to use a most unusual and fascinating ‘‘histone-mimicking’’ mechanism for binding to LSD1.

RESULTS Figure 1. Histone Demethylation by LSD1 (A) Scheme of the LSD1-catalyzed amine oxidation reaction. This enzyme acts on mono- and dimethylated Lys4 of histone H3 and is able to subsequently Structural Analysis of SNAIL1 Recognition by LSD1 remove two methyl groups from a dimethylated substrate. The reactants We used a combination of biochemical and structural experi- involved in the simulated system are highlighted in red and correspond to ments to investigate the interaction between LSD1 and the the protein-bound FADH (Figure 2), the histone H3 N-terminal peptide transcription factor SNAIL1, which was originally discovered by (Figure 1B), and O2. Lin et al. (2010). On the basis of a weak sequence similarity (B) Sequence alignment of the N-terminal residues of histone H3 and the between SNAIL1 and the histone H3 N-terminal tail (in essence, N-terminal sequences of an evolutionary-related family of C2H2 zinc-finger transcription factors that includes SNAIL1 (Barrallo-Gimeno and Nieto, a conserved pattern of positively charged residues) (Figure 1B), 2009). OVO-like1 is an epidermal proliferation/differentiation factor homolo- the authors of this study proposed that SNAIL1 could bind to gous to a protein originally identified in Drosophila melanogaster ovary cells LSD1 in the same site as the histone H3 substrate, i.e., in the (Nair et al., 2006). Scratch proteins are expressed in the nervous system in catalytic site. This model for SNAIL1-LSD1 interactions implied both developing and adult cells (Marıń and Nieto, 2006). Growth factor that SNAIL1 should inhibit LSD1 enzymatic activity. Consistently, independence 1 (gfi1) is a gene repressor involved in hematopoiesis whose we found that a 20-amino-acid peptide corresponding to SNAIL1 expression was already demonstrated to be regulated by LSD1-containing N-terminal residues effectively inhibits LSD1-CoREST. In more complexes (Saleque et al., 2007). Insulinoma-associated 1 (insm1) was originally isolated from neuroendocrine tumor cells and data suggest a role in detail, fitting of the enzyme initial velocities to the equation for differentiation of both neural and pancreatic precursors (Lukowski et al., competitive inhibition resulted in a Ki values of 0.21 ± 0.07 mM 2006). Conserved residues are highlighted in green (identity) or magenta (using a monomethyl-Lys4 H3-peptide as substrate) and 0.22 ± (similarity). Lys4 of LSD1 is indicated in blue. 0.09 mM (using a dimethylated substrate), indicating a rather tight binding. Likewise, we found that also LSD2 binds the site? Do the very different enzyme substrates, a small molecule SNAIL1 peptide (albeit with lower affinity; Ki value of 2.22 ± such as oxygen and a large protein complex such as the nucle- 0.36 mM), which is in line with the previously observed similarities osome, interfere with each other? What are the molecular mech- in the binding properties of LSD1 and LSD2 (Karytinos et al., 2009). Thus, the possibility exists that, in addition to LSD1, SNAIL1 might interact and represent a protein partner also for LSD2. To dissect the mechanism of SNAIL1 recognition by LSD1, we soaked LSD1-CoREST crystals in a solution containing the SNAIL1 peptide used for the inhibition studies (Figure 1B) and we determined the crystal structure of the ternary complex at 3.0 A˚ resolution (Figures 3A and 3B and Table 1). Inspection of the unbiased electron density map (Figure 3B) allowed us to trace the polypeptide chain for the N-terminal nine residues of the SNAIL1 sequence whereas residues 10–20 of the peptide could not be identified in the electron density most likely because they lack an ordered conformation. Peptide binding does not induce any conformational change in the active site as compared to the ligand-free structure (root-mean-square deviation [RMSD] = 0.4 A˚ for the 799 equivalent Ca atoms of LSD1-CoR- Figure 2. Chemical Representation of the Reduced Flavin Adenine EST). The peptide occupies the open cleft that has been shown Dinucleotide (FADH–) Molecule The C4a and C5a atoms relevant for the present study are labeled. The flavin is to form the binding site for the H3 tail (Forneris et al., 2007). The N facing the viewer with its re-side (the si-side is on the opposite side). See also terminus (residues 1–4) adopts a helical turn conformation, Figure S5. closely resembling that of the first residues of the H3 tail (Figures

