Structure Of C-terminal Domain Research Articles

The C-terminal domain of the Uup protein is a DNA-binding coiled coil motif

The bacterial Uup protein belongs to the REG subfamily of soluble ATP-binding cassette (ABC) ATPases, and is implicated in precise excision of transposons. In Escherichia coli, the uup gene encodes a 72 kDa polypeptide that comprises two ABC domains, separated by a linker region, and a 12kDa C-terminal domain (CTD). Uup binds double-stranded DNA with no sequence specificity, and we previously demonstrated that the CTD domain is a crucial region that participates in DNA-binding activity. We report herein the NMR structure of Uup CTD, consisting of an intramolecular antiparallel two-stranded coiled coil motif. Structural comparison with analogous coiled coil domains reveals that Uup CTD contains an atypical 3(10)-helix in the α-hairpin region that contributes to the hydrophobic core. Using NMR titration experiments, we identified residues of the CTD domain involved in the binding to double-stranded DNA. These residues are located on two opposite surfaces at the base of the coiled coil, formed by the N- and C-terminal extremities, where a strictly conserved proline residue induces an overwinding of the coiled coil. Finally, preliminary analysis of NMR spectra recorded on distinct Uup constructs precludes a fully flexible positioning of the CTD domain in full-length Uup. These structural data are the first reported for a non-ATPase domain within ABC REG subfamily.

Structural Basis of Recruitment of DNA Polymerase ζ by Interaction between REV1 and REV7 Proteins

REV1, REV3, and REV7 are pivotal proteins in translesion DNA synthesis, which allows DNA synthesis even in the presence of DNA damage. REV1 and REV3 are error-prone DNA polymerases and function as inserter and extender polymerases in this process, respectively. REV7 interacts with both REV1 and REV3, acting as an adaptor that functionally links the two, although the structural basis of this collaboration remains unclear. Here, we show the crystal structure of the ternary complex, composed of the C-terminal domain of human REV1, REV7, and a REV3 fragment. The REV1 C-terminal domain adopts a four-helix bundle that interacts with REV7. A linker region between helices 2 and 3, which is conserved among mammals, interacts with the β-sheet of REV7. Remarkably, the REV7-binding interface is distinct from the binding site of DNA polymerase η or κ. Thus, the REV1 C-terminal domain might facilitate polymerase switching by providing a scaffold for both inserter and extender polymerases to bind. Our structure reveals the basis of DNA polymerase ζ (a complex of REV3 and REV7) recruitment to the stalled replication fork and provides insight into the mechanism of polymerase switching.

Serine phosphorylation and proline isomerization in RNAP II CTD control recruitment of Nrd1

Recruitment of appropriate RNA processing factors to the site of transcription is controlled by post-translational modifications of the C-terminal domain (CTD) of RNA polymerase II (RNAP II). Here, we report the solution structure of the Ser5 phosphorylated (pSer5) CTD bound to Nrd1. The structure reveals a direct recognition of pSer5 by Nrd1 that requires the cis conformation of the upstream pSer5-Pro6 peptidyl-prolyl bond of the CTD. Mutations at the complex interface diminish binding affinity and impair processing or degradation of noncoding RNAs. These findings underpin the interplay between covalent and noncovalent changes in the CTD structure that constitute the CTD code.

Unfolding the Bridge between Transcription and Translation

Transcription antiterminator RfaH alternates between closed (inactive) and open (activated) conformation. In this issue of Cell, Burmann et al. show that opening is accompanied by dramatic all-α to all-β refolding of its C-terminal domain. Each of the folds has a distinct function: all-α-fold acts as a specificity determinant, directing RfaH to a small subset of operons, whereas the all-β-fold recruits ribosome, thereby coupling RfaH-stimulated transcription to translation.

