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• W2057123121 abstract "Histone demethylases LSD1 and LSD2 (KDM1A/B) catalyze the oxidative demethylation of Lys4 of histone H3. We used molecular dynamics simulations to probe the diffusion of the oxygen substrate. Oxygen can reach the catalytic center independently from the presence of a bound histone peptide, implying that LSD1 can complete subsequent demethylation cycles without detaching from the nucleosomal particle. The simulations highlight the role of a strictly conserved active-site Lys residue providing general insight into the enzymatic mechanism of oxygen-reacting flavoenzymes. The crystal structure of LSD1-CoREST bound to a peptide of the transcription factor SNAIL1 unravels a fascinating example of molecular mimicry. The SNAIL1 N-terminal residues bind to the enzyme active-site cleft, effectively mimicking the H3 tail. This finding predicts that other members of the SNAIL/Scratch transcription factor family might associate to LSD1/2. The combination of selective histone-modifying activity with the distinct recognition mechanisms underlies the biological complexity of LSD1/2. Histone demethylases LSD1 and LSD2 (KDM1A/B) catalyze the oxidative demethylation of Lys4 of histone H3. We used molecular dynamics simulations to probe the diffusion of the oxygen substrate. Oxygen can reach the catalytic center independently from the presence of a bound histone peptide, implying that LSD1 can complete subsequent demethylation cycles without detaching from the nucleosomal particle. The simulations highlight the role of a strictly conserved active-site Lys residue providing general insight into the enzymatic mechanism of oxygen-reacting flavoenzymes. The crystal structure of LSD1-CoREST bound to a peptide of the transcription factor SNAIL1 unravels a fascinating example of molecular mimicry. The SNAIL1 N-terminal residues bind to the enzyme active-site cleft, effectively mimicking the H3 tail. This finding predicts that other members of the SNAIL/Scratch transcription factor family might associate to LSD1/2. The combination of selective histone-modifying activity with the distinct recognition mechanisms underlies the biological complexity of LSD1/2. LSD1/2 histone demethylases function in many transcriptional processes Molecular dynamics provide key insight about enzyme processivity The transcription factor SNAIL1 binds to LSD1 by a molecular mimicry mechanism LSD1/2 function as multiple docking sites for chromatin proteins Large chromatin complexes finely regulate eukaryotic gene expression and are selectively recruited to DNA sequences by specific transcription factors. The post-translational modifications on the histone N-terminal tails protruding from the nucleosomal particle play fundamental roles in gene expression by dictating an epigenetic code that flags the activation or repression status of a gene (Jenuwein and Allis, 2001Jenuwein T. Allis C.D. Translating the histone code.Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7203) Google Scholar). These histone marks are recognized by transcription factors and are dynamically regulated by specific histone-modifying enzymes (Ruthenburg et al., 2007Ruthenburg A.J. Li H. Patel D.J. Allis C.D. Multivalent engagement of chromatin modifications by linked binding modules.Nat. Rev. Mol. Cell Biol. 2007; 8: 983-994Crossref PubMed Scopus (778) Google Scholar). Methylation of histone Lys residues is catalyzed by histone methyl-transferases, a process thought to be irreversible for decades. This view was challenged by the discovery of the first histone lysine demethylase, lysine-specific demethylase 1 (LSD1 or KDM1A, according to the newly adopted nomenclature) (Shi et al., 2004Shi Y. Lan F. Matson C. Mulligan P. Whetstine J.R. Cole P.A. Casero R.A. Shi Y. Histone demethylation mediated by the nuclear amine oxidase homolog LSD1.Cell. 2004; 119: 941-953Abstract Full Text