Figure 3. X-ray structure of LSD1-CoREST in complex with the SNAIL1 peptide (A) Overall ribbon representation of the ternary complex of LSD1 (cyan), CoREST (blue), and the SNAIL1 peptide (orange). The FAD cofactor is in yellow sticks. (B) Fitting of the SNAIL1 peptide (carbons in orange) into the unbiased electron density map contoured at 1.2 s calculated with weighted 2Fo-Fc coefﬁcients. Color-coding and orientation are as in (A). (C) Binding of the SNAIL1 peptide in the LSD1 active site. Color-coding and orientation are as in (A) with H-bonds shown as dashed lines. Red labels are used to highlight residues in

direct contact with oxygen pathways in O2-bound simulations. (D) Superposition between SNAIL1 (orange) and histone H3 (gray) peptides. Conserved peptide side chains (Figure 1B) are drawn in stick representation. The FAD cofactor and the Lys661 side chain are shown as reference. The water bridging Lys661 and ﬂavin N5 (as observed in the unbound enzyme structure that has been solved at higher resolution) are also displayed (Yang et al., 2006). See also Figure S1.

and establishes similar interactions (with Cys360, Asp375, and Glu379) as Arg8 of H3. Likewise, Lys8 of SNAIL1 falls in the same solvent-exposed position observed for Lys9 of H3. Taken together, these ﬁndings highlight three key points: (1) the cavity of LSD1 speciﬁcally recognizes the N-terminal amino group of the peptide ligands; (2) the conserved pattern of positively charged groups and small hydroxyl side chains shared by SNAIL1 and H3 N-terminal tails enable them to bind to LSD1 in a similar conformation; and (3) SNAIL1 N-terminal residues act as a mimic of H3 being able to effectively bind to the enzyme active-site cleft.

Conformational Dynamics of Free and Substrate-Bound LSD1-CoREST Complex Systems We extended our studies on peptide recognition in LSD1-CoREST by analyzing conformational dynamics of the LSD1-CoREST system by MD 3C and 3D). In particular, the N-terminal amino group of Pro1 and simulations in solution. This study was based on two 50-ns- the side chains of Arg2 and Ser3 bind deeply into the cleft and long MD trajectories using LSD1-CoREST crystallographic coor- establish several H-bonding interactions with the surrounding dinates (Yang et al., 2006) in the ligand-free state (hereafter protein residues, in an arrangement almost identical to that referred to as ‘‘unbound’’) and in complex (‘‘bound’’) with the exhibited by the Ala1-Arg2-Thr3 residues of the H3 peptide. histone H3 peptide used in the crystallographic studies (Figure 3) Thus, Pro1 binds to the carbonyl of Ala539 at the C terminus of in which Lys4 is replaced by Met (Lys4Met mutation was previ- a-helix 524–540 whereas Arg2 interacts with Asp553 and ously shown to greatly increase binding affinity) (Forneris et al., Asp556. This binding conformation positions Phe4 of SNAIL1 2007). Figure 4 summarizes the analysis of the backbone Ca in front of the flavin to occupy a location corresponding to that atom-positional RMSD values of LSD1-CoREST MD trajectory of Lys4 of the H3 tail (Figure 3D). Phe4 snugly fits in its binding snapshots from the X-ray structures. After a first initial phase of niche by making edge-to-face interactions with the rings of the about 4 ns—during which the most flexible loop regions relax flavin cofactor and Tyr761. The conformation of the SNAIL1 in solution—RMSDs fluctuate stably. Overall, CoREST and the peptide deviates from that of the H3 tail after residues 4–5 to LSD1 tower domain is somewhat more flexible compared to compensate for the deletion of one residue compared to the LSD1, largely owing to relative motion between the CoREST histone sequence (Figures 1B, 3C, and 3D). However, the SANT2 domain and the LSD1 tower domain (Figure 3A). Differ- comparative analysis of the H3 and SNAIL1 complexes clearly ences in molecular fluctuations between LSD1 unbound and indicates that Arg7 of SNAIL1 occupies the same position bound are moderate (see Figure S1 available online) and

