The Orange Carotenoid Protein (OCP) is a water-soluble protein that governs photoprotection in many cyanobacteria. The 35 kDa OCP is structurally and functionally modular, consisting of an N-terminal effector domain (NTD) and a C-terminal regulatory domain (CTD); a carotenoid spans the two domains. The CTD is a member of the ubiquitous Nuclear Transport Factor-2 (NTF2) superfamily (pfam02136). With the increasing availability of cyanobacterial genomes, bioinformatic analysis has revealed the existence of a new family of proteins, homologs to the CTD, the C-terminal domain-like carotenoid proteins (CCPs). Here we purify holo-CCP2 directly from cyanobacteria and establish that it natively binds canthaxanthin (CAN). We use small-angle X-ray scattering (SAXS) to characterize the structure of this carotenoprotein in two distinct oligomeric states. A single carotenoid molecule spans the two CCPs in the dimer. Our analysis with X-ray footprinting-mass spectrometry (XFMS) identifies critical residues for carotenoid binding that likely contribute to the extreme red shift (ca. 80 nm) of the absorption maximum of the carotenoid bound by the CCP2 dimer and a further 10 nm shift in the tetramer form. These data provide the first structural description of carotenoid binding by a protein consisting of only an NTF2 domain.
- MeSH
- Bacterial Proteins chemistry ultrastructure MeSH
- Canthaxanthin chemistry MeSH
- Crystallography, X-Ray MeSH
- Scattering, Small Angle MeSH
- Nucleocytoplasmic Transport Proteins chemistry genetics ultrastructure MeSH
- Protein Domains genetics MeSH
- Cyanobacteria chemistry ultrastructure MeSH
- Protein Binding drug effects MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Brd2 is a member of the bromodomain extra terminal (BET) protein family, which consists of four chromatin-interacting proteins that regulate gene expression. Each BET protein contains two N-terminal bromodomains, which recognize acetylated histones, and the C-terminal protein-protein interaction domain. Using a genome-wide screen, we identify 1450 genes whose transcription is regulated by Brd2. In addition, almost 290 genes change their alternative splicing pattern upon Brd2 depletion. Brd2 is specifically localized at promoters of target genes, and our data show that Brd2 interaction with chromatin cannot be explained solely by histone acetylation. Using coimmunoprecipitation and live-cell imaging, we show that the C-terminal part is crucial for Brd2 association with chromatin. Live-cell microscopy also allows us to map the average binding time of Brd2 to chromatin and quantify the contributions of individual Brd2 domains to the interaction with chromatin. Finally, we show that bromodomains and the C-terminal domain are equally important for transcription and splicing regulation, which correlates with the role of these domains in Brd2 binding to chromatin.
- MeSH
- Alternative Splicing MeSH
- Chromatin metabolism MeSH
- Transcription, Genetic MeSH
- Genome, Human * MeSH
- HeLa Cells MeSH
- Histones genetics metabolism MeSH
- Humans MeSH
- Promoter Regions, Genetic MeSH
- Protein Serine-Threonine Kinases genetics metabolism MeSH
- Gene Expression Regulation * MeSH
- Recombinant Fusion Proteins genetics metabolism MeSH
- Signal Transduction MeSH
- Protein Structure, Tertiary MeSH
- Protein Binding MeSH
- Microscopy, Video MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't 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
- Cisplatin pharmacology MeSH
- DNA metabolism MeSH
- Financing, Organized MeSH
- Antibodies, Monoclonal immunology MeSH
- Tumor Suppressor Protein p53 genetics chemistry immunology MeSH
- Oxidation-Reduction MeSH
- DNA Damage drug effects MeSH
- Sequence Deletion MeSH
- Protein Structure, Tertiary MeSH
- Protein Binding MeSH
- Binding Sites MeSH
Interleukin-1α (IL-1α) is a proinflammatory cytokine and a key player in host immune responses in higher eukaryotes. IL-1α has pleiotropic effects on a wide range of cell types, and it has been extensively studied for its ability to contribute to various autoimmune and inflammation-linked disorders, including rheumatoid arthritis, Alzheimer's disease, systemic sclerosis and cardiovascular disorders. Interestingly, a significant proportion of IL-1α is translocated to the cell nucleus, in which it interacts with histone acetyltransferase complexes. Despite the importance of IL-1α, little is known regarding its binding targets and functions in the nucleus. We took advantage of the histone acetyltransferase (HAT) complexes being evolutionarily conserved from yeast to humans and the yeast SAGA complex serving as an epitome of the eukaryotic HAT complexes. Using gene knock-out technique and co-immunoprecipitation of the IL-1α precursor with TAP-tagged subunits of the yeast HAT complexes, we mapped the IL-1α-binding site to the HAT/Core module of the SAGA complex. We also predicted the 3-D structure of the IL-1α N-terminal domain, and by employing structure similarity searches, we found a similar structure in the C-terminal regulatory region of the catalytic subunit of the AMP-activated/Snf1 protein kinases, which interact with HAT complexes both in mammals and yeast, respectively. This finding is further supported with the ability of the IL-1α precursor to partially rescue growth defects of snf1Δ yeast strains on media containing 3-Amino-1,2,4-triazole (3-AT), a competitive inhibitor of His3. Finally, the careful evaluation of our data together with other published data in the field allows us to hypothesize a new function for the ADA complex in SAGA complex assembly.