Structure of the Cx43 C-terminal domain bound to tubulin

Structure of the Cx43 C-terminal domain bound to tubulin

Structural Basis for Telomerase RNA Recognition and RNP Assembly by the Holoenzyme La Family Protein p65

Telomerase is a ribonucleoprotein complex essential for maintenance of telomere DNA at linear chromosome ends. The catalytic core of Tetrahymena telomerase comprises a ternary complex of telomerase RNA (TER), telomerase reverse transcriptase (TERT), and the essential La family protein p65. NMR and crystal structures of p65 C-terminal domain and its complex with stem IV of TER reveal that RNA recognition is achieved by a combination of single- and double-stranded RNA binding, which induces a 105° bend in TER. The domain is a cryptic, atypical RNA recognition motif with a disordered C-terminal extension that forms an α helix in the complex necessary for hierarchical assembly of TERT with p65-TER. This work provides the first structural insight into biogenesis and assembly of TER with a telomerase-specific protein. Additionally, our studies define a structurally homologous domain (xRRM) in genuine La and LARP7 proteins and suggest a general mode of RNA binding for biogenesis of their diverse RNA targets.

DNA Polymerase δ and ζ Switch by Sharing Accessory Subunits of DNA Polymerase δ

Translesion DNA synthesis is an important branch of the DNA damage tolerance pathway that assures genomic integrity of living organisms. The mechanisms of DNA polymerase (Pol) switches during lesion bypass are not known. Here, we show that the C-terminal domain of the Pol ζ catalytic subunit interacts with accessory subunits of replicative DNA Pol δ. We also show that, unlike other members of the human B-family of DNA polymerases, the highly conserved and similar C-terminal domains of Pol δ and Pol ζ contain a [4Fe-4S] cluster coordinated by four cysteines. Amino acid changes in Pol ζ that prevent the assembly of the [4Fe-4S] cluster abrogate Pol ζ function in UV mutagenesis. On the basis of these data, we propose that Pol switches at replication-blocking lesions occur by the exchange of the Pol δ and Pol ζ catalytic subunits on a preassembled complex of accessory proteins retained on DNA during translesion DNA synthesis.

Solution NMR Structure, Backbone Dynamics, and Heme-Binding Properties of a Novel Cytochrome c Maturation Protein CcmE from Desulfovibrio vulgaris

Cytochrome c maturation protein E, CcmE, plays an integral role in the transfer of heme to apocytochrome c in many prokaryotes and some mitochondria. A novel subclass featuring a heme-binding cysteine has been identified in archaea and some bacteria. Here we describe the solution NMR structure, backbone dynamics, and heme binding properties of the soluble C-terminal domain of Desulfovibrio vulgaris CcmE, dvCcmE'. The structure adopts a conserved β-barrel OB fold followed by an unstructured C-terminal tail encompassing the CxxxY heme-binding motif. Heme binding analyses of wild-type and mutant dvCcmE' demonstrate the absolute requirement of residue C127 for noncovalent heme binding in vitro.

Structure of human Mad1 C-terminal domain reveals its involvement in kinetochore targeting

The spindle checkpoint prevents aneuploidy by delaying anaphase onset until all sister chromatids achieve proper microtubule attachment. The kinetochore-bound checkpoint protein complex Mad1-Mad2 promotes the conformational activation of Mad2 and serves as a catalytic engine of checkpoint signaling. How Mad1 is targeted to kinetochores is not understood. Here, we report the crystal structure of the conserved C-terminal domain (CTD) of human Mad1. Mad1 CTD forms a homodimer and, unexpectedly, has a fold similar to those of the kinetochore-binding domains of Spc25 and Csm1. Nonoverlapping Mad1 fragments retain detectable kinetochore targeting. Deletion of the CTD diminishes, does not abolish, Mad1 kinetochore localization. Mutagenesis studies further map the functional interface of Mad1 CTD in kinetochore targeting and implicate Bub1 as its receptor. Our results indicate that CTD is a part of an extensive kinetochore-binding interface of Mad1, and rationalize graded kinetochore targeting of Mad1 during checkpoint signaling.

