The heme-based oxygen sensor protein AfGcHK is a globin-coupled histidine kinase in the soil bacterium Anaeromyxobacter sp. Fw109-5. Its C-terminal functional domain exhibits autophosphorylation activity induced by oxygen binding to the heme-Fe(II) complex located in the oxygen-sensing N-terminal globin domain. A detailed understanding of the signal transduction mechanisms in heme-containing sensor proteins remains elusive. Here, we investigated the role of the globin domain's dimerization interface in signal transduction in AfGcHK. We present a crystal structure of a monomeric imidazole-bound AfGcHK globin domain at 1.8 Å resolution, revealing that the helices of the WT globin dimer are under tension and suggesting that Tyr-15 plays a role in both this tension and the globin domain's dimerization. Biophysical experiments revealed that whereas the isolated WT globin domain is dimeric in solution, the Y15A and Y15G variants in which Tyr-15 is replaced with Ala or Gly, respectively, are monomeric. Additionally, we found that although the dimerization of the full-length protein is preserved via the kinase domain dimerization interface in all variants, full-length AfGcHK variants bearing the Y15A or Y15G substitutions lack enzymatic activity. The combined structural and biophysical results presented here indicate that Tyr-15 plays a key role in the dimerization of the globin domain of AfGcHK and that globin domain dimerization is essential for internal signal transduction and autophosphorylation in this protein. These findings provide critical insights into the signal transduction mechanism of the histidine kinase AfGcHK from Anaeromyxobacter.

• MeSH
• bakteriální proteiny chemie   metabolismus   MeSH
• fosforylace MeSH
• globiny chemie   metabolismus   MeSH
• histidinkinasa chemie   metabolismus   MeSH
• konformace proteinů, alfa-helix MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• molekulární modely MeSH
• multimerizace proteinu MeSH
• Myxococcales chemie   metabolismus   MeSH
• proteinové domény MeSH
• signální transdukce MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

It was shown previously that the p53 protein can recognize DNA modified with antitumor agent cisplatin (cisPt-DNA). Here, we studied p53 binding to the cisPt-DNA using p53 deletion mutants and via modulation of the p53-DNA binding by changes of the protein redox state. Isolated p53 C-terminal domain (CTD) bound to the cisPt-DNA with a significantly higher affinity than to the unmodified DNA. On the other hand, p53 constructs involving the core domain but lacking the C-terminal DNA binding site (CTDBS) exhibited only small binding preference for the cisPt-DNA. Oxidation of cysteine residues within the CD of posttranslationally unmodified full length p53 did not affect its ability to recognize cisPt-DNA. Blocking of the p53 CTDBS by a monoclonal antibody Bp53-10.1 resulted in abolishment of the isolated CTD binding to the cisPt-DNA. Our results demonstrate a crucial role of the basic region of the p53 CTD (aa 363-382) in the cisPt-DNA recognition.

• MeSH
• cisplatina farmakologie   MeSH
• DNA metabolismus   MeSH
• financování organizované MeSH
• monoklonální protilátky imunologie   MeSH
• nádorový supresorový protein p53 genetika   chemie   imunologie   MeSH
• oxidace-redukce MeSH
• poškození DNA účinky léků   MeSH
• sekvenční delece MeSH
• terciární struktura proteinů MeSH
• vazba proteinů MeSH
• vazebná místa MeSH

The nine-amino-acid transactivation domains (9aaTAD) was identified in numerous transcription factors including Gal4, p53, E2A, MLL, c-Myc, N-Myc, and also in SP, KLF, and SOX families. Most of the 9aaTAD domains interact with the KIX domain of transcription mediators MED15 and CBP to activate transcription. The NFkB activation domain occupied the same position on the KIX domain as the 9aaTADs of MLL, E2A, and p53. Binding of the KIX domain is established by the two-point interaction involving 9aaTAD positions p3-4 and p6-7. The NFkB primary binding region (positions p3-4) is almost identical with MLL and E2A, but secondary NFkB binding region differs by the position and engages the distal NFkB region p10-11. Thus, the NFkB activation domain is five amino acids longer than the other 9aaTADs. The NFkB activation domain includes an additional region, which we called the Omichinski Insert extending activation domain length to 14 amino acids. By deletion, we demonstrated that Omichinski Insert is an entirely non-essential part of NFkB activation domain. In summary, we recognized the NFkB activation domain as prolonged 9aaTAD conserved in evolution from humans to amphibians.

