Interaction Of C-terminal Domain Research Articles

Control of the RNA polymerase II phosphorylation state in promoter regions by CTD interaction domain-containing proteins RPRD1A and RPRD1B

RNA polymerase II (RNAP II) C-terminal domain (CTD) phosphorylation is important for various transcription-related processes. Here, we identify by affinity purification and mass spectrometry three previously uncharacterized human CTD-interaction domain (CID)-containing proteins, RPRD1A, RPRD1B and RPRD2, which co-purify with RNAP II and three other RNAP II-associated proteins, RPAP2, GRINL1A and RECQL5, but not with the Mediator complex. RPRD1A and RPRD1B can accompany RNAP II from promoter regions to 3'-untranslated regions during transcription in vivo, predominantly interact with phosphorylated RNAP II, and can reduce CTD S5- and S7-phosphorylated RNAP II at target gene promoters. Thus, the RPRD proteins are likely to have multiple important roles in transcription.

Architecture of a Full-length Retroviral Integrase Monomer and Dimer, Revealed by Small Angle X-ray Scattering and Chemical Cross-linking

We determined the size and shape of full-length avian sarcoma virus (ASV) integrase (IN) monomers and dimers in solution using small angle x-ray scattering. The low resolution data obtained establish constraints for the relative arrangements of the three component domains in both forms. Domain organization within the small angle x-ray envelopes was determined by combining available atomic resolution data for individual domains with results from cross-linking coupled with mass spectrometry. The full-length dimer architecture so revealed is unequivocally different from that proposed from x-ray crystallographic analyses of two-domain fragments, in which interactions between the catalytic core domains play a prominent role. Core-core interactions are detected only in cross-linked IN tetramers and are required for concerted integration. The solution dimer is stabilized by C-terminal domain (CTD-CTD) interactions and by interactions of the N-terminal domain in one subunit with the core and CTD in the second subunit. These results suggest a pathway for formation of functional IN-DNA complexes that has not previously been considered and possible strategies for preventing such assembly.

The interaction of azurin and C-terminal domain of p53 is mediated by nucleic acids

Azurin is bacterial protein, which was been reported to promote cancer cell death in vitro. The interaction of azurin and p53 is important for the cytotoxic effect of azurin towards cancer cells. In this study, it was found that nucleic acids mediated the interaction of azurin and the C-terminal domain of p53 (residues 352–393). The results provide novel insight into the interaction, and raising the possibility that the allosteric regulation of C-terminus of p53 by nucleic acids play an important role in the interaction of p53 with azurin. Meanwhile an elongated expressed product of azurin was cloned and purified, which was found to have stronger interaction with C-terminal domain of p53. Cytotoxicity studies showed that the cytotoxic effect of this elongated expressed product of azurin was stronger than wild-type azurin. The difference found in the cytotoxic effect of azurin with various sequence may provide valuable insight for finding more effective anticancer peptides.

3D architecture of DNA Pol α reveals the functional core of multi-subunit replicative polymerases

Eukaryotic DNA replication requires the coordinated activity of the multi-subunit DNA polymerases: Pol α, Pol δ and Pol ɛ. The conserved catalytic and regulatory B subunits associate in a constitutive heterodimer that represents the functional core of all three replicative polymerases. Here, we combine X-ray crystallography and electron microscopy (EM) to describe subunit interaction and 3D architecture of heterodimeric yeast Pol α. The crystal structure of the C-terminal domain (CTD) of the catalytic subunit bound to the B subunit illustrates a conserved mechanism of accessory factor recruitment by replicative polymerases. The EM reconstructions of Pol α reveal a bilobal shape with separate catalytic and regulatory modules. Docking of the B–CTD complex in the EM reconstruction shows that the B subunit is tethered to the polymerase domain through a structured but flexible linker. Our combined findings provide a structural template for the common functional architecture of the three major replicative DNA polymerases.