Full Text PDF PubMed Scopus (2857) Google Scholar, Forneris et al., 2005Forneris F. Binda C. Vanoni M. Mattevi A. Battaglioli E. Histone demethylation catalyzed by LSD1 is a flavin-dependent oxidative process.FEBS Lett. 2005; 579: 2203-2207Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). This enzyme acts on mono- and dimethylated Lys4 of histone H3 through a FAD-dependent oxidative process (Figure 1A ). LSD1 is often associated to the histone deacetylases (HDAC) 1 and 2 and to the corepressor protein CoREST, which tightly binds to LSD1 enhancing both its stability and enzymatic activity. A number of studies have indicated that LSD1-CoREST interacts with various protein complexes involved in gene regulation and chromatin modification (Forneris et al., 2008Forneris F. Binda C. Battaglioli E. Mattevi A. LSD1: oxidative chemistry for multifaceted functions in chromatin regulation.Trends Biochem. Sci. 2008; 33: 181-189Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, Mosammaparast and Shi, 2010Mosammaparast N. Shi Y. Reversal of histone methylation: biochemical and molecular mechanisms of histone demethylases.Annu. Rev. Biochem. 2010; 79: 155-179Crossref PubMed Scopus (408) Google Scholar). Especially relevant for our investigations is the finding that LSD1 is recruited to target gene promoters by interacting with the N-terminal SNAG domain of SNAIL1, a master regulator of the epithelial-mesenchymal transition, which is at the heart of many morphogenetic events including the establishment of tumor invasiveness (Lin et al., 2010Lin Y. Wu Y. Li J. Dong C. Ye X. Chi Y.-I. Evers B.M. Zhou B.P. The SNAG domain of Snail1 functions as a molecular hook for recruiting lysine-specific demethylase 1.EMBO J. 2010; 29: 1803-1816Crossref PubMed Scopus (232) Google Scholar) (Figure 1B). A further addition to this biological complexity has been the discovery of LSD2 (or KDM1B), a mammalian homolog of LSD1 (Karytinos et al., 2009Karytinos A. Forneris F. Profumo A. Ciossani G. Battaglioli E. Binda C. Mattevi A. A novel mammalian flavin-dependent histone demethylase.J. Biol. Chem. 2009; 284: 17775-17782Crossref PubMed Scopus (191) Google Scholar). LSD2 exhibits the same H3-Lys4 demethylase activity as LSD1 but it functions in distinct transcriptional complexes with specific biological functions (Ciccone et al., 2009Ciccone D.N. Su H. Hevi S. Gay F. Lei H. Bajko J. Xu G. Li E. Chen T. KDM1B is a histone H3K4 demethylase required to establish maternal genomic imprints.Nature. 2009; 461: 415-418Crossref PubMed Scopus (367) Google Scholar, Fang et al., 2010Fang R. Barbera A.J. Xu Y. Rutenberg M. Leonor T. Bi Q. Lan F. Mei P. Yuan G.-C. Lian C. et al.Human LSD2/KDM1b/AOF1 regulates gene transcription by modulating intragenic H3K4me2 methylation.Mol. Cell. 2010; 39: 222-233Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, Yang et al., 2010Yang Z. Jiang J. Stewart D.M. Qi S. Yamane K. Li J. Zhang Y. Wong J. AOF1 is a histone H3K4 demethylase possessing demethylase activity-independent repression function.Cell Res. 2010; 20: 276-287Crossref PubMed Scopus (47) Google Scholar). LSD1 and LSD2 are distinguished from the histone demethylases of JmjC class that have been identified in the last years. The JmjC enzymes display wider substrate specificity by acting 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., 2010Horton J.R. Upadhyay A.K. Qi H.H. Zhang X. Shi Y. Cheng X. Enzymatic and structural insights for substrate specificity of a family of jumonji histone lysine demethylases.Nat. Struct. Mol. Biol. 2010; 17: 38-43Crossref PubMed Scopus (276) Google Scholar, Tsukada et al., 2006Tsukada Y.-i. Fang J. Erdjument-Bromage H. Warren M.E. Borchers C.H. Tempst P. Zhang Y. Histone demethylation by a family of JmjC domain-containing proteins.Nature. 