Table 1. Data Collection and Refinement Statistics for This first set of simulations indicated that binding of the histone LSD1-CoREST- SNAIL1 Peptide Complex H3 N-terminal peptide has only local packing effects. No signif- Space group I222 icant impact on the overall dynamics of the LSD1-CoREST complex as well as of LSD1 and CoREST individual partners Unit cell axes (A˚ ) 119.2, 181.5, 234.4 was observed over the simulated timescales. The binding site Resolution (A˚ ) 3.0 for the histone tail on LSD1-CoREST is essentially preorganized a,b Rsym (%) 9.8 (53.7) to host and stabilize the peptide in the observed folded confor- b Completeness (%) 99.7 (100.0) mation (Forneris et al., 2007). This is consistent with the X-ray Unique reflections 50,937 data on the SNAIL1 complex. Also in that case, the bound Redundancy 3.6 (3.7) peptide seems to adapt its conformation to the shape of the I/sb 9.5 (1.9) binding cleft whose H-bond acceptors function as anchoring No. of atoms protein/FAD/ligandc 6286/53/77 elements for the positively charged groups of the peptide (Figures 3C and 3D). Average B value for ligand atoms (A˚ 2) 75.2 d Rcryst (%) 21.2 d Oxygen Diffusion into Free and Substrate-Bound Rfree (%) 24.6 LSD1-CoREST Complex ˚ RMS bond length (A) 0.014 Having inspected the structure and dynamics of peptide binding, RMS bond angles () 1.49 we investigated the diffusion of O2 molecules into the active site RMS: root-mean-squareP P of LSD1-CoREST to probe the mechanisms of oxygen migration a j j th Rsym = Ii / Ii, where Ii is the intensity of i observation and and binding in this enzymatic system (Figure 1A). The integration is the mean intensity of the reflection. of biochemical and structural experiments with powerful MD b Values in parentheses are for reflections in the highest resolution shell. simulations has already shown to be an effective strategy to c The final model consists of residues 171–836 of LSD1, a FAD molecule, investigate the relationship between enzyme dynamics and residues 308–440P of CoREST,P and residues 1–9 of the SNAIL1 peptide. d oxygen biocatalysis (Baron et al., 2009a, 2009b; Saam et al., Rcryst = jFobs Fcalcj/ jFobsj where Fobs and Fcalc are the observed and calculated structure factor amplitudes, respectively. The set of 2007, 2010). Our computational approach intended to address a crucial question about LSD1 molecular function: does binding reflections used for Rfree calculations and excluded from refinement was extracted from the structure factor file relative to the PDB entry of the histone peptide in the active site cleft interfere with the 2V1D. diffusion of molecular oxygen into the active center? Two sets of five independent MD simulations were initialized based, respectively, on the X-ray structures of the unbound and histone changes on substrate binding are limited to the side chain motion peptide-bound LSD1-CoREST complexes and were analyzed to of residues in direct contact with the H3 peptide (not shown). probe the effect of peptide binding on oxygen diffusion. The FAD These observations are further supported by analysis along was set to be in the reduced FAD form (FADH)(Figure 2)asitis the trajectories of the backbone Ca atoms of the scaled size- the case for the enzymatic state that undergoes reoxidation independent similarity parameter (Maiorov and Crippen, 1995), following Lys demethylation (Figure 1A). After equilibration, as described in Figure S2. The ensemble averaged rSC values 100 O2 molecules were added to the bulk water in each system are 0.2 and 0.3 for LSD1 and CoREST, respectively, with no according to a nonarbitrary procedure that avoids biasing differences between unbound and bound simulations. their starting positions. We name these 10 simulation runs

Figure 4. Backbone Ca Atom-Positional RMSD of LSD1-CoREST MD Trajectory Snapshots Time series (left) and normalized probability distri- butions (right) of the RMSD values were calculated either for all Ca atoms of the complex (top) or for the individual LSD1 or CoREST subunits (middle and bottom) from both unbound and bound 50 ns simulations using the X-ray structures as reference (Yang et al., 2006; Forneris et al., 2007). Structures were superimposed using all backbone Ca atoms for which RMSD values are reported. Corresponding time series and distribu- tions of the scaled size-independent similarity parameter (Maiorov and Crippen, 1995) are reported in Figure S2.

