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ABSTRACT: Modular serine proteases are central to the complement cascade of the mammalian humoral immune system. These proteases form protein complexes through multi-domain interactions to achieve their proteolytic activity. We review the structural insights into complement initiation by auto-activation of the hetero-tetrameric proteases of the large danger-recognition protein complexes, amplification and labelling of particles by the formation and activity of C3 convertases, and regulation by convertase dissociation and degradation to prevent 'bystander' damage to healthy host cells and tissues. The data reveal that complex formation and large domain-domain rearrangements underlie the proteolytic reactions of the complement cascade, which enables the host to recognize and clear invading microbes and host debris from its blood and fluids surrounding tissues.
Current Opinion in Structural Biology 05/2012; 22(3):333-41. · 9.42 Impact Factor
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ABSTRACT: Activation of the complement cascade induces inflammatory responses and marks cells for immune clearance. In the central complement-amplification step, a complex consisting of surface-bound C3b and factor B is cleaved by factor D to generate active convertases on targeted surfaces. We present crystal structures of the pro-convertase C3bB at 4 angstrom resolution and its complex with factor D at 3.5 angstrom resolution. Our data show how factor B binding to C3b forms an open "activation" state of C3bB. Factor D specifically binds the open conformation of factor B through a site distant from the catalytic center and is activated by the substrate, which displaces factor D's self-inhibitory loop. This concerted proteolytic mechanism, which is cofactor-dependent and substrate-induced, restricts complement amplification to C3b-tagged target cells.
Science 12/2010; 330(6012):1816-20. · 31.20 Impact Factor
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Claudia Binda,
Sergio Valente,
Mauro Romanenghi,
Simona Pilotto,
Roberto Cirilli,
Aristotele Karytinos,
Giuseppe Ciossani,
Oronza A Botrugno, Federico Forneris,
Maria Tardugno,
Dale E Edmondson,
Saverio Minucci,
Andrea Mattevi,
Antonello Mai
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ABSTRACT: LSD1 and LSD2 histone demethylases are implicated in a number of physiological and pathological processes, ranging from tumorigenesis to herpes virus infection. A comprehensive structural, biochemical, and cellular study is presented here to probe the potential of these enzymes for epigenetic therapies. This approach employs tranylcypromine as a chemical scaffold for the design of novel demethylase inhibitors. This drug is a clinically validated antidepressant known to target monoamine oxidases A and B. These two flavoenzymes are structurally related to LSD1 and LSD2. Mechanistic and crystallographic studies of tranylcypromine inhibition reveal a lack of selectivity and differing covalent modifications of the FAD cofactor depending on the enantiomeric form. These findings are pharmacologically relevant, since tranylcypromine is currently administered as a racemic mixture. A large set of tranylcypromine analogues were synthesized and screened for inhibitory activities. We found that the common evolutionary origin of LSD and MAO enzymes, despite their unrelated functions and substrate specificities, is reflected in related ligand-binding properties. A few compounds with partial enzyme selectivity were identified. The biological activity of one of these new inhibitors was evaluated with a cellular model of acute promyelocytic leukemia chosen since its pathogenesis includes aberrant activities of several chromatin modifiers. Marked effects on cell differentiation and an unprecedented synergistic activity with antileukemia drugs were observed. These data demonstrate that these LSD1/2 inhibitors are of potential relevance for the treatment of promyelocytic leukemia and, more generally, as tools to alter chromatin state with promise of a block of tumor progression.
Journal of the American Chemical Society 04/2010; 132(19):6827-33. · 9.91 Impact Factor
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ABSTRACT: A variety of chromatin remodeling complexes are thought to orchestrate transcriptional programs that lead neuronal precursors from earliest commitment to terminal differentiation. Here we show that mammalian neurons have a specialized chromatin remodeling enzyme arising from a neurospecific splice variant of LSD1/KDM1, histone lysine specific demethylase 1, whose demethylase activity on Lys4 of histone H3 has been related to gene repression. We found that alternative splicing of LSD1 transcript generates four full-length isoforms from combinatorial retention of two identified exons: the 4 aa exon E8a is internal to the amine oxidase domain, and its inclusion is restricted to the nervous system. Remarkably, the expression of LSD1 splice variants is dynamically regulated throughout cortical development, particularly during perinatal stages, with a progressive increase of LSD1 neurospecific isoforms over the ubiquitous ones. Notably, the same LSD1 splice dynamics can be fairly recapitulated in cultured cortical neurons. Functionally, LSD1 isoforms display in vitro a comparable demethylase activity, yet the inclusion of the sole exon E8a reduces LSD1 repressor activity on a reporter gene. Additional distinction among isoforms is supported by the knockdown of neurospecific variants in cortical neurons resulting in the inhibition of neurite maturation, whereas overexpression of the same variants enhances it. Instead, perturbation of LSD1 isoforms that are devoid of the neurospecific exon elicits no morphogenic effect. Collectively, results demonstrate that the arousal of neuronal LSD1 isoforms pacemakes early neurite morphogenesis, conferring a neurospecific function to LSD1 epigenetic activity.