2006; 439: 811-816Crossref PubMed Scopus (1441) Google Scholar). Conversely, LSD1/2 are flavoenzymes that use FAD to oxidatively demethylate their substrate. The reduced flavin, generated on Lys demethylation, is reoxidized by molecular oxygen (O2) with the production of hydrogen peroxide in addition to formaldehyde (Figures 1A and 2) (Forneris et al., 2005Forneris F. Binda C. Vanoni M. Mattevi A. Battaglioli E. Histone demethylation catalyzed by LSD1 is a flavin-dependent oxidative process.FEBS Lett. 2005; 579: 2203-2207Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, Shi et al., 2004Shi Y. Lan F. Matson C. Mulligan P. Whetstine J.R. Cole P.A. Casero R.A. Shi Y. Histone demethylation mediated by the nuclear amine oxidase homolog LSD1.Cell. 2004; 119: 941-953Abstract Full Text Full Text PDF PubMed Scopus (2857) Google Scholar). This peculiar hydrogen-peroxide generating activity of LSD1/2 represents an important aspect because the reaction product and its reactive oxygen species are potentially dangerous in the context of the chromatin environment. An intriguing hypothesis is that they might have a signaling role in cellular processes, further expanding the biological roles and functions of LSD1/2 (Amente et al., 2010Amente S. Bertoni A. Morano A. Lania L. Avvedimento E.V. Majello B. LSD1-mediated demethylation of histone H3 lysine 4 triggers Myc-induced transcription.Oncogene. 2010; 29: 3691-3702Crossref PubMed Scopus (110) Google Scholar, Forneris et al., 2008Forneris F. Binda C. Battaglioli E. Mattevi A. LSD1: oxidative chemistry for multifaceted functions in chromatin regulation.Trends Biochem. Sci. 2008; 33: 181-189Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, Winterbourn, 2008Winterbourn C.C. Reconciling the chemistry and biology of reactive oxygen species.Nat. Chem. Biol. 2008; 4: 278-286Crossref PubMed Scopus (1511) Google Scholar). Here, we study the mechanisms and processes of molecular recognition in LSD1 with a focus on the recognition of substrates and protein ligands. How does oxygen diffuse into the catalytic site? Do the very different enzyme substrates, a small molecule such as oxygen and a large protein complex such as the nucleosome, interfere with each other? What are the molecular mechanisms 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., 2008Forneris F. Binda C. Battaglioli E. Mattevi A. LSD1: oxidative chemistry for multifaceted functions in chromatin regulation.Trends Biochem. Sci. 2008; 33: 181-189Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, Mosammaparast and Shi, 2010Mosammaparast N. Shi Y. Reversal of histone methylation: biochemical and molecular mechanisms of histone demethylases.Annu. Rev. Biochem. 2010; 79: 155-179Crossref PubMed Scopus (408) Google Scholar)? 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 mechanisms 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. We used a combination of biochemical and structural experiments to investigate the interaction between LSD1 and the transcription factor SNAIL1, which was originally discovered by Lin et al., 2010Lin Y. Wu Y. Li J. Dong C. Ye X. Chi Y.-I. Evers B.M. Zhou B.P. The SNAG domain of Snail1 functions as a molecular hook for recruiting lysine-specific demethylase 1.EMBO J. 2010; 29: 1803-1816Crossref PubMed Scopus (232) Google Scholar. On the basis of a weak sequence similarity between SNAIL1 and the histone H3 N-terminal tail (in essence, a conserved pattern of positively charged residues) (Figure 1B), the authors of this study proposed that SNAIL1 could bind to LSD1 in the same site as the histone H3 substrate, i.e., in the catalytic site. This model for SNAIL1-LSD1 interactions implied that SNAIL1 should inhibit LSD1 enzymatic activity. Consistently, we found that a 20-amino-acid peptide corresponding to SNAIL1 N-terminal residues effectively inhibits LSD1-CoREST. In more detail, fitting of the enzyme initial velocities to the equation for competitive inhibition resulted in a Ki values of 0.21 ± 0.07 mM (using a monomethyl-Lys4 H3-peptide as substrate) and 0.22 ± 0.09 mM (using a dimethylated substrate), indicating a rather tight binding. Likewise, we found that also LSD2 binds the SNAIL1 peptide (albeit with lower affinity; Ki value of 2.22 ± 0.36 μM), which is in line with the previously observed similarities in the binding properties of LSD1 and LSD2 (Karytinos et al., 2009Karytinos A. Forneris F. Profumo A. Ciossani G. Battaglioli E. Binda C. Mattevi A. A novel mammalian flavin-dependent histone demethylase.J. Biol. Chem. 2009; 284: 17775-17782Crossref PubMed Scopus (191) Google Scholar). 