• MeSH
• aktivace transkripce MeSH
• aminokyseliny * metabolismus   MeSH
• lidé MeSH
• nádorový supresorový protein p53 * metabolismus   MeSH
• NF-kappa B metabolismus   MeSH
• sekvence aminokyselin MeSH
• transkripční faktory metabolismus   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The universal nine-amino-acid transactivation domains (9aaTADs) have been identified in numerous transcription activators. Here, we identified the conserved 9aaTAD motif in all nine members of the specificity protein (SP) family. Previously, the Sp1 transcription factor has been defined as a glutamine-rich activator. We showed by amino acid substitutions that the glutamine residues are completely dispensable for 9aaTAD function and are not conserved in the SP family. We described the origin and evolutionary history of 9aaTADs. The 9aaTADs of the ancestral Sp2 gene became inactivated in early chordates. We next discovered that an accumulation of valines in 9aaTADs inactivated their transactivation function and enabled their strict conservation during evolution. Subsequently, in chordates, Sp2 has duplicated and created new paralogs, Sp1, Sp3, and Sp4 (the SP1-4 clade). During chordate evolution, the dormancy of the Sp2 activation domain lasted over 100 million years. The dormant but still intact ancestral Sp2 activation domains allowed diversification of the SP1-4 clade into activators and repressors. By valine substitution in the 9aaTADs, Sp1 and Sp3 regained their original activator function found in ancestral lower metazoan sea sponges. Therefore, the vertebrate SP1-4 clade could include both repressors and activators. Furthermore, we identified secondary 9aaTADs in Sp2 introns present from fish to primates, including humans. In the gibbon genome, introns containing 9aaTADs were used as exons, which turned the Sp2 gene into an activator. Similarly, we identified introns containing 9aaTADs used conditionally as exons in the (SP family-unrelated) transcription factor SREBP1, suggesting that the intron-9aaTAD reservoir is a general phenomenon.

• MeSH
• aktivace transkripce MeSH
• duplikace genu MeSH
• fylogeneze MeSH
• introny * genetika   MeSH
• lidé MeSH
• molekulární evoluce * MeSH
• regulace genové exprese * MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie MeSH
• transkripční faktor Sp2 * antagonisté a inhibitory   genetika   metabolismus   MeSH
• valin genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH

The universal nine-amino-acid transactivation domains (9aaTADs) have been identified in numerous transcription activators. Here, we identified the conserved 9aaTAD motif in all nine members of the specificity protein (SP) family. Previously, the Sp1 transcription factor has been defined as a glutamine-rich activator. We showed by amino acid substitutions that the glutamine residues are completely dispensable for 9aaTAD function and are not conserved in the SP family. We described the origin and evolutionary history of 9aaTADs. The 9aaTADs of the ancestral Sp2 gene became inactivated in early chordates. We next discovered that an accumulation of valines in 9aaTADs inactivated their transactivation function and enabled their strict conservation during evolution. Subsequently, in chordates, Sp2 has duplicated and created new paralogs, Sp1, Sp3, and Sp4 (the SP1-4 clade). During chordate evolution, the dormancy of the Sp2 activation domain lasted over 100 million years. The dormant but still intact ancestral Sp2 activation domains allowed diversification of the SP1-4 clade into activators and repressors. By valine substitution in the 9aaTADs, Sp1 and Sp3 regained their original activator function found in ancestral lower metazoan sea sponges. Therefore, the vertebrate SP1-4 clade could include both repressors and activators. Furthermore, we identified secondary 9aaTADs in Sp2 introns present from fish to primates, including humans. In the gibbon genome, introns containing 9aaTADs were used as exons, which turned the Sp2 gene into an activator. Similarly, we identified introns containing 9aaTADs used conditionally as exons in the (SP family-unrelated) transcription factor SREBP1, suggesting that the intron-9aaTAD reservoir is a general phenomenon.