Figure 5. Successful Oxygen Diffusion Paths in the LSD1-CoREST Complex

Time series of the O2 – C4a distance are displayed with different colors (green, yellow, red, blue, and black) for independent MD simulations in O2-saturated ˚ conditions (O2-unbound and O2-bound). The dashed horizontal line deﬁnes the ﬂavin active site as a sphere of 7 A radius centered on C4a. See Figure 2 for the atom numbering of FADH. See also Figure S3.

‘‘O2-unbound’’ and ‘‘O2-bound’’ (peptide-free and peptide- four O2-bound), O2 diffusion pathways clearly converge above bound, respectively) and distinguish them by color-coding in Fig- the re-face of FADH where Lys661 is located (Figure 6; Fig- ure 5 and Figure 6. These runs were propagated for different ure S4). We find that oxygen molecules diffuse toward the flavin ˚ simulation periods, till any O2 molecule was observed entering edge and, once within 5–7 A distance from the flavin C4a-N5- the LSD1 active site and arriving in close contact with the flavin, C5a atoms (Figure 2), they transiently displace the water that and were terminated when such O2 molecule would leave the bridges the Lys661 side chain to flavin N5 (Figure 3D). In this active site (Figure 5). Paths carrying O2 molecules inside the way, oxygen molecules can intercalate between the Lys661 protein though not in proximity of the flavin (distance > 7 A˚ ) side-chain amino group and bound H3 peptide to directly were observed as well, but were excluded from further analysis. contact (4–5 A˚ distances) either the edge or the re-face of the No relevant differences were observed on the simulated time- cofactor, depending on the trajectory (Figures 2 and 6). The scales between conditions of O2-saturation and absence of statistical analysis of the O2 FADH encounter events further oxygen regarding LSD1-CoREST dynamics and deviation from indicates a shift toward smaller values of the C4a O2 distances the X-ray reference structures (Yang et al., 2006; Forneris with respect to the corresponding values for C5a atom, implying et al., 2007) (see also Figure S3). Of a total of 500 O2 trajectories that C4a is the site for preferential (but not exclusive) approach per system, these simulations allowed collecting 10 complete (Figure S5). spontaneous diffusion pathways that bring O2 molecules at The MD simulations highlight the role of Lys661 as the ‘‘entry close distance from FADH starting from random configurations residue’’ for oxygen into the active center. This amino acid is in the bulk solvent (Figure 5). A total of about 83 and 50 ns MD engaged in a water-mediated interaction with the flavin ring (Fig- sampling times was required for O2-unbound and O2-bound ure 3D). This peculiar Lys-water-flavin triad is a highly conserved simulations, respectively, to observe a total of 10 spontaneous feature, which characterizes the amine oxidase flavoenzymes diffusion routes. Successful paths typically cover overall that share the same folding topology as the catalytic domain of distances of about 20–60 A˚ from the protein surface to the flavin LSD1 (Binda et al., 1999, 2002). Lys661 is known to be essential and display a stepwise behavior from the time of entrance for enzyme function in LSD1 and polyamine oxidase (Shi et al., through the protein surface (Figure 6). In fact, O2 molecules 2004; Polticelli et al., 2005). Moreover, studies on sarcosine may temporarily reside on the surface and/or generally visit oxidase indicated that mutations of this Lys to neutral residues several cavities along each of these paths. Similar diffusion drastically reduce (up to 9000-fold) the reactivity of reduced routes were observed in the unbound and bound simulations flavin with oxygen (Jorns et al., 2010; Zhao et al., 2008). Taken (not shown), suggesting that H3 peptide binding has a minor together, these data suggest a 2-fold role of the conserved Lys effect on the migration of O2 molecules in full agreement with residue in oxygen reaction: (1) a gating function to channel the kinetic data (Forneris et al., 2006). oxygen molecules to the catalytic site (Baron et al., 2009a); and (2) a catalytic function in oxygen activation possibly through Insights about the Reaction of LSD1-CoREST the electrostatic stabilization of the superoxide-flavin semiqui- with Oxygen none pair, which is thought to transiently form during the elec-