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 Å 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 Å for the 799 equivalent Cα atoms of LSD1-CoREST). The peptide occupies the open cleft that has been shown to form the binding site for the H3 tail (Forneris et al., 2007Forneris F. Binda C. Adamo A. Battaglioli E. Mattevi A. Structural basis of LSD1-CoREST selectivity in histone H3 recognition.J. Biol. Chem. 2007; 282: 20070-20074Crossref PubMed Scopus (167) Google Scholar). The N terminus (residues 1–4) adopts a helical turn conformation, closely resembling that of the first residues of the H3 tail (Figures 3C and 3D). In particular, the N-terminal amino group of Pro1 and the side chains of Arg2 and Ser3 bind deeply into the cleft and establish several H-bonding interactions with the surrounding protein residues, in an arrangement almost identical to that exhibited by the Ala1-Arg2-Thr3 residues of the H3 peptide. Thus, Pro1 binds to the carbonyl of Ala539 at the C terminus of α-helix 524–540 whereas Arg2 interacts with Asp553 and Asp556. This binding conformation positions Phe4 of SNAIL1 in front of the flavin to occupy a location corresponding to that of Lys4 of the H3 tail (Figure 3D). Phe4 snugly fits in its binding niche by making edge-to-face interactions with the rings of the flavin cofactor and Tyr761. The conformation of the SNAIL1 peptide deviates from that of the H3 tail after residues 4–5 to compensate for the deletion of one residue compared to the histone sequence (Figures 1B, 3C, and 3D). However, the comparative analysis of the H3 and SNAIL1 complexes clearly indicates that Arg7 of SNAIL1 occupies the same position 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.Table 1Data Collection and Refinement Statistics for LSD1-CoREST- SNAIL1 Peptide ComplexSpace groupI222Unit cell axes (Å)119.2, 181.5, 234.4Resolution (Å)3.0Rsym (%)aRsym = ∑|Ii − <I>|/∑Ii, where Ii is the intensity of ith observation and <I> is the mean intensity of the reflection., bValues in parentheses are for reflections in the highest resolution shell.9.8 (53.7)Completeness (%)bValues in parentheses are for reflections in the highest resolution shell.99.7 (100.0)Unique reflections50,937Redundancy3.6 (3.7)I/σbValues in parentheses are for reflections in the highest resolution shell.9.5 (1.9)No. of atoms protein/FAD/ligandcThe final model consists of residues 171–836 of LSD1, a FAD molecule, residues 308–440 of CoREST, and residues 1–9 of the SNAIL1 peptide.6286/53/77Average B value for ligand atoms (Å2)75.2Rcryst (%)dRcryst = ∑|Fobs − Fcalc|/∑|Fobs| where Fobs and Fcalc are the observed and calculated structure factor amplitudes, respectively. The set of reflections used for Rfree calculations and excluded from refinement was extracted from the structure factor file relative to the PDB entry 2V1D.21.2Rfree (%)dRcryst = ∑|Fobs − Fcalc|/∑|Fobs| where Fobs and Fcalc are the observed and calculated structure factor amplitudes, respectively. The set of reflections used for Rfree calculations and excluded from refinement was extracted from the structure factor file relative to the PDB entry 2V1D.24.6RMS bond length (Å)0.014RMS bond angles (°)1.49RMS: root-mean-squarea Rsym = ∑|Ii − <I>|/∑Ii, where Ii is the intensity of ith observation and <I> is the mean intensity of the reflection.b Values in parentheses are for reflections in the highest resolution shell.c The final model consists