The BARD1 C-Terminal Domain Structure and Interactions with Polyadenylation Factor CstF-50

The BARD1 C-Terminal Domain Structure and Interactions with Polyadenylation Factor CstF-50

The BARD1 C-terminal domain structure and interactions with polyadenylation factor CstF-50.

The BARD1 N-terminal RING domain binds BRCA1 while the BARD1 C-terminal ankyrin and tandem BRCT repeat domains bind CstF-50 to modulate mRNA processing and RNAP II stability in response to DNA damage. Here we characterize the BARD1 structural biochemistry responsible for CstF-50 binding. The crystal structure of the BARD1 BRCT domain uncovers a degenerate phosphopeptide binding pocket lacking the key arginine required for phosphopeptide interactions in other BRCT proteins. Small angle X-ray scattering together with limited proteolysis results indicates that ankyrin and BRCT domains are linked by a flexible tether and do not adopt a fixed orientation relative to one another. Protein pull-down experiments utilizing a series of purified BARD1 deletion mutants indicate that interactions between the CstF-50 WD-40 domain and BARD1 involve the ankyrin-BRCT linker but do not require ankyrin or BRCT domains. The structural plasticity imparted by the ANK-BRCT linker helps to explain the regulated assembly of different protein BARD1 complexes with distinct functions in DNA damage signaling including BARD1-dependent induction of apoptosis plus p53 stabilization and interactions. BARD1 architecture and plasticity imparted by the ANK-BRCT linker are suitable to allow the BARD1 C-terminus to act as a hub with multiple binding sites to integrate diverse DNA damage signals directly to RNA polymerase.

Mrtl—A translation/localization regulatory protein encoded within the human c‐myc locus and distributed throughout the endoplasmic and nucleoplasmic reticular network

mrtl (myc-related translation/localization regulatory factor) is a previously uncharacterized protein synthesized from the first open reading frame contained within the human c-myc P0 transcript, approximately 800 nucleotides upstream of the Myc coding sequence. The mrtl protein, 114 amino acids in length, is projected to contain an N-terminal transmembrane domain and a highly charged C-terminal interaction domain with homology to numerous RNA-binding proteins. Using monoclonal antibodies raised against the hydrophilic C-terminal domain, endogenous mrtl was visualized in human breast tumor cell lines and primary mammary epithelial cells at the nuclear envelope and contiguous endoplasmic/nucleoplasmic reticulum. mrtl colocalizes and coimmunoprecipitates with translation initiation factor eIF2alpha and the 40S ribosomal protein RACK1, and appears capable of binding specifically to the c-myc RNA. Inducible ectopic overexpression of wild-type mrtl interferes with the function of endogenous mrtl, which results in loss of Myc from the nucleus. Furthermore, treatment of cells with a peptide derived from the C-terminal domain displaces endogenous mrtl and causes a dramatic reduction in total cellular Myc protein levels. Together with our previous work demonstrating complete loss of tumorigenicity in association with ectopic expression of the c-myc P0 5'-UTR (containing the mrtl coding sequence), these results suggest that mrtl may serve an important function in regulating Myc translation and localization to the nucleus, perhaps ultimately contributing to the role of the c-myc locus in oncogenesis.

Andersen‐Tawil Syndrome: Management Challenges During Pregnancy, Labor, and Delivery

Andersen-Tawil syndrome (ATS) is characterized by ventricular arrhythmias, hypokalemic periodic paralysis and developmental anomalies. It is caused by mutations in the KCNJ2 gene that encodes for the alpha-subunit of Kir2.1, a K(+) channel responsible for cardiac repolarization. Providing effective therapy to reduce arrhythmia burden and risk of sudden death is challenging, especially in the context of pregnancy and childbirth. We report a case of a pregnant 27-year-old woman with an R218W mutation in the C-terminal interaction domain of KCNJ2 causing ATS. Regular cardiac and obstetric assessments were performed for the duration of the pregnancy, which carried to term and delivered successfully with potassium replacement and intravenous beta blockade. ATS is a rare and potentially lethal condition in which there is considerable genetic and phenotypic heterogeneity. Effective management strategies are directed at reducing symptoms, arrhythmia burden and sudden cardiac death. This case illustrates the challenges and approach to management of patients with ATS who are pregnant and undergo childbirth.