Journal of Neuroscience 02/2010; 30(7):2521-32. · 7.11 Impact Factor
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ABSTRACT: Haspin, a nuclear and chromosome-associated serine/threonine (S/T) kinase, is responsible for mitotic phosphorylation of Thr-3 of histone H3. Haspin bears recognizable similarity to the eukaryotic protein kinase (ePK) fold, but its sequence is highly divergent and there is therefore considerable interest in its structural organization. We report the 2.15-A crystal structure of the kinase domain of human Haspin. The ePK fold of Haspin contains an array of insertions and deletions. The structure illustrates how Haspin escapes the classical activation scheme of most other kinases. The alphaC helix, which bears a conserved glutamate that is essential for catalysis, adopts its final active conformation within the small lobe of the kinase. It is sandwiched between an alpha-helical insertion that precedes the kinase domain, and the activation segment, which adopts an unprecedented conformation. The activation segment, which does not contain phosphorylatable residues, packs against an unusually structured alphaEF helix. Significantly extruded from the core of the fold, it forms an extensive plateau, hosting several residues implicated in substrate binding. Overall, the structure of the Haspin kinase domain reveals an active conformation that is poised for substrate recognition and phosphorylation in the absence of external regulators.
Proceedings of the National Academy of Sciences 11/2009; 106(48):20204-9. · 9.68 Impact Factor
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ABSTRACT: Lysine-specific demethylase 1 (LSD1) is an enzyme that removes methyl groups from mono- and dimethylated Lys4 of histone H3, a post-translational modification associated with gene activation. Human LSD1 was the first histone demethylase to be discovered and this enzymatic activity is conserved among eukaryotes. LSD1 has been identified in a number of chromatin-remodeling complexes that control gene transcription and its demethylase activity has also been linked to pathological processes including tumorigenesis. The 852-residue sequence of LSD1 comprises an amine oxidase domain which identifies a family of enzymes that catalyze the FAD-dependent oxidation of amine substrates ranging from amino acids to aromatic neurotransmitters. Among these proteins, LSD1 is peculiar in that it acts on a protein substrate in the nuclear environment of chromatin-remodeling complexes. This functional divergence occurred during evolution from the eubacteria to eukaryotes by acquisition of additional domains such as the SWIRM domain. The N-terminal part of LSD1, predicted to be disordered, contains linear motifs that might represent functional sites responsible for the association of this enzyme with a variety of transcriptional protein complexes. LSD1 shares structural features with other flavin amine oxidases, including the overall fold of the amine oxidase domain region and details in the active site that are relevant for amine substrate oxidation.
FEBS Journal 08/2009; 276(16):4304-12. · 3.79 Impact Factor
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Riccardo Baron,
Conor Riley,
Pirom Chenprakhon,
Kittisak Thotsaporn,
Remko T Winter,
Andrea Alfieri, Federico Forneris,
Willem J H van Berkel,
Pimchai Chaiyen,
Marco W Fraaije,
Andrea Mattevi,
J Andrew McCammon
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ABSTRACT: Dioxygen (O(2)) and other gas molecules have a fundamental role in a variety of enzymatic reactions. However, it is only poorly understood which O(2) uptake mechanism enzymes employ to promote efficient catalysis and how general this is. We investigated O(2) diffusion pathways into monooxygenase and oxidase flavoenzymes, using an integrated computational and experimental approach. Enhanced-statistics molecular dynamics simulations reveal spontaneous protein-guided O(2) diffusion from the bulk solvent to preorganized protein cavities. The predicted protein-guided diffusion paths and the importance of key cavity residues for oxygen diffusion were verified by combining site-directed mutagenesis, rapid kinetics experiments, and high-resolution X-ray structures. This study indicates that monooxygenase and oxidase flavoenzymes employ multiple funnel-shaped diffusion pathways to absorb O(2) from the solvent and direct it to the reacting C4a atom of the flavin cofactor. The difference in O(2) reactivity among dehydrogenases, monooxygenases, and oxidases ultimately resides in the fine modulation of the local environment embedding the reactive locus of the flavin.