of residues 171–836 of LSD1, a FAD molecule, residues 308–440 of CoREST, and residues 1–9 of the SNAIL1 peptide.d Rcryst = ∑|Fobs − Fcalc|/∑|Fobs| where Fobs and Fcalc are the observed and calculated structure factor amplitudes, respectively. The set of reflections used for Rfree calculations and excluded from refinement was extracted from the structure factor file relative to the PDB entry 2V1D. Open table in a new tab RMS: root-mean-square We extended our studies on peptide recognition in LSD1-CoREST by analyzing conformational dynamics of the LSD1-CoREST system by MD simulations in solution. This study was based on two 50-ns-long MD trajectories using LSD1-CoREST crystallographic coordinates (Yang et al., 2006Yang M. Gocke C.B. Luo X. Borek D. Tomchick D.R. Machius M. Otwinowski Z. Yu H. Structural basis for CoREST-dependent demethylation of nucleosomes by the human LSD1 histone demethylase.Mol. Cell. 2006; 23: 377-387Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar) in the ligand-free state (hereafter referred to as “unbound”) and in complex (“bound”) with the histone H3 peptide used in the crystallographic studies (Figure 3) in which Lys4 is replaced by Met (Lys4Met mutation was previously shown to greatly increase binding affinity) (Forneris et al., 2007Forneris F. Binda C. Adamo A. Battaglioli E. Mattevi A. Structural basis of LSD1-CoREST selectivity in histone H3 recognition.J. Biol. Chem. 2007; 282: 20070-20074Crossref PubMed Scopus (167) Google Scholar). Figure 4 summarizes the analysis of the backbone Cα atom-positional RMSD values of LSD1-CoREST MD trajectory snapshots from the X-ray structures. After a first initial phase of about 4 ns—during which the most flexible loop regions relax in solution—RMSDs fluctuate stably. Overall, CoREST and the LSD1 tower domain is somewhat more flexible compared to LSD1, largely owing to relative motion between the CoREST SANT2 domain and the LSD1 tower domain (Figure 3A). Differences in molecular fluctuations between LSD1 unbound and bound are moderate (see Figure S1 available online) and changes on substrate binding are limited to the side chain motion of residues in direct contact with the H3 peptide (not shown). These observations are further supported by analysis along the trajectories of the backbone Cα atoms of the scaled size-independent similarity parameter (Maiorov and Crippen, 1995Maiorov V.N. Crippen G.M. Size-independent comparison of protein 3-dimensional structures.proteins. 1995; 22: 273-283Crossref PubMed Scopus (97) Google Scholar), as described in Figure S2. The ensemble averaged ρSC values are 0.2 and 0.3 for LSD1 and CoREST, respectively, with no differences between unbound and bound simulations. This first set of simulations indicated that binding of the histone H3 N-terminal peptide has only local packing effects. No significant impact on the overall dynamics of the LSD1-CoREST complex as well as of LSD1 and CoREST individual partners was observed over the simulated timescales. The binding site for the histone tail on LSD1-CoREST is essentially preorganized to host and stabilize the peptide in the observed folded conformation (Forneris et al., 2007Forneris F. Binda C. Adamo A. Battaglioli E. Mattevi A. Structural basis of LSD1-CoREST selectivity in histone H3 recognition.J. Biol. Chem. 2007; 282: 20070-20074Crossref PubMed Scopus (167) Google Scholar). This is consistent with the X-ray data on the SNAIL1 complex. Also in that case, the bound peptide seems to adapt its conformation to the shape of the binding cleft whose H-bond acceptors function as anchoring elements for the positively charged groups of the peptide (Figures 3C and 3D). Having inspected the structure and dynamics of peptide binding, we investigated the diffusion of O2 molecules into the active site of LSD1-CoREST to probe the mechanisms of oxygen migration and binding in this enzymatic system (Figure 1A). The integration of biochemical