The Nrd1–Nab3–Sen1 termination complex interacts with the Ser5-phosphorylated RNA polymerase II C-terminal domain

RNA polymerase II (Pol II) in Saccharomyces cerevisiae can terminate transcription via several pathways. To study how a mechanism is chosen, we analyzed recruitment of Nrd1, which cooperates with Nab3 and Sen1 to terminate small nucleolar RNAs and other short RNAs. Budding yeast contains three C-terminal domain (CTD) interaction domain (CID) proteins, which bind the CTD of the Pol II largest subunit. Rtt103 and Pcf11 act in mRNA termination, and both preferentially interact with CTD phosphorylated at Ser2. The crystal structure of the Nrd1 CID shows a fold similar to that of Pcf11, but Nrd1 preferentially binds to CTD phosphorylated at Ser5, the form found proximal to promoters. This indicates why Nrd1 cross-links near 5′ ends of genes and why the Nrd1–Nab3–Sen1 termination pathway acts specifically at short Pol II–transcribed genes. Nrd1 recruitment to genes involves a combination of interactions with CTD and Nab3.

Monitoring Protein-Protein Interactions between the Mammalian Integral Membrane Transporters and PDZ-interacting Partners Using a Modified Split-ubiquitin Membrane Yeast Two-hybrid System

PDZ-binding motifs are found in the C-terminal tails of numerous integral membrane proteins where they mediate specific protein-protein interactions by binding to PDZ-containing proteins. Conventional yeast two-hybrid screens have been used to probe protein-protein interactions of these soluble C termini. However, to date no in vivo technology has been available to study interactions between the full-length integral membrane proteins and their cognate PDZ-interacting partners. We previously developed a split-ubiquitin membrane yeast two-hybrid (MYTH) system to test interactions between such integral membrane proteins by using a transcriptional output based on cleavage of a transcription factor from the C terminus of membrane-inserted baits. Here we modified MYTH to permit detection of C-terminal PDZ domain interactions by redirecting the transcription factor moiety from the C to the N terminus of a given integral membrane protein thus liberating their native C termini. We successfully applied this "MYTH 2.0" system to five different mammalian full-length renal transporters and identified novel PDZ domain-containing partners of the phosphate (NaPi-IIa) and sulfate (NaS1) transporters that would have otherwise not been detectable. Furthermore this assay was applied to locate the PDZ-binding domain on the NaS1 protein. We showed that the PDZ-binding domain for PDZK1 on NaS1 is upstream of its C terminus, whereas the two interacting proteins, NHERF-1 and NHERF-2, bind at a location closer to the N terminus of NaS1. Moreover NHERF-1 and NHERF-2 increased functional sulfate uptake in Xenopus oocytes when co-expressed with NaS1. Finally we used MYTH 2.0 to demonstrate that the NaPi-IIa transporter homodimerizes via protein-protein interactions within the lipid bilayer. In summary, our study establishes the MYTH 2.0 system as a novel tool for interactive proteomics studies of membrane protein complexes.

Structure determination of discoidin II from Dictyostelium discoideum and carbohydrate binding properties of the lectin domain

The social amoeba Dictyostelium discoideum adopts a cohesive stage upon starvation and then produces Discoidin I and II, two proteins able to bind galactose and N-acetyl-galactosamine. The N-terminal domain or discoidin domain (DS) is widely distributed in eukaryotes where it plays a role in extracellular matrix binding while the C-terminal domain displays sequence similarities to invertebrate lectins. We present the first X-ray structures of the wild-type and recombinant Discoidin II in unliganded state and in complex with monosaccharides. The protein forms a homotrimer which presents two binding surfaces situated on the opposite boundaries of the structure. The binding sites of the N-terminal domain contain PEG molecules that could mimics binding of natural ligand. The C-terminal lectin domain interactions with N-acetyl-D-galactosamine and methyl-beta-galactoside are described. The carbohydrate binding sites are located at the interface between monomers. Specificity for galacto configuration can be rationalized since the axial O4 hydroxyl group is involved in several hydrogen bonds with protein side chains. Titration microcalorimetry allowed characterization of affinity and demonstrated the enthalpy-driven character of the interaction. Those results highlight the structural differentiation of the DS domain involved in many cell-adhesion processes from the lectin activity of Dictyostelium discoidins.

