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1.
Proc Natl Acad Sci U S A ; 114(23): 6016-6021, 2017 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-28533407

RESUMO

Double plant homeodomain finger 2 (DPF2) is a highly evolutionarily conserved member of the d4 protein family that is ubiquitously expressed in human tissues and was recently shown to inhibit the myeloid differentiation of hematopoietic stem/progenitor and acute myelogenous leukemia cells. Here, we present the crystal structure of the tandem plant homeodomain finger domain of human DPF2 at 1.6-Å resolution. We show that DPF2 interacts with the acetylated tails of both histones 3 and 4 via bipartite binding pockets on the DPF2 surface. Blocking these interactions through targeted mutagenesis of DPF2 abolishes its recruitment to target chromatin regions as well as its ability to prevent myeloid differentiation in vivo. Our findings suggest that the histone binding of DPF2 plays an important regulatory role in the transcriptional program that drives myeloid differentiation.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Histonas/química , Histonas/metabolismo , Células Mieloides/citologia , Células Mieloides/metabolismo , Acetilação , Diferenciação Celular/fisiologia , Cromatina/química , Cromatina/metabolismo , Cristalografia por Raios X , Hematopoese/fisiologia , Humanos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Fatores de Transcrição
2.
Science ; 352(6283): aaf1015, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-27081075

RESUMO

The nuclear pore complex (NPC) controls the transport of macromolecules between the nucleus and cytoplasm, but its molecular architecture has thus far remained poorly defined. We biochemically reconstituted NPC core protomers and elucidated the underlying protein-protein interaction network. Flexible linker sequences, rather than interactions between the structured core scaffold nucleoporins, mediate the assembly of the inner ring complex and its attachment to the NPC coat. X-ray crystallographic analysis of these scaffold nucleoporins revealed the molecular details of their interactions with the flexible linker sequences and enabled construction of full-length atomic structures. By docking these structures into the cryoelectron tomographic reconstruction of the intact human NPC and validating their placement with our nucleoporin interactome, we built a composite structure of the NPC symmetric core that contains ~320,000 residues and accounts for ~56 megadaltons of the NPC's structured mass. Our approach provides a paradigm for the structure determination of similarly complex macromolecular assemblies.


Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Poro Nuclear/ultraestrutura , Mapas de Interação de Proteínas , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Microscopia Crioeletrônica , Cristalografia por Raios X , Citoplasma/metabolismo , Tomografia com Microscopia Eletrônica , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Dados de Sequência Molecular , Poro Nuclear/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo
3.
J Mol Biol ; 426(14): 2605-16, 2014 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-24846647

RESUMO

Tubulin protomers undergo an extensive array of post-translational modifications to tailor microtubules to specific tasks. One such modification, the acetylation of lysine 40 of α-tubulin, located in the lumen of microtubules, is associated with stable, long-living microtubule structures. MEC-17 was recently identified as the acetyltransferase that mediates this event. We have determined the crystal structure of the catalytic core of human MEC-17 in complex with its cofactor acetyl-CoA at 1.7Å resolution. The structure reveals that the MEC-17 core adopts a canonical Gcn5-related N-acetyltransferase (GNAT) fold that is decorated with extensive surface loops. An enzymatic analysis of 33 MEC-17 surface mutants identifies hot-spot residues for catalysis and substrate recognition. A large, evolutionarily conserved hydrophobic surface patch that is critical for enzymatic activity is identified, suggesting that specificity is achieved by interactions with the α-tubulin substrate that extend outside of the modified surface loop. An analysis of MEC-17 mutants in Caenorhabditis elegans shows that enzymatic activity is dispensable for touch sensitivity.