Proceedings of the National Academy of Sciences 07/2009; 106(26):10603-8. · 9.68 Impact Factor
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ABSTRACT: In living organisms, genes encoding proteins that contain flavins as a prosthetic group constitute approximately 2-3% of the total. The fluorescence of flavin cofactors in these proteins is a property that is widely employed for biochemical characterisation. Here, we present a modified Thermofluor approach called ThermoFAD (Thermofluor-adapted flavin ad hoc detection system), which simplifies identification of optimal purification and storage conditions as well as high-affinity ligands. In this technique, the flavin cofactor is used as an intrinsic probe to monitor protein folding and stability, taking advantage of the different fluorescent properties of flavin-containing proteins between the folded and denatured state. The main advantage of the method is that it allows a large amount of biochemical data to be obtained using very small amounts of protein sample and standard laboratory equipment. We have explored several cases that demonstrate the reliability and versatility of this technique when applied to globular flavoenzymes, membrane-anchored flavoproteins, and macromolecular complexes. The information gathered from ThermoFAD analysis can be very valuable for any biochemical and biophysical analysis, including crystallisation. The method is likely to be applicable to other classes of proteins that possess endogenous fluorescent cofactors and prosthetic groups.
FEBS Journal 06/2009; 276(10):2833-40. · 3.79 Impact Factor
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ABSTRACT: Methylation of Lys residues on histone proteins is a well known and extensively characterized epigenetic mark. The recent discovery of lysine-specific demethylase 1 (LSD1) demonstrated that lysine methylation can be dynamically controlled. Among the histone demethylases so far identified, LSD1 has the unique feature of functioning through a flavin-dependent amine oxidation reaction. Data base analysis reveals that mammalian genomes contain a gene (AOF1, for amine-oxidase flavin-containing domain 1) that is homologous to the LSD1-coding gene. Here, we demonstrate that the protein encoded by AOF1 represents a second mammalian flavin-dependent histone demethylase, named LSD2. The new demethylase is strictly specific for mono- and dimethylated Lys4 of histone H3, recognizes a long stretch of the H3 N-terminal tail, senses the presence of additional epigenetic marks on the histone substrate, and is covalently inhibited by tranylcypromine. As opposed to LSD1, LSD2 does not form a biochemically stable complex with the C-terminal domain of the corepressor protein CoREST. Furthermore, LSD2 contains a CW-type zinc finger motif with potential zinc-binding sites that are not present in LSD1. We conclude that mammalian LSD2 represents a new flavin-dependent H3-Lys4 demethylase that features substrate specificity properties highly similar to those of LSD1 but is very likely to be part of chromatin-remodeling complexes that are distinct from those involving LSD1.
Journal of Biological Chemistry 05/2009; 284(26):17775-82. · 4.77 Impact Factor
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ABSTRACT: Many cellular reactions involve both hydrophobic and hydrophilic molecules that reside within the chemically distinct environments defined by the phospholipid-based membranes and the aqueous lumens of cytoplasm and organelles. Enzymes performing this type of reaction are required to access a lipophilic substrate located in the membranes and to catalyze its reaction with a polar, water-soluble compound. Here, we explore the different binding strategies and chemical tricks that enzymes have developed to overcome this problem. These reactions can be catalyzed by integral membrane proteins that channel a hydrophilic molecule into their active site, as well as by water-soluble enzymes that are able to capture a lipophilic substrate from the phospholipid bilayer. Many chemical and biological aspects of this type of enzymology remain to be investigated and will require the integration of protein chemistry with membrane biology.