Structural Characterization of the Transmembrane Domain from Subunit e of Yeast F1Fo-ATP Synthase: A Helical GXXXG Motif Located Just under the Micelle Surface

F1Fo-ATP synthase is a large multiprotein complex, including at least 10 subunits in the membrane-bound Fo-sector. One of these Fo proteins is subunit e (Su e), involved in the stable dimerization of F1Fo-ATP synthase, and required for the establishment of normal cristae membrane architecture. As a step toward enabling structure-function studies of the Fo-sector, the Su e transmembrane region was structurally characterized in micelles. Based on a series of NMR and CD (circular dichroism) studies, a structural model of the Su e/micelle complex was constructed, indicating Su e is largely helical, and emerges from the micelle with Arg20 near the phosphate head groups. Su e only adopts this folded conformation in the context of the micelle, and is essentially disordered in DMSO, water or trifluoroethanol/water. Within the micelle the C-terminal Ala10-Arg20 stretch is helical, while the region N-terminal may be transiently helical, based on negative CSI (chemical shift index) values. The Ala10-Arg20 helix contains the G14XXXG18 motif, which has been proposed to play an important role in dimer formation with another protein from the Fo-sector. The Gly on the C-terminal end of this motif (Gly18) is slightly more mobile than the more buried Gly14, based on NMR order parameter measurements (Gly14 S2 = 0.950; Gly18 S2 = 0.895). Only one Su e transmembrane peptide is bound per micelle, and micelles are 22-23 A in diameter, composed of 51 +/- 4 dodecylphosphocholine detergent molecules. Although there is no evidence for Su e homodimerization via the transmembrane domain, potentially synergistic roles for N-terminal (membrane) and C-terminal (soluble) domain interactions may still occur. Furthermore, the presence of a buried charged residue (Arg7) suggests there may be interactions with other Fo-sector protein(s) that stabilize this charge, and possibly drive the folding of the N-terminal 9 residues of the transmembrane domain.

Novel toxicity of the unedited GluR2 AMPA receptor subunit dependent on surface trafficking and increased Ca 2+-permeability

RNA editing modifies the GluR2 AMPA receptor subunit pore loop at the Q/R site and limits receptor Ca 2+ permeability. Editing failure is implicated in neurodegenerative diseases, including amyotrophic lateral sclerosis. We show that channels with unedited GluR2 are highly toxic in cultured hippocampal neurons. Toxicity exceeds that of other Ca 2+-permeable AMPA receptor types and is influenced by agonist binding site mutations, ability to desensitize, and extracellular Ca 2+ levels. Significantly, toxicity also depends on GluR2's constitutive surface trafficking, a function dependent on GluR2 C-terminal domain interaction with NSF, a specialized chaperone. We have exploited the interaction between unedited GluR2 and NSF to reduce GluR2 surface levels. We show that a peptide that blocks the GluR2-NSF interaction reduces unedited GluR2 toxicity by reducing receptor surface expression. Peptide block of trafficking provides a model for design of drugs to reduce unedited GluR2 excitotoxicity in neurodegenerative diseases that result from editing failure.