Assuntos
Acetiltransferases/química , Acetiltransferases/metabolismo , Acetilcoenzima A/química , Acetilcoenzima A/metabolismo , Acetiltransferases/genética , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Cristalografia por Raios X , Humanos , Interações Hidrofóbicas e Hidrofílicas , Proteínas dos Microtúbulos , Dados de Sequência Molecular , Mutação , Conformação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Tubulina (Proteína)/metabolismo
4.
J Mol Biol ; 426(3): 526-41, 2014 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-24120939

RESUMO

SIRT1 is a NAD(+)-dependent deacetylase that plays important roles in many cellular processes. SIRT1 activity is uniquely controlled by a C-terminal regulatory segment (CTR). Here we present crystal structures of the catalytic domain of human SIRT1 in complex with the CTR in an open apo form and a closed conformation in complex with a cofactor and a pseudo-substrate peptide. The catalytic domain adopts the canonical sirtuin fold. The CTR forms a ß hairpin structure that complements the ß sheet of the NAD(+)-binding domain, covering an essentially invariant hydrophobic surface. The apo form adopts a distinct open conformation, in which the smaller subdomain of SIRT1 undergoes a rotation with respect to the larger NAD(+)-binding subdomain. A biochemical analysis identifies key residues in the active site, an inhibitory role for the CTR, and distinct structural features of the CTR that mediate binding and inhibition of the SIRT1 catalytic domain.


Assuntos
NAD/metabolismo , Elementos Reguladores de Transcrição , Sirtuína 1/química , Sirtuína 1/metabolismo , Acetilação , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação Proteica
5.
J Mol Biol ; 419(5): 330-46, 2012 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-22480613

RESUMO

The cytoplasmic filament nucleoporins of the nuclear pore complex (NPC) are critically involved in nuclear export and remodeling of mRNA ribonucleoprotein particles and are associated with various human malignancies. Here, we report the crystal structure of the Nup98 C-terminal autoproteolytic domain, frequently missing from leukemogenic forms of the protein, in complex with the N-terminal domain of Nup82 and the C-terminal tail fragment of Nup159. The Nup82 ß propeller serves as a noncooperative binding platform for both binding partners. Interaction of Nup98 with Nup82 occurs through a reciprocal exchange of loop structures. Strikingly, the same Nup98 groove promiscuously interacts with Nup82 and Nup96 in a mutually excusive fashion. Simultaneous disruption of both Nup82 interactions in yeast causes severe defects in mRNA export, while the severing of a single interaction is tolerated. Thus, the cytoplasmic filament network of the NPC is robust, consistent with its essential function in nucleocytoplasmic transport.


Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/química , Poro Nuclear/química , Proteínas Oncogênicas/química , Animais , Cristalografia por Raios X , Citoplasma/metabolismo , Humanos , Camundongos , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/química
6.
Proc Natl Acad Sci U S A ; 106(42): 17693-8, 2009 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-19805193

RESUMO

The heptameric Nup84 complex constitutes an evolutionarily conserved building block of the nuclear pore complex. Here, we present the crystal structure of the heterotrimeric Sec13 x Nup145C x Nup84 complex, the centerpiece of the heptamer, at 3.2-A resolution. Nup84 forms a U-shaped alpha-helical solenoid domain, topologically similar to two other members of the heptamer, Nup145C and Nup85. The interaction between Nup84 and Nup145C is mediated via a hydrophobic interface located in the kink regions of the two solenoids that is reinforced by additional interactions of two long Nup84 loops. The Nup84 binding site partially overlaps with the homo-dimerization interface of Nup145C, suggesting competing binding events. Fitting of the elongated Z-shaped heterotrimer into electron microscopy (EM) envelopes of the heptamer indicates that structural changes occur at the Nup145C x Nup84 interface. Docking the crystal structures of all heptamer components into the EM envelope constitutes a major advance toward the completion of the structural characterization of the Nup84 complex.


Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/química , Poro Nuclear/química , Proteínas de Saccharomyces cerevisiae/química , Sítios de Ligação , Ligação Competitiva , Cristalografia por Raios X , Modelos Moleculares , Complexos Multiproteicos/química , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Saccharomyces cerevisiae/química
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