Architecture of a Dodecameric Bacterial Replicative Helicase

Hexameric DnaB helicases are often loaded at DNA replication forks by interacting with the initiator protein DnaA and/or a helicase loader (DnaC in Escherichia coli). These loaders are not universally required, and DnaB from Helicobacter pylori was found to bypass DnaC when expressed in E.coli cells. The crystal structure of Helicobacter pylori DnaB C-terminal domain (HpDnaB-CTD) reveals a large two-helix insertion (named HPI) in the ATPase domain that protrudes away from the RecA fold. Biophysical characterization and electron microscopy (EM) analysis of the full-length protein show that HpDnaB forms head-to-head double hexamers remarkably similar to helicases found in some eukaryotes, archaea, and viruses. The docking of the HpDnaB-CTD structure into EM reconstruction of HpDnaB provides a model that shows how hexamerization of the CTD is facilitated by HPI-HPI interactions. The HpDnaB double-hexamer architecture supports an alternative strategy to load bacterial helicases onto forks in the absence of helicase loaders.

Mg2+/Ca2+ Induced Changes in Structure, Dynamics and Stability of Dream Protein

Downstream Regulatory Element Antagonist Modulator (DREAM) is a member of the neuronal calcium sensor family that controls activity of potassium voltage channels and regulates c-fos and prodynorphine gene transcription in a Ca2+ dependent manner. Here, we have investigated the impact of Mg2+ and Ca2+ binding on the structure, stability and dynamics of DREAM and DREAM C-terminal domain (DREAM-C). Mg2+ binding to the apoDREAM does not alter the secondary or tertiary structure as based on CD and Trp emission spectra whereas the association of Mg2+ to either EF-3 or EF-4 in apoDREAM-C results in an increase in protein secondary structure and alteration of the tertiary structure. Ca2+ binding to either apo- or Mg2+DREAM and apo- or Mg2+DREAM-C triggers larger conformational changes as evident from the blue-shift in emission spectra and the decrease of Stern-Volmer constant. Ca2+ triggered changes in DREAM conformational dynamics were characterized by time-resolved fluorescence. In apoDREAM, single tryptophan residue exhibits two lifetimes (τ1=3.38 ns, f1=73% and τ2=7.72 ns, f2=27%). Ca2+ binding to apo- and Mg2+DREAM leads to the shortening of the first lifetime and decrease of the fractional contribution (τ1 = 1.75 ns, f1 = 47% and τ2 = 7. ns, f2 = 53%). Furthermore, the impact of Ca2+/Mg2+ on DREAM stability was determined in equilibrium folding studies. The binding of Ca2+ increase the protein stability by ∼ 7 kcal mol−1 whereas the impact of Mg2+ on DREAM stability is significantly smaller, ∼ 1 kcal mol−1. The stability of the C terminal domain in both apo and Ca2+ bound form is significantly smaller compared to the full length protein, suggesting that the inter-domain interactions significantly contribute to the structural and functional properties of DREAM.

Atg8 Transfer from Atg7 to Atg3: A Distinctive E1-E2 Architecture and Mechanism in the Autophagy Pathway

Atg7 is a noncanonical, homodimeric E1 enzyme that interacts with the noncanonical E2 enzyme, Atg3, to mediate conjugation of the ubiquitin-like protein (UBL) Atg8 during autophagy. Here we report that the unique N-terminal domain of Atg7 (Atg7(NTD)) recruits a unique "flexible region" from Atg3 (Atg3(FR)). The structure of an Atg7(NTD)-Atg3(FR) complex reveals hydrophobic residues from Atg3 engaging a conserved groove in Atg7, important for Atg8 conjugation. We also report the structure of the homodimeric Atg7 C-terminal domain, which is homologous to canonical E1s and bacterial antecedents. The structures, SAXS, and crosslinking data allow modeling of a full-length, dimeric (Atg7~Atg8-Atg3)(2) complex. The model and biochemical data provide a rationale for Atg7 dimerization: Atg8 is transferred in trans from the catalytic cysteine of one Atg7 protomer to Atg3 bound to the N-terminal domain of the opposite Atg7 protomer within the homodimer. The studies reveal a distinctive E1~UBL-E2 architecture for enzymes mediating autophagy.