Lipid-induced Extension of Apolipoprotein E Helix 4 Correlates with Low Density Lipoprotein Receptor Binding Ability

Apolipoprotein E (apoE) serves as a ligand for the low density lipoprotein receptor (LDLR) only when bound to lipid. The N-terminal domain of lipid-free apoE exists as globular 4-helix bundle that is conferred with LDLR recognition ability after undergoing a lipid binding-induced conformational change. To investigate the structural basis for this phenomenon, site-directed spin label electron paramagnetic resonance spectroscopy experiments were conducted, focusing on the region near the C-terminal end of helix 4 (Ala-164). Using C112S apoE-N-terminal as template, a series of single cysteine substitution variants (at sequence positions 161, 165, 169, 173, 176, and 181) were produced, isolated, and labeled with the nitroxide probe, methane thiosulfonate. Electron paramagnetic resonance analysis revealed that lipid association induced fixed secondary structure in a region of the molecule known to exist as random coil in the lipid-free state. In a complementary approach, site-directed fluorescence analysis using an environmentally sensitive probe indicated that the lipid-induced transition of this region of the protein to alpha helix was accompanied by relocation to a more hydrophobic environment. In studies with full-length apoE single Cys variants, a similar random coil to stable backbone transition was observed, consistent with the concept that lipid interaction induced an extension of helix 4 beyond the boundary defining its lipid-free conformation. This structural transition likely represents a key conformational change necessary for manifestation of the LDLR recognition properties of apoE.

MDMX Overexpression Prevents p53 Activation by the MDM2 Inhibitor Nutlin

The p53 tumor suppressor plays a key role in maintaining genomic stability and protection against malignant transformation. MDM2 and MDMX are both p53-binding proteins that regulate p53 stability and activity. Recent development of the MDM2 inhibitor Nutlin 3 has greatly facilitated functional analysis of MDM2-p53 binding. We found that although MDMX is homologous to MDM2 and binds to the same region on p53 N terminus, Nutlin does not disrupt p53-MDMX interaction. The ability of Nutlin to activate p53 is compromised in tumor cells overexpressing MDMX. Combination of Nutlin with MDMX siRNA resulted in synergistic activation of p53 and growth arrest. These results suggest that MDMX is also a valid target for p53 activation in tumor cells. Development of novel compounds that are MDMX-specific or optimized for dual-inhibition of MDM2 and MDMX are necessary to achieve full activation of p53 in tumor cells.

Structural basis for the inhibition of insulin-like growth factors by insulin-like growth factor-binding proteins

Insulin-like growth factor-binding proteins (IGFBPs) control bioavailability, activity, and distribution of insulin-like growth factor (IGF)1 and -2 through high-affinity IGFBP/IGF complexes. IGF-binding sites are found on N- and C-terminal fragments of IGFBPs, the two conserved domains of IGFBPs. The relative contributions of these domains to IGFBP/IGF complexation has been difficult to analyze, in part, because of the lack of appropriate three-dimensional structures. To analyze the effects of N- and C-terminal domain interactions, we determined several x-ray structures: first, of a ternary complex of N- and C-terminal domain fragments of IGFBP4 and IGF1 and second, of a "hybrid" ternary complex using the C-terminal domain fragment of IGFBP1 instead of IGFBP4. We also solved the binary complex of the N-terminal domains of IGFBP4 and IGF1, again to analyze C- and N-terminal domain interactions by comparison with the ternary complexes. The structures reveal the mechanisms of IGF signaling regulation via IGFBP binding. This finding supports research into the design of IGFBP variants as therapeutic IGF inhibitors for diseases of IGF disregulation. In IGFBP4, residues 1-38 form a rigid disulphide bond ladder-like structure, and the first five N-terminal residues bind to IGF and partially mask IGF residues responsible for the type 1 IGF receptor binding. A high-affinity IGF1-binding site is located in a globular structure between residues 39 and 82. Although the C-terminal domains do not form stable binary complexes with either IGF1 or the N-terminal domain of IGFBP4, in the ternary complex, the C-terminal domain contacts both and contributes to blocking of the IGF1 receptor-binding region of IGF1.