Can we identify an orientation model for the approach of O2 tron-transfer process underlying flavin reoxidation (Mattevi, molecules to the flavin cofactor? Answering this question is of 2006; Jorns et al., 2010). basic importance to understand the mechanism of the oxidative half-reaction that follows Lys demethylation (Figure 1A). To DISCUSSION identify preferential models for the approach of O2 molecules to the reduced flavin cofactor, we analyzed the successful O2 LSD1 was originally identified as H3-Ly4 demethylase and this is diffusion pathways using the statistics obtained from all 10 inde- the only activity that, so far, has been demonstrated in vitro (For- pendent simulations. In eight of them (four O2-unbound + neris et al., 2008; van Essen et al., 2010). The three-dimensional

bind the N-terminal sequences of other proteins, such as SNAIL1, that are only distantly related to H3. In more detail, they identify the elements that determine peptide recognition: (1) the conformation of the bound peptide whose N-terminal helical-turn binds deeply into the LSD1 active site; (2) the conserved pattern of positively charged and small polar side chains that are all involved in specific H-bond interactions; (3) the peptide N-terminus that is specifically recognized by the enzyme; and (4) the high degree of preorganization of the binding cleft as indicated by the lack of detectable conformational changes in LSD1-CoREST crystal structure on peptide binding as well as be the overall stability of the active-site conformation in all MD simulations (Figures 1B, 3C, and 3D). The emerging general conclusion is that the conformation of LSD1 associated to CoREST is tailored for binding the N-terminal residues of a peptide ligand with the side chain of its fourth amino acid point- ing toward and in direct contact with the flavin ring of the cofactor. It remains to be seen if association of LSD1 with other protein partners can induce conformational changes that switch the binding specificity of the enzyme. The comparative analysis of the SNAIL1 and H3 peptide binding reveals a further intriguing feature: the folded conformation adopted by the peptides shields the flavin from the solvent by forming a sort of plug that occludes the active site cleft (Fig- ure 3)(Forneris et al., 2007; Yang et al., 2007). This observation raises the question of the possible interference exerted by the bound peptide on the diffusion and reaction of molecular oxygen, the electron-acceptor substrate required to regenerate the oxidized state of the flavin (Figure 1A). To investigate this point, we used MD to simulate oxygen diffusion. The key observation from these studies is that oxygen reaches the active site following multiple routes that converge to a residue that acts Figure 6. O2 Spontaneous Diffusion into the Bound LSD1-CoREST Complex as entry point for admission of oxygen into the catalytic site

