Sojin An

Crystal Structure of the Human Histone Methyltransferase ASH1L Catalytic Domain and Its Implications for the Regulatory Mechanism

Absent, small, or homeotic disc1 (Ash1) is a trithorax group histone methyltransferase that is involved in gene activation. Although there are many known histone methyltransferases, their regulatory mechanisms are poorly understood. Here, we present the crystal structure of the human ASH1L catalytic domain, showing its substrate binding pocket blocked by a loop from the post-SET domain. In this configuration, the loop limits substrate access to the active site. Mutagenesis of the loop stimulates ASH1L histone methyltransferase activity, suggesting that ASH1L activity may be regulated through the loop from the post-SET domain. In addition, we show that human ASH1L specifically methylates histone H3 Lys-36. Our data implicate that there may be a regulatory mechanism of ASH1L histone methyltransferases.

STRUCTURAL AND MECHANISTIC INSIGHTS INTO HEMOGLOBIN-CATALYZED HYDROGEN SULFIDE OXIDATION AND THE FATE OF POLYSULFIDE PRODUCTS.

Hydrogen sulfide is a cardioprotective signaling molecule but is toxic at elevated concentrations. Red blood cells can synthesize H2S but, lacking organelles, cannot dispose of H2S via the mitochondrial sulfide oxidation pathway. We have recently shown that at high sulfide concentrations, ferric hemoglobin oxidizes H2S to a mixture of thiosulfate and iron-bound polysulfides in which the latter species predominates. Here, we report the crystal structure of human hemoglobin containing low spin ferric sulfide, the first intermediate in heme-catalyzed sulfide oxidation. The structure provides molecular insights into why sulfide is susceptible to oxidation in human hemoglobin but is stabilized against it in HbI, a specialized sulfide-carrying hemoglobin from a mollusk adapted to life in a sulfide-rich environment. We have also captured a second sulfide bound at a postulated ligand entry/exit site in the α-subunit of hemoglobin, which, to the best of our knowledge, represents the first direct evidence for this site being used to access the heme iron. Hydrodisulfide, a postulated intermediate at the junction between thiosulfate and polysulfide formation, coordinates ferric hemoglobin and, in the presence of air, generated thiosulfate. At low sulfide/heme iron ratios, the product distribution between thiosulfate and iron-bound polysulfides was approximately equal. The iron-bound polysulfides were unstable at physiological glutathione concentrations and were reduced with concomitant formation of glutathione persulfide, glutathione disulfide, and H2S. Hence, although polysulfides are unlikely to be stable in the reducing intracellular milieu, glutathione persulfide could serve as a persulfide donor for protein persulfidation, a posttranslational modification by which H2S is postulated to signal.

Mis16 Independently Recognizes Histone H4 and the CENP-ACnp1-Specific Chaperone Scm3sp

CENP-A is a centromere-specific histone H3 variant that is required for kinetochore assembly and accurate chromosome segregation. For it to function properly, CENP-A must be specifically localized to centromeres. In fission yeast, Scm3sp and the Mis18 complex, composed of Mis16, Eic1, and Mis18, function as a CENP-A(Cnp1)-specific chaperone and a recruiting factor, respectively, and together ensure accurate delivery of CENP-A(Cnp1) to centromeres. Although how Scm3sp specifically recognizes CENP-A(Cnp1) has been revealed recently, the recruiting mechanism of CENP-A(Cnp1) via the Mis18 complex remains unknown. In this study, we have determined crystal structures of Schizosaccharomyces japonicus Mis16 alone and in complex with the helix 1 of histone H4 (H4α1). Crystal structures followed by mutant analysis and affinity pull-downs have revealed that Mis16 recognizes both H4α1 and Scm3sp independently within the CENP-A(Cnp1)/H4:Scm3sp complex. This observation suggests that Mis16 gains CENP-A(Cnp1) specificity by recognizing both Scm3sp and histone H4. Our studies provide insights into the molecular mechanisms underlying specific recruitment of CENP-A(Cnp1)/H4:Scm3sp into centromeres.