- MeSH
- Models, Biological MeSH
- Cell Nucleus metabolism MeSH
- Gene Knockout Techniques MeSH
- Histone Acetyltransferases metabolism MeSH
- Immunoprecipitation MeSH
- Interleukin-1alpha chemistry metabolism MeSH
- Humans MeSH
- Protein Subunits metabolism MeSH
- Protein Serine-Threonine Kinases chemistry metabolism MeSH
- AMP-Activated Protein Kinases chemistry metabolism MeSH
- Protein Precursors chemistry metabolism MeSH
- Saccharomyces cerevisiae Proteins metabolism MeSH
- Saccharomyces cerevisiae metabolism MeSH
- Signal Transduction MeSH
- Structural Homology, Protein MeSH
- Subcellular Fractions metabolism MeSH
- Protein Structure, Tertiary MeSH
- Trans-Activators metabolism MeSH
- Protein Binding MeSH
- Binding Sites MeSH
- Computational Biology MeSH
- Structure-Activity Relationship MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Influenza viruses can cause severe respiratory infections in humans, leading to nearly half a million deaths worldwide each year. Improved antiviral drugs are needed to address the threat of development of novel pandemic strains. Current therapeutic interventions target three key proteins in the viral life cycle: neuraminidase, the M2 channel and RNA-dependent-RNA polymerase. Protein-protein interactions between influenza polymerase subunits are potential new targets for drug development. Using a newly developed assay based on AlphaScreen technology, we screened a peptide panel for protein-protein interaction inhibitors to identify a minimal PB1 subunit-derived peptide that retains high inhibition potential and can be further modified. Here, we present an X-ray structure of the resulting decapeptide bound to the C-terminal domain of PA polymerase subunit from pandemic isolate A/California/07/2009 H1N1 at 1.6 Å resolution and discuss its implications for the design of specific, potent influenza polymerase inhibitors.
- MeSH
- Antiviral Agents pharmacology MeSH
- Protein Interaction Domains and Motifs drug effects physiology MeSH
- Crystallization MeSH
- Humans MeSH
- RNA-Dependent RNA Polymerase chemistry metabolism MeSH
- Protein Binding MeSH
- Viral Proteins antagonists & inhibitors chemistry metabolism MeSH
- Influenza A Virus, H1N1 Subtype drug effects enzymology metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Phosphorylation patterns of the C-terminal domain (CTD) of largest subunit of RNA polymerase II (called the CTD code) orchestrate the recruitment of RNA processing and transcription factors. Recent studies showed that not only serines and tyrosines but also threonines of the CTD can be phosphorylated with a number of functional consequences, including the interaction with yeast transcription termination factor, Rtt103p. Here, we report the solution structure of the Rtt103p CTD-interacting domain (CID) bound to Thr4 phosphorylated CTD, a poorly understood letter of the CTD code. The structure reveals a direct recognition of the phospho-Thr4 mark by Rtt103p CID and extensive interactions involving residues from three repeats of the CTD heptad. Intriguingly, Rtt103p's CID binds equally well Thr4 and Ser2 phosphorylated CTD A doubly phosphorylated CTD at Ser2 and Thr4 diminishes its binding affinity due to electrostatic repulsion. Our structural data suggest that the recruitment of a CID-containing CTD-binding factor may be coded by more than one letter of the CTD code.
- MeSH
- Phosphorylation MeSH
- Transcription, Genetic MeSH
- Protein Kinases metabolism MeSH
- Proteolysis MeSH
- RNA Polymerase II chemistry metabolism MeSH
- Saccharomyces cerevisiae Proteins chemistry metabolism MeSH
- Serine metabolism MeSH
- Protein Structure, Tertiary MeSH
- Threonine chemistry metabolism MeSH
- Transcription Factors chemistry metabolism MeSH
- Tyrosine metabolism MeSH
- Protein Binding MeSH
- Publication type
- Journal Article MeSH
Transcription elongation factor Spt6 associates with RNA polymerase II (RNAP II) via a tandem SH2 (tSH2) domain. The mechanism and significance of the RNAP II-Spt6 interaction is still unclear. Recently, it was proposed that Spt6-tSH2 is recruited via a newly described phosphorylated linker between the Rpb1 core and its C-terminal domain (CTD). Here, we report binding studies with isolated tSH2 of Spt6 (Spt6-tSH2) and Spt6 lacking the first unstructured 297 residues (Spt6ΔN) with a minimal CTD substrate of two repetitive heptads phosphorylated at different sites. The data demonstrate that Spt6 also binds the phosphorylated CTD, a site that was originally proposed as a recognition epitope. We also show that an extended CTD substrate harboring 13 repetitive heptads of the tyrosine-phosphorylated CTD binds Spt6-tSH2 and Spt6ΔN with tighter affinity than the minimal CTD substrate. The enhanced binding is achieved by avidity originating from multiple phosphorylation marks present in the CTD. Interestingly, we found that the steric effects of additional domains in the Spt6ΔN construct partially obscure the binding of the tSH2 domain to the multivalent ligand. We show that Spt6-tSH2 binds various phosphorylation patterns in the CTD and found that the studied combinations of phospho-CTD marks (1,2; 1,5; 2,4; and 2,7) all facilitate the interaction of CTD with Spt6. Our structural studies reveal a plasticity of the tSH2 binding pockets that enables the accommodation of CTDs with phosphorylation marks in different registers.
- MeSH
- Epitopes genetics MeSH
- Phosphorylation genetics MeSH
- Transcription, Genetic * MeSH
- Histone Chaperones genetics MeSH
- RNA Polymerase II genetics MeSH
- Saccharomyces cerevisiae Proteins genetics MeSH
- Saccharomyces cerevisiae genetics MeSH
- Amino Acid Sequence genetics MeSH
- src Homology Domains genetics MeSH
- Transcriptional Elongation Factors genetics MeSH
- Protein Binding genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