(A–C) (A) Overall location, (B) top view, and (C) side view of the O2 diffusion (Lys661) (see Figures 3 and 6). This concept of ‘‘multiple path- pathways displayed with color-coding corresponding that of the right panel ways to an entry point’’ is emerging as the common feature high- of Figure 5. For graphical purposes the blue pathway is displayed in Figure S4. lighted by computational and biophysical studies on several The bound histone H3 tail (gray coil) and LSD1 (cyan coil) are also shown. oxygen-reacting enzymes and finds support in mutagenesis (D) Side view of the time-dependent representations along the simulation time: data (Baron et al., 2009a; Jorns et al., 2010). Most importantly, O2 molecules are colored from red (entrance into the protein matrix) to blue in the case of LSD1, this model for diffusion implies that O2 (exit from the active site). All displayed paths conduct O2 molecules to the C4a-N5 locus of the FADH-reduced flavin cofactor (yellow sticks). Residues can reach the flavin cofactor even in the presence of the bound Lys661 (orange sticks) and Tyr761 (purple sticks) are also highlighted. H3 peptide as clearly confirmed by the MD simulations of oxygen trajectories in the peptide-bound enzyme (Figures 5– 7). This structure of the enzyme in complex with an H3 N-terminal feature becomes very insightful when analyzed in light of the peptide (Forneris et al., 2007) displayed a substrate binding kinetic properties of the LSD1-CoREST reaction, showing that mode that is fully consistent with this specificity. However, the reactivity of oxygen with the reduced flavin is virtually unaf- several in vivo evidences indicated that LSD1 might also act on fected by peptide binding and that the Km values for the mono- H3-Lys9 as well as on a nonhistone substrate such as p53 and and dimethylated substrates are very similar (Shi et al., 2004; that a switch in substrate specificity might be promoted by the Forneris et al., 2005, 2006). Taken together, these kinetic and association with other proteins (Nair et al., 2010; Huang et al., computational data strongly suggest that that LSD1 can function 2007). Thus, probing the binding properties and conformational processively, i.e., the possibility for oxygen to bind and react dynamics is especially relevant to fully understand the biology while a peptide is bound to the active site, enabling the enzyme of LSD1. The discovery of the tight association between LSD1 to complete subsequent demethylation cycles without detach- and N-terminal residues of the SNAIL1 transcription factor was ing from the nucleosomal particle. particularly insightful because of the peculiar mode of associa- The binding mode exhibited by the SNAIL1 peptide has also tion proposed to underlie this protein complex (Lin et al., far-reaching implications for the functions of LSD1-CoREST 2010). Furthermore, we could see that this interaction occurs and LSD2 in chromatin biology. SNAIL1 is part of a large family also in vitro using recombinant proteins, paving the way to our of transcription factors with key roles in development and onco- molecular dynamics and structural investigations. These studies genesis (the so-called SNAIL/Scratch superfamily) (Figure 1B) demonstrate that the active site of LSD1-CoREST complex can (Barrallo-Gimeno and Nieto, 2009; Lin et al., 2010; Marıń and