• MeSH
• aktivace transkripce MeSH
• duplikace genu MeSH
• fylogeneze MeSH
• introny genetika   MeSH
• lidé MeSH
• molekulární evoluce * MeSH
• regulace genové exprese * MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie MeSH
• transkripční faktor Sp2 antagonisté a inhibitory   genetika   metabolismus   MeSH
• valin genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

UNLABELLED: The hexameric lattice of an immature retroviral particle consists of Gag polyprotein, which is the precursor of all viral structural proteins. Lentiviral and alpharetroviral Gag proteins contain a peptide sequence called the spacer peptide (SP), which is localized between the capsid (CA) and nucleocapsid (NC) domains. SP plays a critical role in intermolecular interactions during the assembly of immature particles of several retroviruses. Published models of supramolecular structures of immature particles suggest that in lentiviruses and alpharetroviruses, SP adopts a rod-like six-helix bundle organization. In contrast, Mason-Pfizer monkey virus (M-PMV), a betaretrovirus that assembles in the cytoplasm, does not contain a distinct SP sequence, and the CA-NC connecting region is not organized into a clear rod-like structure. Nevertheless, the CA-NC junction comprises a sequence critical for assembly of immature M-PMV particles. In the present work, we characterized this region, called the SP-like domain, in detail. We provide biochemical data confirming the critical role of the M-PMV SP-like domain in immature particle assembly, release, processing, and infectivity. Circular dichroism spectroscopy revealed that, in contrast to the SP regions of other retroviruses, a short SP-like domain-derived peptide (SPLP) does not form a purely helical structure in aqueous or helix-promoting solution. Using 8-Å cryo-electron microscopy density maps of immature M-PMV particles, we prepared computational models of the SP-like domain and indicate the structural features required for M-PMV immature particle assembly. IMPORTANCE: Retroviruses such as HIV-1 are of great medical importance. Using Mason-Pfizer monkey virus (M-PMV) as a model retrovirus, we provide biochemical and structural data confirming the general relevance of a short segment of the structural polyprotein Gag for retrovirus assembly and infectivity. Although this segment is critical for assembly of immature particles of lentiviruses, alpharetroviruses, and betaretroviruses, the organization of this domain is strikingly different. A previously published electron microscopic structure of an immature M-PMV particle allowed us to model this important region into the electron density map. The data presented here help explain the different packing of the Gag segments of various retroviruses, such as HIV, Rous sarcoma virus (RSV), and M-PMV. Such knowledge contributes to understanding the importance of this region and its structural flexibility among retroviral species. The region might play a key role in Gag-Gag interactions, leading to different morphological pathways of immature particle assembly.

• MeSH
• cirkulární dichroismus MeSH
• elektronová kryomikroskopie MeSH
• konformace proteinů MeSH
• Masonův-Pfizerův opičí virus fyziologie   MeSH
• molekulární modely MeSH
• nukleokapsida - proteiny chemie   genetika   metabolismus   ultrastruktura   MeSH
• sestavení viru * MeSH
• uvolnění viru z buňky MeSH
• virové plášťové proteiny chemie   genetika   metabolismus   ultrastruktura   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

While the three-dimensional structures of heme- and flavin-binding domains of the NOS isoforms have been determined, the structures of the holoenzymes remained elusive. Application of electron cryo-microscopy and structural modeling of the bovine endothelial nitric oxide synthase (eNOS) holoenzyme produced detailed models of the intact holoenzyme in the presence and absence of Ca(2+)/calmodulin (CaM). These models accommodate the cross-electron transfer from the reductase in one monomer to the heme in the opposite monomer. The heme domain acts as the anchoring dimeric structure for the entire enzyme molecule, while the FMN domain is activated by CaM to move flexibly to bridge the distance between the reductase and oxygenase domains. Our results indicate that the key regulatory role of CaM involves the stabilization of structural intermediates and precise positioning of the pivot for the FMN domain tethered shuttling motion to accommodate efficient and rapid electron transfer in the homodimer of eNOS.