Two Blades of the [Ex]Scissor

Boelens and coworkers (Tripsianes et al., 2005) present the structure of the heterodimeric complex of the C-terminal interaction domains of the human XPF/ERCC1 structure-specific endonuclease. The authors also provide new insights into the mechanism of XPF/ERCC1 by characterizing its DNA binding properties.

Redox-Dependent Structural Differences in Putidaredoxin Derived from Homologous Structure Refinement via Residual Dipolar Couplings,

Structural differences in the [2Fe-2S] ferredoxin, putidaredoxin (Pdx), from the camphor hydroxylation pathway of Pseudomonas putida have been investigated as a function of oxidation state of the iron cluster. Pdx is involved in biological electron transfer to cytochrome P450(cam) (CYP101). Redox-dependent differences have been observed previously for Pdx in terms of binding affinities to CYP101, NMR spectral differences, and dynamic properties. To further characterize these differences, structure refinement of both oxidized and reduced Pdx has been carried out using a hybrid approach utilizing paramagnetic distance restraints and NMR orientational restraints in the form of backbone (15)N residual dipolar couplings. Use of these new restraints has improved the structure of oxidized Pdx considerably over the earlier solution NMR structure without RDC restraints, with the new structure now much closer in overall fold to the recently published X-ray crystal structures. We now observe better defined relative orientations of the major secondary structure elements as also of the conformation of the metal binding loop region. Extension of this approach to structure calculation of reduced Pdx has identified structural differences that are primarily localized for residues in the C-terminal interaction domain consisting of the functionally important residue Trp 106 and regions near the metal binding loop in Pdx. These redox-dependent structural differences in Pdx correlate to dynamic changes observed before and may be linked to differences in binding and electron transfer properties between oxidized and reduced Pdx.

Is the Prion Domain of Soluble Ure2p Unstructured?

The [URE3] prion is a self-propagating amyloid form of the Ure2 protein of Saccharomyces cerevisiae. Deletions in the C-terminal nitrogen regulation domain of Ure2p increase the frequency with which the N-terminal prion domain polymerizes into the prion form, suggesting that the C-terminus stabilizes the prion domain or that the structured C-terminal region sterically impairs amyloid formation. We find by in vivo two-hybrid analysis no evidence of interaction of prion domain and C-terminal domain. Furthermore, surface plasmon resonance spectrometry shows no evidence of interaction of prion domain and C-terminal domain, and cleavage at a specific site between the domains frees the two fragments. Our NMR analysis indicates that most residues of the prion domain are in fact disordered in the soluble form of Ure2p. Deleting the tether holding the C-terminal structured region to the amyloid core does not impair prion formation, arguing against steric impairment of amyloid formation. These results suggest that the N-terminal prion domain is unstructured in the soluble protein and does not have a specific interaction with the C-terminus.

Alternative splicing generates multiple SMRT transcripts encoding conserved repressor domains linked to variable transcription factor interaction domains.

Silencing mediator for retinoid and thyroid hormone receptor (SMRT) and nuclear receptor corepressor protein (NCoR) are corepressors that interact with a range of transcription factors. They both consist of N-terminal repressor domains that associate with histone deacetylases and C-terminal interaction domains (IDs) that contain CoRNR box motifs. These motifs mediate the interaction between corepressors and nuclear receptors (NRs), such as the retinoid and thyroid hormone receptors. However, whilst NCoR produces a single transcript during Xenopus development, xSMRT is subject to alternative splicing at four sites in the 3' part of the transcript, the region encoding the C-terminal IDs. Although this provides the potential to produce 16 different transcripts, only five isoforms are found in early embryos. The sites of alternative splicing predict that the resultant isoforms will differ in their ability to interact with NRs, as one site varies the number of CoRNR boxes, the second site changes the sequence flanking CoRNR box-1 and the other sites delete amino acid residues between CoRNR boxes 1 and 2 and so alter the critical spacing between these motifs. SMRT and NCoR therefore represent paralogues in which one form, SMRT, has evolved the ability to generate multiple isoforms whereas the other, NCoR, is invariant in Xenopus development.