Nieto, 2006). Alignment of the N-terminal sequences of their peptide (Figure 1B) (Thermo Electron Corporation) was tested in inhibition common SNAG domain reveals that the residues that are key studies by enzymatic assays in the presence of varied concentrations m for binding to LSD1, as gathered from the SNAIL1-LSD1-CoR- (2–100 M) of H3 substrates. The best fit was obtained with the equation describing a competitive inhibition. EST crystal structure, are conserved among these SNAIL1- Crystals of LSD1-CoREST were grown as previously published (Forneris related proteins. This feature predicts that other (if not all) et al., 2007) and were soaked in a cryoprotectant solution containing transcription factors of the SNAIL/Scratch family are likely to 0.3 mM SNAIL1 peptide for 3 hr and then flash-cooled in a stream of gaseous associate to LSD1 (and possibly LSD2) following the same nitrogen at 100 K. Crystals were obtained by both cocrystallization and soak- molecular mechanism highlighted by SNAIL1. Consistently, a ing and many of them had to be screened at beam-lines of SOLEIL, ESRF, SLS member of this transcription factor family (gfi) (Figure 1B) has synchrotrons due to poorly reproducible diffraction. The best data set was measured on a crystal obtained by soaking. Data processing and scaling been already shown to exert its regulatory roles in hematopoiesis (Table 1) were carried out using MOSFLM (Leslie, 1999) and programs of the partly through the recruitment of LSD1 to specific target DNA CCP4 package (CCP4, 1994). The structure of the LSD1-CoREST complex sequences (Saleque et al., 2007). An intriguing feature of this (Yang et al., 2006) (PDB entry 2IW5) was used as initial model for refinement mode of function—mimicking the histone peptide—is that it after removal of all water atoms. Unbiased 2Fo-Fc and Fo-Fc maps were makes these proteins inherently able to inhibit LSD1/2. The used to manually build the protein-bound peptide inhibitor (Figure 3B). Crystal- lographic refinement (Table 1) was performed with Refmac5 (Murshudov et al., competitive inhibition constant Ki measured for the SNAIL1 peptide (0.2 mM) indicates a relatively tight binding when com- 1997) and manual rebuilding was done with Coot (Emsley and Cowtan, 2004). m Pictures were produced with PyMol (www.pymol.org). Crystallographic coor- pared to the Km values of 3-4 M the methylated H3 peptides dinates have been deposited with the Protein Data Bank (2y48). (Forneris et al., 2006). Clearly, the N-terminal residues of SNAIL1 must be released from the LSD1 active site to make the enzyme Molecular Model and Computational Procedure catalytically active. Consistently, Lin et al. have shown that, Conformational dynamics in solution was investigated for two (peptide-bound in vivo, nucleosomes can indeed displace SNAIL1 so that, after and peptide-free) LSD1-CoREST complex systems noncovalently bound to being targeted to the SNAIL1-binding DNA sequences, LSD1 FADH . The spontaneous, explicit diffusion of dioxygen in these two systems can exert its demethylase activity (Lin et al., 2010). An obvious was separately addressed based on 10 independent MD simulations in fascinating question for future studies will be to evaluate the O2-saturated conditions (O2-unbound and O2-bound), a successful approach for oxygen-consuming enzymes (Baron et al., 2009a, 2009b). Note that in the potential regulatory role played by SNAIL1 and related transcrip- present study, O2 molecules have physical masses, thus MD timescales tion factors as endogenous inhibitors of LSD1 and LSD2. are realistic. Initial configurations for the LSD1-CoREST-FADH- complexes, The mix of flexibility and processivity highlighted by our the ions, and the crystallographic water sites were taken from PDB 2IW5 studies provides a molecular rationale for the dual (catalysis (Yang et al., 2006); H3 peptide coordinates from PDB 2V1D (Forneris et al., versus binding) activity of LSD1-CoREST that can act as a 2007). Initial configurations were solvated in (pre-equilibrated) boxes large histone-modifying enzyme as well as multidocking element for enough to avoid interactions between mirror images under rectangular peri- odic boundary conditions along the entire MD trajectory. For O -unbound a number of functionally and structurally distinct protein partners 2 and O2-bound simulations, 100 O2-molecules were substituted (after 5 ns (Forneris et al., 2008; Mosammaparast and Shi, 2010; Shi et al., equilibration) to randomly chosen water molecules (enforcing minimum recip- 2004). The (demethylated) nucleosomal particle(s) and transcrip- rocal distances and minimum distances from any protein atom of 1.0 nm). All tion factors such as SNAIL1 have their primary anchoring site in systems were neutralized with (unbound: 2; bound: 6) Cl ions and contained the active site cleft whereas other proteins (such as HDACs) will (unbound: 60106; bound: 60093; O2-unbound: 60006; O2-bound: 59993) associate to other regions of the LSD1-CoREST complex water molecules. All trajectories in explicit water were generated and analyzed in double (possibly including the tower domain). The challenge for the precision using the GROMACS 4.0.4 software (Hess et al., 2008). Force field future will be to translate these findings to the complexity of parameters and charges were set from the 53A6 GROMOS force field (Oos- the crowded chromatin environment. tenbrink et al., 2004) to reproduce the experimental condition of apparent neutral pH. GROMOS compatible SPC water model (Berendsen, 1981) and ion parameters (A˚ qvist, 1990) were employed. Newton’s equations of motion EXPERIMENTAL PROCEDURES were integrated using the leap-frog algorithm (Ryckaert et al., 1977) with a 2-fs time-step. The P-LINCS numerical algorithm (Hess, 2008) was applied Biochemical and Crystallographic Studies to constrain all bond lengths, excluding water molecules that were kept rigid All chemicals were purchased from Sigma-Aldrich unless specified. Escheri- using the SETTLE analytical algorithm (Miyamoto and Kollman, 1992). All chia coli overexpression, purification, and crystallization of the human simulations were carried out in the N,p,T canonical ensemble (300 K; 1 LSD1-CoREST complex were carried out following previously published Atm) by separately coupling the temperature of LSD1, COREST, FADH, procedures (Forneris et al., 2007). A His-tagged recombinant form of LSD1 solvent, (and O2) degrees of freedom to a heat bath using the Nose´ -Hoover comprising residues 171–836 was copurified with a GST-tagged CoREST thermostat (Hoover, 1989; Nose´ , 1984) and the Parrinello-Rahman pressure protein (residues 308–440) by tandem-affinity chromatography followed by coupling (Parrinello and Rahman, 1981). Full treatment of electrostatic inter- gel filtration on a Superdex200 column (GE Healthcare). Recombinant mouse actions was achieved using a smooth particle mesh Ewald approximation LSD2 was purified as described (Karytinos et al., 2009). The enzymatic activity (Essmann et al., 1995). Nonbonded interactions were shifted (Baron et al., of purified and homogeneous samples LSD1-CoREST and LSD2 were 2007) to zero at a distance of 1.4 nm, recalculated every time-step in the measured on a 21-residue H3 peptides mono- and dimethylated at Lys4 range 0.0–0.8 nm and every five time-steps in the range 0.8–1.4 nm, using (Thermo Electron Corporation) by the peroxidase-coupled assay at 25C using a twin-range cutoff scheme (van Gunsteren and Berendsen, 1990). Trajec- a Cary 100 UV/Vis spectrophotometer (Varian Inc.) (Forneris et al., 2005). tory snapshots were extracted every 10 ps for analysis along 50 ns of