Structural Basis for the Activation of Innate Immune Pattern-Recognition Receptor RIG-I by Viral RNA

RIG-I is a key innate immune pattern-recognition receptor that triggers interferon expression upon detection of intracellular 5'triphosphate double-stranded RNA (5'ppp-dsRNA) of viral origin. RIG-I comprises N-terminal caspase activation and recruitment domains (CARDs), a DECH helicase, and a C-terminal domain (CTD). We present crystal structures of the ligand-free, autorepressed, and RNA-bound, activated states of RIG-I. Inactive RIG-I has an open conformation with the CARDs sequestered by a helical domain inserted between the two helicase moieties. ATP and dsRNA binding induce a major rearrangement to a closed conformation in which the helicase and CTD bind the blunt end 5'ppp-dsRNA with perfect complementarity but incompatibly with continued CARD binding. We propose that after initial binding of 5'ppp-dsRNA to the flexibly linked CTD, co-operative tight binding of ATP and RNA to the helicase domain liberates the CARDs for downstream signaling. These findings significantly advance our molecular understanding of the activation of innate immune signaling helicases.

Substrate tRNA Recognition Mechanism of a Multisite-specific tRNA Methyltransferase, Aquifex aeolicus Trm1, Based on the X-ray Crystal Structure

Archaeal and eukaryotic tRNA (N(2),N(2)-guanine)-dimethyltransferase (Trm1) produces N(2),N(2)-dimethylguanine at position 26 in tRNA. In contrast, Trm1 from Aquifex aeolicus, a hyper-thermophilic eubacterium, modifies G27 as well as G26. Here, a gel mobility shift assay revealed that the T-arm in tRNA is the binding site of A. aeolicus Trm1. To address the multisite specificity, we performed an x-ray crystal structure study. The overall structure of A. aeolicus Trm1 is similar to that of archaeal Trm1, although there is a zinc-cysteine cluster in the C-terminal domain of A. aeolicus Trm1. The N-terminal domain is a typical catalytic domain of S-adenosyl-l-methionine-dependent methyltransferases. On the basis of the crystal structure and amino acid sequence alignment, we prepared 30 mutant Trm1 proteins. These mutant proteins clarified residues important for S-adenosyl-l-methionine binding and enabled us to propose a hypothetical reaction mechanism. Furthermore, the tRNA-binding site was also elucidated by methyl transfer assay and gel mobility shift assay. The electrostatic potential surface models of A. aeolicus and archaeal Trm1 proteins demonstrated that the distribution of positive charges differs between the two proteins. We constructed a tRNA-docking model, in which the T-arm structure was placed onto the large area of positive charge, which is the expected tRNA-binding site, of A. aeolicus Trm1. In this model, the target G26 base can be placed near the catalytic pocket; however, the nucleotide at position 27 gains closer access to the pocket. Thus, this docking model introduces a rational explanation of the multisite specificity of A. aeolicus Trm1.

Structure of the C-terminal domain of the MutL homolog fromN. gonorrhoeae

Structure of the C-terminal domain of the MutL homolog from<i>N. gonorrhoeae</i>

Crystal structure of the mismatch-specific thymine glycosylase domain of human methyl-CpG-binding protein MBD4

Methyl-CpG (mCpG) binding domain protein 4 (MBD4) is a member of mammalian DNA glycosylase superfamily. It contains an amino-proximal methyl-CpG binding domain (MBD) and a C-terminal mismatch-specific glycosylase domain, which is an important molecule believed to be involved in maintaining of genome stability. Herein, we determined the crystal structure of C-terminal glycosylase domain of human MBD4. And the structural alignments of other helix-hairpin-helix (HhH) DNA glycosylases show that the human MBD4 glycosylase domain has the similar active site and the catalytic mechanisms as others. But the different residues in the N-terminal of domain result in the change of charge distribution on the surface of the protein, which suggest the different roles that may relate some diseases.