Structure-based nuclear import mechanism of histones H3 and H4 mediated by Kap123

Kap123, a major karyopherin protein of budding yeast, recognizes the nuclear localization signals (NLSs) of cytoplasmic histones H3 and H4 and translocates them into the nucleus during DNA replication. Mechanistic questions include H3- and H4-NLS redundancy toward Kap123 and the role of the conserved diacetylation of cytoplasmic H4 (K5ac and K12ac) in Kap123-mediated histone nuclear translocation. Here, we report crystal structures of full-length Kluyveromyces lactis Kap123 alone and in complex with H3- and H4-NLSs. Structures reveal the unique feature of Kap123 that possesses two discrete lysine-binding pockets for NLS recognition. Structural comparison illustrates that H3- and H4-NLSs share at least one of two lysine-binding pockets, suggesting that H3- and H4-NLSs are mutually exclusive. Additionally, acetylation of key lysine residues at NLS, particularly H4-NLS diacetylation, weakens the interaction with Kap123. These data support that cytoplasmic histone H4 diacetylation weakens the Kap123-H4-NLS interaction thereby facilitating histone Kap123-H3-dependent H3:H4/Asf1 complex nuclear translocation.

MMOD-induced structural changes of hydroxylase in soluble methane monooxygenase

Soluble methane monooxygenase in methanotrophs converts methane to methanol under ambient conditions1-3. The maximum catalytic activity of hydroxylase (MMOH) is achieved via interplay of its regulatory protein (MMOB) and reductase4-6. An additional auxiliary protein, MMOD, is believed to function as an inhibitor of the catalytic activity of MMOH; however, the mechanism of its action remains unknown7,8. Herein, we report the crystal structure of MMOH–MMOD complex from Methylosinus sporium strain 5 (2.6 Å), which illustrates that two molecules of MMOD associate symmetrically with the canyon region of MMOH in a manner similar to MMOB, indicating that MMOD competes with MMOB for MMOH recognition. Further, MMOD binding disrupts the geometry of the di-iron centre and opens the substrate access channel. Notably, the electron density of 1,6-hexanediol at the substrate access channel mimics products of sMMO in hydrocarbon oxidation. The crystal structure of MMOH–MMOD unravels the inhibitory mechanism by which MMOD suppresses the MMOH catalytic activity, and reveals how hydrocarbon substrates/products access to the di-iron centre.

Mis16 Switches Function from a Histone H4 Chaperone to a CENP-ACnp1-Specific Assembly Factor through Eic1 Interaction

The Mis18 complex, composed of Mis16, Eic1, and Mis18 in fission yeast, selectively deposits the centromere-specific histone H3 variant, CENP-ACnp1, at centromeres. How the intact Mis18 holo-complex oligomerizes and how Mis16, a well-known ubiquitous histone H4 chaperone, plays a centromere-specific role in the Mis18 holo-complex, remain unclear. Here, we report the stoichiometry of the intact Mis18 holo-complex as (Mis16)2:(Eic1)2:(Mis18)4 using analytical ultracentrifugation. We further determine the crystal structure of Schizosaccharomyces pombe Mis16 in complex with the C-terminal portion of Eic1 (Eic1-CT). Notably, Mis16 accommodates Eic1-CT through the binding pocket normally occupied by histone H4, indicating that Eic1 and H4 compete for the same binding site, providing a mechanism for Mis16 to switch its binding partner from histone H4 to Eic1. Thus, our analyses not only determine the stoichiometry of the intact Mis18 holo-complex but also uncover the molecular mechanism by which Mis16 plays a centromere-specific role through Eic1 association.

Structural insights into the architecture of human Importin4_histone H3/H4_Asf1a complex and its histone H3 tail binding

Importin4 transports histone H3/H4 in complex with Asf1a to the nucleus for chromatin assembly. Importin4 recognizes the nuclear localization sequence located at the N-terminal tail of histones. Here, we analyzed the structures and interactions of human Importin4, histones and Asf1a by cross-linking mass spectrometry, X-ray crystallography, negative-stain electron microscopy, small-angle X-ray scattering and integrative modeling. The XL-MS data showed that the C-terminal region of Importin4 interacts extensively with the histone H3 tail. We determined the crystal structure of the C-terminal region of Importin4 bound to the histone H3 peptide, thus revealing that the acidic path in Importin4 accommodates the histone H3 tail and that histone H3 Lys14 is the primary residue interacting with Importin4. Furthermore, the molecular architecture of the Importin4_histone H3/H4_Asf1a complex was produced through an integrative modeling approach. Overall, this work provides structural insights into how Importin4 recognizes histones and their chaperone complex.