Brieﬂy, reactions were started by adding 2 ml of protein solution (40 mM protein unbound or bound simulations or periods of varying length of 10 O2-unbound in 50 mM potassium phosphate buffer pH 7.5 and 5% (w/v) glycerol) to and O2-bound simulations (totaling about 83 and 50 ns sampling, respec- reaction mixtures (150 ml) consisting of 50 mM HEPES/NaOH buffer pH 7.5, tively). O2-diffusion pathways to the FADH cofactor were analyzed as in 0.1 mM 4-aminoantipyrine, 1 mM 3,5-dichloro-2-hydroxybenzenesulfonic Baron et al. (2009b) with the VMD software (Humphrey et al., 1996). Addi- acid, 0.35 mM horseradish peroxidase, and variable concentrations tional computational details are reported as Supplemental Experimental (2–100 mM) of mono- or dimethylated H3-K4 peptides. The 20-residue SNAIL1 Procedures.

ACCESSION NUMBERS Fang, R., Barbera, A.J., Xu, Y., Rutenberg, M., Leonor, T., Bi, Q., Lan, F., Mei, P., Yuan, G.-C., Lian, C., et al. (2010). Human LSD2/KDM1b/AOF1 regulates Coordinates have been deposited with the Protein Data Bank (accession code gene transcription by modulating intragenic H3K4me2 methylation. Mol. Cell 2y48). 39, 222–233. Forneris, F., Binda, C., Vanoni, M., Mattevi, A., and Battaglioli, E. (2005). SUPPLEMENTAL INFORMATION Histone demethylation catalyzed by LSD1 is a flavin-dependent oxidative process. FEBS Lett. 579, 2203–2207. Supplemental Information includes Supplemental Experimental Procedures Forneris, F., Binda, C., Dall’Aglio, A., Fraaije, M.W., Battaglioli, E., and Mattevi, and five figures and can be found with this article online at doi:10.1016/j.str. A. (2006). A highly specific mechanism of histone H3-K4 recognition by histone 2011.01.001. demethylase LSD1. J. Biol. Chem. 281, 35289–35295. Forneris, F., Binda, C., Adamo, A., Battaglioli, E., and Mattevi, A. (2007). ACKNOWLEDGMENTS Structural basis of LSD1-CoREST selectivity in histone H3 recognition. J. Biol. Chem. 282, 20070–20074. Work at the University of Pavia was supported by Fondazione Cariplo, MIUR- Forneris, F., Binda, C., Battaglioli, E., and Mattevi, A. (2008). LSD1: oxidative PRIN08, Regione Lombardia-ASTIL, and Associazione Italiana Ricerca sul chemistry for multifaceted functions in chromatin regulation. Trends Cancro. Work at UCSD was supported partly by NSF, NIH, HHMI, CTBP, Biochem. Sci. 33, 181–189. NBCR, and the NSF Supercomputer Centers. Hess, B. (2008). P-LINCS: a parallel linear constraint solver for molecular simulation. J. Chem. Theory Comput. 4, 116–122. Received: October 4, 2010 Hess, B., Kutzner, C., van der Spoel, D., and Lindahl, E. (2008). GROMACS 4: Revised: November 20, 2010 Algorithms for highly efficient, load-balanced, and scalable molecular simula- Accepted: January 4, 2011 tion. J. 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