Crystal structure of the C-terminal domain of human DNA primase large subunit

DNA polymerases cannot synthesize DNA without a primer, and DNA primase is the only specialized enzyme capable of de novo synthesis of short RNA primers. In eukaryotes, primase functions within a heterotetrameric complex in concert with a tightly bound DNA polymerase α (Pol α). In humans, the Pol α part is comprised of a catalytic subunit (p180) and an accessory subunit B (p70), and the primase part consists of a small catalytic subunit (p49) and a large essential subunit (p58). The latter subunit participates in primer synthesis, counts the number of nucleotides in a primer, assists the release of the primer-template from primase and transfers it to the Pol α active site. Recently reported crystal structures of the C-terminal domains of the yeast and human enzymes’ large subunits provided critical information related to their structure, possible sites for binding of nucleotides and template DNA, as well as the overall organization of eukaryotic primases. However, the structures also revealed a difference in the folding of their proposed DNA-binding fragments, raising the possibility that yeast and human proteins are functionally different. Here we report new structure of the C-terminal domain of the human primase p58 subunit. This structure exhibits a fold similar to a fold reported for the yeast protein but different than a fold reported for the human protein. Based on a comparative analysis of all three C-terminal domain structures, we propose a mechanism of RNA primer length counting and dissociation of the primer-template from primase by a switch in conformation of the ssDNA-binding region of p58.

Helicase-Primase Interactions and their Structural Studies in Helicobacter Pylori

In eubacterial DNA replication initiation complex, two proteins of prime foci are DnaB helicase, a homohexameric motor protein which unwinds duplex DNA, and DnaG primase, a DNA dependent RNA polymerase which synthesizes oligonucleotide primers for DNA replication.In our study we have found the H. pylori DnaB helicase activity being stimulated by H. pylori DnaG primase, suggesting the presence of helicase-primase cohort at replication fork. We have reported the crystal structure of NTD of HpDnaB helicase at 2.2 A resolution. The structural details of NTD besides SPR studies suggest that the linker region between NTD and C-terminal helicase domain plays a vital role in physiological assembly of NTD dimers, and strong interaction with C-terminal domain (CTD) of primase. The sequence analysis of the linker region reveals that they should form four helix bundles. The surface charge distribution on the primase binding surface of NTD's of various helicases shows that the DnaB-DnaG interaction and stability of the complex appears to be charge dependent.Experimental phasing (SAD) was used to solve the crystal structure of HpDnaG primase CTD from 1.7 A diffraction data. The structure is helical in nature like the other two eubacterial DnaG primase CTD structures of Bacillus stearothermophilus and Escherichia coli. Structural comparison suggests that different orientation of helical hairpin in the DnaG primase CTD may play an important role in the regulation of primase activity.

TFPI-Beta Is An Effective Inhibitor of TF-FVIIa Mediated Coagulation and Tumor Cell Invasion