Science 08/2008; 321(5886):213-6. · 31.20 Impact Factor
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ABSTRACT: Three years after its discovery, lysine-specific demethylase 1 remains at the forefront of chromatin research. Its demethylase activity on Lys4 of histone H3 supports its role in gene repression. By contrast, the biochemical mechanisms underlying lysine-specific demethylase 1 involvement in transcriptional activation are not firmly established. Structural studies highlight a specific binding site for the histone H3 N-terminal tail and a catalytic machinery that is closely related to that of other flavin-dependent amine oxidases. These insights are crucial for the development of demethylation inhibitors. Furthermore, the exploration of putative non-histone substrates and potential signaling roles of hydrogen peroxide produced by the demethylation reaction could lead to new paradigms in chromatin biology.
Trends in Biochemical Sciences 05/2008; 33(4):181-9. · 10.85 Impact Factor
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ABSTRACT: Alditol oxidase (AldO) from Streptomyces coelicolor A3(2) is a soluble monomeric flavin-dependent oxidase that performs selective oxidation of the terminal primary hydroxyl group of several alditols. Here, we report the crystal structure of the recombinant enzyme in its native state and in complex with both six-carbon (mannitol and sorbitol) and five-carbon substrates (xylitol). AldO shares the same folding topology of the members of the vanillyl-alcohol oxidase family of flavoenzymes and exhibits a covalently linked FAD which is located at the bottom of a funnel-shaped pocket that forms the active site. The high resolution of the three-dimensional structures highlights a well-defined hydrogen-bonding network that tightly constrains the substrate in the productive conformation for catalysis. Substrate binding occurs through a lock-and-key mechanism and does not induce conformational changes with respect to the ligand-free protein. A network of charged residues is proposed to favor catalysis through stabilization of the deprotonated form of the substrate. A His side chain acts as back door that "pushes" the substrate-reactive carbon atom toward the N5-C4a locus of the flavin. Analysis of the three-dimensional structure reveals possible pathways for diffusion of molecular oxygen and a small cavity on the re side of the flavin that may host oxygen during FAD reoxidation. These features combined with the tight shape of the catalytic site provide insights into the mechanism of AldO-mediated regioselective oxidation reactions and its substrate specificity.
Biochemistry 02/2008; 47(3):978-85. · 3.42 Impact Factor
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ABSTRACT: Histone demethylase LSD1 regulates transcription by demethylating Lys(4) of histone H3. The crystal structure of the enzyme in complex with CoREST and a substrate-like peptide inhibitor highlights an intricate network of interactions and a folded conformation of the bound peptide. The core of the peptide structure is formed by Arg(2), Gln(5), and Ser(10), which are engaged in specific intramolecular H-bonds. Several charged side chains on the surface of the substrate-binding pocket establish electrostatic interactions with the peptide. The three-dimensional structure predicts that methylated Lys(4) binds in a solvent inaccessible position in front of the flavin cofactor. This geometry is fully consistent with the demethylation reaction being catalyzed through a flavin-mediated oxidation of the substrate amino-methyl group. These features dictate the exquisite substrate specificity of LSD1 and provide a structural framework to explain the fine tuning of its catalytic activity and the active role of CoREST in substrate recognition.