• MeSH
• alosterická regulace MeSH
• flavinmononukleotid chemie   MeSH
• hem chemie   MeSH
• holoenzymy chemie   MeSH
• kalmodulin chemie   metabolismus   MeSH
• kinetika MeSH
• oxidace-redukce MeSH
• skot MeSH
• synthasa oxidu dusnatého, typ III chemie   metabolismus   MeSH
• terciární struktura proteinů MeSH
• transport elektronů MeSH
• vápník chemie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Many patients with chronic myeloid leukemia in deep remission experience return of clinical disease after withdrawal of tyrosine kinase inhibitors (TKIs). This suggests signaling of inactive BCR-ABL, which allows the survival of cancer cells, and relapse. We show that TKI treatment inhibits catalytic activity of BCR-ABL, but does not dissolve BCR-ABL core signaling complex, consisting of CRKL, SHC1, GRB2, SOS1, cCBL, p85a-PI3K, STS1 and SHIP2. Peptide microarray and co-immunoprecipitation results demonstrate that CRKL binds to proline-rich regions located in C-terminal, intrinsically disordered region of BCR-ABL, that SHC1 requires pleckstrin homology, src homology and tyrosine kinase domains of BCR-ABL for binding, and that BCR-ABL sequence motif located in disordered region around phosphorylated tyrosine 177 mediates binding of three core complex members, i.e., GRB2, SOS1, and cCBL. Further, SHIP2 binds to the src homology and tyrosine kinase domains of BCR-ABL and its inositol phosphatase activity contributes to BCR-ABL-mediated phosphorylation of SHC1. Together, this study characterizes protein-protein interactions within the BCR-ABL core complex and determines the contribution of particular BCR-ABL domains to downstream signaling. Understanding the structure and dynamics of BCR-ABL interactome is critical for the development of drugs targeting integrity of the BCR-ABL core complex.

• MeSH
• adaptorové proteiny signální transdukční metabolismus   MeSH
• aminokyselinové motivy MeSH
• bcr-abl fúzní proteiny chemie   genetika   metabolismus   MeSH
• chronická myeloidní leukemie metabolismus   patologie   MeSH
• čipová analýza proteinů MeSH
• fosfatidylinositol-3,4,5-trisfosfát-5-fosfatasy metabolismus   MeSH
• fosforylace MeSH
• HEK293 buňky MeSH
• inhibitory proteinkinas farmakologie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• pyrimidiny farmakologie   MeSH
• signální transdukce účinky léků   MeSH
• vazba proteinů účinky léků   MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH

Many patients with chronic myeloid leukemia in deep remission experience return of clinical disease after withdrawal of tyrosine kinase inhibitors (TKIs). This suggests signaling of inactive BCR-ABL, which allows the survival of cancer cells, and relapse. We show that TKI treatment inhibits catalytic activity of BCR-ABL, but does not dissolve BCR-ABL core signaling complex, consisting of CRKL, SHC1, GRB2, SOS1, cCBL, p85a-PI3K, STS1 and SHIP2. Peptide microarray and co-immunoprecipitation results demonstrate that CRKL binds to proline-rich regions located in C-terminal, intrinsically disordered region of BCR-ABL, that SHC1 requires pleckstrin homology, src homology and tyrosine kinase domains of BCR-ABL for binding, and that BCR-ABL sequence motif located in disordered region around phosphorylated tyrosine 177 mediates binding of three core complex members, i.e., GRB2, SOS1, and cCBL. Further, SHIP2 binds to the src homology and tyrosine kinase domains of BCR-ABL and its inositol phosphatase activity contributes to BCR-ABL-mediated phosphorylation of SHC1. Together, this study characterizes protein-protein interactions within the BCR-ABL core complex and determines the contribution of particular BCR-ABL domains to downstream signaling. Understanding the structure and dynamics of BCR-ABL interactome is critical for the development of drugs targeting integrity of the BCR-ABL core complex.

• MeSH
• adaptorové proteiny signální transdukční metabolismus   MeSH
• aminokyselinové motivy MeSH
• bcr-abl fúzní proteiny chemie   genetika   metabolismus   MeSH
• chronická myeloidní leukemie metabolismus   patologie   MeSH
• čipová analýza proteinů MeSH
• fosfatidylinositol-3,4,5-trisfosfát-5-fosfatasy metabolismus   MeSH
• fosforylace MeSH
• HEK293 buňky MeSH
• inhibitory proteinkinas farmakologie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• pyrimidiny farmakologie   MeSH
• signální transdukce * účinky léků   MeSH
• src homologní domény MeSH
• transformující protein 1 obsahující src homologní doménu 2 metabolismus   MeSH
• vazba proteinů účinky léků   MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

• MeSH
• hem chemie   MeSH
• hydrolasy chemie   metabolismus   MeSH
• Ramanova spektroskopie MeSH
• systém (enzymů) cytochromů P-450 chemie   metabolismus   MeSH