AbstractAbstract 347Tissue Factor Pathway Inhibitor (TFPI) is the primary physiological inhibitor of TF-fVIIa, the in vivo activator of blood coagulation. TFPI is an alternatively spliced protein with two major isoforms, TFPI-alpha and TFPI-beta. These isoforms differ in their C-terminal domain structure and their mechanism for cell surface attachment with TFPI-alpha indirectly associating with the endothelial surface through binding to a GPI-floated protein while TFPI-beta is directly attached to a GPI-float. In addition, the isoforms are differentially expressed in mouse tissues, with TFPI-alpha present in placenta, embryo and platelets, while TFPI-beta is the predominant isoform in adult vascular beds. However, TFPI-beta has ∼20-fold decreased activity when compared to TFPI-alpha in solution phase plasma clotting assays. We hypothesized that TFPI-beta will have more physiologically relevant activity when associated with a cell surface instead of in solution phase. To test this hypothesis, a CHO cell system that allows measurement of the anti-TF activity of different cell surface forms of TFPI in both in vitro and in vivo assays was developed. CHO cells were stably transfected with TF creating CHO-TF cells. In contrast to wild type CHO cells, CHO-TF cells 1) generate fXa in vitro in the presence of fVIIa, fX and calcium ions, 2) migrate through matrigel in transwell assays, and 3) produce tumors in the lungs of SCID mice following tail vein injection. The ability of solution phase TFPI-alpha and TFPI-beta to inhibit TF-fVIIa mediated fXa generation on the surface of the CHO-TF cells was examined with amidolytic assays. The Ki(final) for TFPI-alpha and TFPI-beta were 5.81 nM and 21.6 nM, respectively, confirming that solution phase TFPI-beta has decreased inhibitory activity. To examine the activity of cell surface associated TFPI-beta, CHO-TF cells were co-transfected with either TFPI-beta or equal amounts of an altered form of TFPI, called K1K2K3-GPI that is similar to TFPI-alpha but lacks its basic C-terminal region. Forms of TFPI containing the basic C-terminal region with a GPI-float could not be studied because they were not expressed by the CHO cells. CHO-TF cells expressing TFPI-beta had equal inhibitory activity to that observed in CHO-TF cells with K1K2K3-GPI suggesting that TFPI-beta is a potent inhibitor of TF activity when associated with the cell surface. The activity of cell surface associated TFPI-beta was further examined in transwell migration assays. The number of cells migrating through matrigel in multiple 20X fields was averaged. In this assay, cell surface associated TFPI-beta was a potent inhibitor of TF activity. The results are as follows: wild type CHO cells—6.6+/−4.1; CHO-TF cells—98.0+/−32.5; CHO-TF cells with TFPI-beta—9.05+/−7.0; CHO-TF cells with K1K2K3-GPI—43.5+/−21.5. Migration of CHO-TF cells was blocked using argatroban, an active site directed inhibitor of thrombin (15.0+/−4.5 CHO-TF cells migrated in the presence of 100 micromolar argatroban), demonstrating that the likely mechanism for the TF-mediated cell migration is generation of thrombin with cellular activation through cleavage of protease activated receptors. In the SCID tumor model, the severity of lung tumor burden was graded as 1–4 by a pathologist blinded to the cell type injected, n=5-8 per group. Average scores were: wild type CHO cells—1.17; CHO-TF cells 2.80; CHO-TF cells with TFPI-beta—2.14; demonstrating that cell surface associated TFPI-beta is an effective inhibitor of TF activity in vivo. Thus, while TFPI-beta has limited anticoagulant activity when examined in solution phase assays in vitro, evaluation of cell surface associated TFPI-beta reveals that it is a highly effective inhibitor of TF-fVIIa activity both in vitro and in vivo. Disclosures:Mast:Novo Nordisk: Research Funding; Siemens: Speakers Bureau.

X-ray Structures of Human Galectin-9 C-terminal Domain in Complexes with a Biantennary Oligosaccharide and Sialyllactose

Galectin-9, a tandem-repeat-type β-galactoside-specific animal lectin with two carbohydrate recognition domains (CRDs) at the N- and C-terminal ends, is involved in chemoattraction, apoptosis, and the regulation of cell differentiation and has anti-allergic effects. Its ability to recognize carbohydrates is essential for its biological functions. Human galectin-9 (hG9) has high affinity for branched N-glycan-type oligosaccharides (dissociation constants of 0.16-0.70 μM) and linear β1-3-linked poly-N-acetyllactosamines (0.09-8.3 μM) and significant affinity for the α2-3-sialylated oligosaccharides (17-34 μM). Further, its N-terminal CRD (hG9N) and C-terminal CRD (hG9C) differ in specificity. To elucidate this unique feature of hG9, x-ray structures of hG9C in the free form and in complexes with N-acetyllactosamine, the biantennary pyridylaminated oligosaccharide, and α2-3-sialyllactose were determined. They are the first x-ray structural analysis of C-terminal CRD of the tandem-repeat-type galectin. The results clearly revealed the mechanism by which branched and α2-3-sialylated oligosaccharides are recognized and explained the difference in specificity between hG9N and hG9C. Based on structural comparisons with other galectins, we propose that the wide entrance for ligand binding and the shallow binding site of hG9C are favorable for branched oligosaccharides and that Arg(221) is responsible for recognizing sialylated oligosaccharides.