Journal of Biological Chemistry 08/2007; 282(28):20070-4. · 4.77 Impact Factor
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ABSTRACT: Alditol oxidase is a 45 kDa enzyme containing a covalently bound FAD cofactor. This oxidase efficiently oxidizes a range of alditols to the corresponding aldoses. Owing to its substrate range and regioselectivity, this enzyme is an interesting candidate for biotechnological applications. Crystals of alditol oxidase from Streptomyces coelicolor A3(2) were obtained by the hanging-drop vapour-diffusion method and diffracted to 1.1 A resolution. The crystals belong to space group C2, with unit-cell parameters a = 107, b = 68, c = 58 A, beta = 94 degrees. Crystals of seleno-L-methionine-labelled alditol oxidase were obtained after seeding the crystallization drops with native microcrystals and showed a diffraction limit of 2.4 A.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 01/2007; 62(Pt 12):1298-300. · 0.51 Impact Factor
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ABSTRACT: Human lysine-specific demethylase (LSD1) is a chromatin-modifying enzyme that specifically removes methyl groups from mono- and dimethylated Lys4 of histone H3 (H3-K4). We used a combination of in vivo and in vitro experiments to characterize the substrate specificity and recognition by LSD1. Biochemical assays on histone peptides show that essentially all epigenetic modifications on the 21 N-terminal amino acids of histone H3 cause a significant reduction in enzymatic activity. Replacement of Lys4 with Arg greatly enhances binding affinity, and a histone peptide incorporating this mutation has a strong inhibitory power. Conversely, a peptide bearing a trimethylated Lys4 is only a weak inhibitor of the enzyme. Rapid kinetics measurements evidence that the enzyme is efficiently reoxidized by molecular oxygen with a second-order rate constant of 9.6x10(3) M-1 s-1, and that the presence of the reaction product does not greatly influence the rate of flavin reoxidation. In vivo experiments provide a correlation between the in vitro inhibitory properties of the tested peptides and their ability of affecting endogenous LSD1 activity. Our results show that epigenetic modifications on histone H3 need to be removed before Lys4 demethylation can efficiently occur. The complex formed by LSD1 with histone deacetylases 1/2 may function as a "double-blade razor" that first eliminates the acetyl groups from acetylated Lys residues and then removes the methyl group from Lys4. We suggest that after H3-K4 demethylation, LSD1 recruits the forthcoming chromatin remodelers leading to the introduction of gene repression marks.
Journal of Biological Chemistry 12/2006; 281(46):35289-95. · 4.77 Impact Factor
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ABSTRACT: Human histone demethylase LSD1 is a flavin-dependent amine oxidase that catalyzes the specific removal of methyl groups from mono- and dimethylated Lys4 of histone H3. The N-terminal tail of H3 is subject to various covalent modifications, and a fundamental question in LSD1 biology is how these epigenetic marks affect the demethylase activity. We show that LSD1 does not have a strong preference for mono- or dimethylated Lys4 of H3. Substrate recognition is not confined to the residues neighboring Lys4, but it requires a sufficiently long peptide segment consisting of the N-terminal 20 amino acids of H3. Electrostatic interactions are an important factor in protein-substrate recognition, as indicated by the high sensitivity of Km to ionic strength. We have probed LSD1 for its ability to demethylate Lys4 in presence of a second modification on the same peptide substrate. Methylation of Lys9 does not affect enzyme catalysis. Conversely, Lys9 acetylation causes an almost 6-fold increase in the Km value, whereas phosphorylation of Ser10 totally abolishes activity. LSD1 is inhibited by a demethylated peptide with an inhibition constant of 1.8 microM, suggesting that LSD1 can bind to H3 independently of Lys4 methylation. LSD1 is a chromatin-modifying enzyme, which is able to read different epigenetic marks on the histone N-terminal tail and can serve as a docking module for the stabilization of the associated corepressor complex(es) on chromatin.
Journal of Biological Chemistry 01/2006; 280(50):41360-5. · 4.77 Impact Factor
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ABSTRACT: Lysine-specific histone demethylase 1 (LSD1) is a very recently discovered enzyme which specifically removes methyl groups from Lys4 of histone 3. We have addressed the functional properties of the protein demonstrating that histone demethylation involves the flavin-catalysed oxidation of the methylated lysine. The nature of the substrate that acts as the electron acceptor required to complete the catalytic cycle was investigated. LSD1 converts oxygen to hydrogen peroxide although this reactivity is not as pronounced as that of other flavin-dependent oxidases. Our findings raise the possibility that in vivo LSD1 might not necessarily function as an oxidase, but it might use alternative electron acceptors.
FEBS Letters 05/2005; 579(10):2203-7. · 3.54 Impact Factor
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ABSTRACT: Activation of the complement cascade induces inflammatory responses and marks cells for immune clearance. In the central complement-amplification step, a complex consisting of surface-bound C3b and factor B is cleaved by factor D to generate active convertases on targeted surfaces. We present crystal structures of the pro-convertase C3bB at 4 angstrom resolution and its complex with factor D at 3.5 angstrom resolution. Our data show how factor B binding to C3b forms an open “activation” state of C3bB. Factor D specifically binds the open conformation of factor B through a site distant from the catalytic center and is activated by the substrate, which displaces factor D’s self-inhibitory loop. This concerted proteolytic mechanism, which is cofactor-dependent and substrate-induced, restricts complement amplification to C3b-tagged target cells.
