Insulin and insulin-like growth factors I and II are closely related protein hormones. Their distinct evolution has resulted in different yet overlapping biological functions with insulin becoming a key regulator of metabolism, whereas insulin-like growth factors (IGF)-I/II are major growth factors. Insulin and IGFs cross-bind with different affinities to closely related insulin receptor isoforms A and B (IR-A and IR-B) and insulin-like growth factor type I receptor (IGF-1R). Identification of structural determinants in IGFs and insulin that trigger their specific signaling pathways is of increasing importance in designing receptor-specific analogs with potential therapeutic applications. Here, we developed a straightforward protocol for production of recombinant IGF-II and prepared six IGF-II analogs with IGF-I-like mutations. All modified molecules exhibit significantly reduced affinity toward IR-A, particularly the analogs with a Pro-Gln insertion in the C-domain. Moreover, one of the analogs has enhanced binding affinity for IGF-1R due to a synergistic effect of the Pro-Gln insertion and S29N point mutation. Consequently, this analog has almost a 10-fold higher IGF-1R/IR-A binding specificity in comparison with native IGF-II. The established IGF-II purification protocol allowed for cost-effective isotope labeling required for a detailed NMR structural characterization of IGF-II analogs that revealed a link between the altered binding behavior of selected analogs and conformational rearrangement of their C-domains.
- MeSH
- Antigens, CD chemistry genetics metabolism MeSH
- Insulin-Like Growth Factor II chemistry genetics metabolism MeSH
- Humans MeSH
- Mutation, Missense MeSH
- Protein Isoforms chemistry genetics metabolism MeSH
- Protein Domains MeSH
- Receptor, IGF Type 1 chemistry genetics metabolism MeSH
- Receptor, Insulin chemistry genetics metabolism MeSH
- Recombinant Proteins chemistry genetics metabolism MeSH
- Amino Acid Substitution MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
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
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
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
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
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