and structural experiments with powerful MD simulations has already shown to be an effective strategy to investigate the relationship between enzyme dynamics and oxygen biocatalysis (Baron et al., 2009aBaron R. McCammon J.A. Mattevi A. The oxygen-binding vs. oxygen-consuming paradigm in biocatalysis: structural biology and biomolecular simulation.Curr. Opin. Struct. Biol. 2009; 19: 672-679Crossref PubMed Scopus (31) Google Scholar, Baron et al., 2009bBaron R. Riley C. Chenprakhon P. Thotsunboundrn K. Winter R.T. Alfieri A. Forneris F. van Berkel W.J. Chaiyen P. Fraaije M.W. et al.Multiple pathways guide oxygen diffusion into flavoenzyme active sites.Proc. Natl. Acad. Sci. USA. 2009; 106: 10603-10608Crossref PubMed Scopus (136) Google Scholar, Saam et al., 2007Saam J. Ivanov I. Walther M. Holzhutter H.G. Kuhn H. Molecular dioxygen enters the active site of 12/15-lipoxygenase via dynamic oxygen access channels.Proc. Natl. Acad. Sci. USA. 2007; 104: 13319-13324Crossref PubMed Scopus (118) Google Scholar, Saam et al., 2010Saam J. Rosini E. Molla G. Schulten K. Pollegioni L. Ghisla S. O2-reactivity of flavoproteins: dynamic access of dioxygen to the active site and role of a H+ relay system in D-amino acid oxidase.J. Biol. Chem. 2010; 285: 24439-24446Crossref PubMed Scopus (49) Google Scholar). Our computational approach intended to address a crucial question about LSD1 molecular function: does binding of the histone peptide in the active site cleft interfere with the 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 peptide-bound LSD1-CoREST complexes and were analyzed to probe the effect of peptide binding on oxygen diffusion. The FAD was set to be in the reduced FAD form (FADH−) (Figure 2) as it is the case for the enzymatic state that undergoes reoxidation following Lys demethylation (Figure 1A). After equilibration, 100 O2 molecules were added to the bulk water in each system according to a nonarbitrary procedure that avoids biasing their starting positions. We name these 10 simulation runs “O2-unbound” and “O2-bound” (peptide-free and peptide-bound, respectively) and distinguish them by color-coding in Figure 5 and Figure 6. These runs were propagated for different simulation periods, till any O2 molecule was observed entering the LSD1 active site and arriving in close contact with the flavin, and were terminated when such O2 molecule would leave the active site (Figure 5). Paths carrying O2 molecules inside the protein though not in proximity of the flavin (distance > 7 Å) were observed as well, but were excluded from further analysis. No relevant differences were observed on the simulated timescales between conditions of O2-saturation and absence of oxygen regarding LSD1-CoREST dynamics and deviation from the X-ray reference structures (Yang et al., 2006Yang M. Gocke C.B. Luo X. Borek D. Tomchick D.R. Machius M. Otwinowski Z. Yu H. Structural basis for CoREST-dependent demethylation of nucleosomes by the human LSD1 histone demethylase.Mol. Cell. 2006; 23: 377-387Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, Forneris et al., 2007Forneris F. Binda C. Adamo A. Battaglioli E. Mattevi A. Structural basis of LSD1-CoREST selectivity in histone H3 recognition.J. Biol. Chem. 2007; 282: 20070-20074Crossref PubMed Scopus (167) Google Scholar) (see also Figure S3). Of a total of 500 O2 trajectories per system, these simulations allowed collecting 10 complete spontaneous diffusion pathways that bring O2 molecules at close distance from FADH− starting from random configurations in the bulk solvent (Figure 5). A total of about 83 and 50 ns MD sampling times was required for O2-unbound and O2-bound simulations, respectively, to observe a total of 10 spontaneous diffusion routes. Successful paths typically cover overall distances of about 20–60 Å from the protein surface to the flavin and display a stepwise behavior from the time of entrance through the protein surface (Figure 6). In fact, O2 molecules may temporarily reside on the surface and/or generally visit several cavities along each of these paths. Similar diffusion routes were observed in the unbound and bound simulations (not shown), suggesting that H3 peptide binding has a minor effect on the migration of O2 molecules in full agreement with the kinetic data (Forneris et al., 2006Forneris F. Binda C. Dall'Aglio A. Fraaije M.W. Battaglioli E. Mattevi A. A highly specific mechanism of histone H3-K4 recognition by histone demethylase LSD1.J. Biol. Chem. 2006; 281: 35289-35295Crossref PubMed Scopus (94) Google Scholar).Figure 6O2 Spontaneous Diffusion into the Bound LSD1-CoREST ComplexShow full caption(A–C) (A) Overall location, (B) top view, and (C) side view of the O2 diffusion pathways displayed with color-coding corresponding that of the right panel of Figure 5. For graphical purposes the blue pathway is displayed in Figure S4. The bound histone H3 tail (gray coil) and LSD1 (cyan coil) are also shown.(D) Side view of the time-dependent representations along the simulation time: O2 molecules are colored from red (entrance into the protein matrix) to blue (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 Lys661 (orange sticks) and Tyr761 (purple sticks) are also highlighted.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A–C) (A) Overall location, (B) top view, and (C) side view of the O2 diffusion pathways displayed with color-coding corresponding that of the right panel of Figure 5. For graphical purposes the blue pathway is displayed in Figure S4. The bound histone H3 tail (gray coil) and LSD1 (cyan coil) are also shown. (D) Side view of the time-dependent representations along the simulation time: O2 molecules are colored from red (entrance into the protein matrix) to blue (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 Lys661 (orange sticks) and Tyr761 (purple sticks) are also highlighted. Can we identify an orientation model for the approach of O2 molecules to the flavin cofactor? Answering this question is of basic importance to understand the mechanism of the oxidative half-reaction that follows Lys demethylation (Figure 1A). To identify preferential models for the approach of O2 molecules to the reduced flavin cofactor, we analyzed the successful O2 diffusion pathways using the statistics obtained from all 10 independent simulations. In eight of them (four O2-unbound + four O2-bound), O2 diffusion pathways clearly converge above the re-face of FADH− where Lys661 is located (Figure 6; Figure S4). We find that oxygen molecules diffuse toward the flavin edge and, once within 5–7 Å distance from the flavin C4a-N5-C5a atoms (Figure 2), they transiently displace the water that bridges the Lys661 side chain to flavin N5 (Figure 3D). In this way, oxygen molecules can intercalate between the Lys661 side-chain amino group and bound H3 peptide to directly contact (∼4–5 Å distances) either the edge or the re-face of the cofactor, depending on the trajectory (Figures 2 and 6). The statistical analysis of the O2 − FADH− encounter events further indicates a shift toward smaller values of the C4a − O2 distances with respect to the corresponding values for C5a atom, implying that C4a is the site for preferential (but not exclusive) approach (Figure S5). The MD simulations highlight the role of Lys661 as the “entry residue” for oxygen into the active center. This amino acid is engaged in a water-mediated interaction with the flavin ring (Figure 3D). This peculiar Lys-water-flavin tr" @default.
• W2057123121 created "2016-06-24" @default.
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• W2057123121 date "2011-02-01" @default.
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• W2057123121 title "Molecular Mimicry and Ligand Recognition in Binding and Catalysis by the Histone Demethylase LSD1-CoREST Complex" @default.
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