Crystal structure of a multifunctional 2-Cys peroxiredoxin heme-binding protein 23 kDa/proliferation-associated gene product

Heme-binding protein 23 kDa (HBP23), a rat isoform of human proliferation-associated gene product (PAG), is a member of the peroxiredoxin family of peroxidases, having two conserved cysteine residues. Recent biochemical studies have shown that HBP23/PAG is an oxidative stress-induced and proliferation-coupled multifunctional protein that exhibits specific bindings to c-Abl protein tyrosine kinase and heme, as well as a peroxidase activity. A 2.6-Å resolution crystal structure of rat HBP23 in oxidized form revealed an unusual dimer structure in which the active residue Cys-52 forms a disulfide bond with conserved Cys-173 from another subunit by C-terminal tail swapping. The active site is largely hydrophobic with partially exposed Cys-173, suggesting a reduction mechanism of oxidized HBP23 by thioredoxin. Thus, the unusual cysteine disulfide bond is involved in peroxidation catalysis by using thioredoxin as the source of reducing equivalents. The structure also provides a clue to possible interaction surfaces for c-Abl and heme. Several significant structural differences have been found from a 1-Cys peroxiredoxin, ORF6, which lacks the C-terminal conserved cysteine corresponding to Cys-173 of HBP23.

A Z-DNA binding domain present in the human editing enzyme, double-stranded RNA adenosine deaminase

Editing of RNA changes the read-out of information from DNA by altering the nucleotide sequence of a transcript. One type of RNA editing found in all metazoans uses double-stranded RNA (dsRNA) as a substrate and results in the deamination of adenosine to give inosine, which is translated as guanosine. Editing thus allows variant proteins to be produced from a single pre-mRNA. A mechanism by which dsRNA substrates form is through pairing of intronic and exonic sequences before the removal of noncoding sequences by splicing. Here we report that the RNA editing enzyme, human dsRNA adenosine deaminase (DRADA1, or ADAR1) contains a domain (Zα) that binds specifically to the left-handed Z-DNA conformation with high affinity (KD = 4 nM). As formation of Z-DNA in vivo occurs 5′ to, or behind, a moving RNA polymerase during transcription, recognition of Z-DNA by DRADA1 provides a plausible mechanism by which DRADA1 can be targeted to a nascent RNA so that editing occurs before splicing. Analysis of sequences related to Zα has allowed identification of motifs common to this class of nucleic acid binding domain.

The solution structure of the Zα domain of the human RNA editing enzyme ADAR1 reveals a prepositioned binding surface for Z-DNA

Double-stranded RNA deaminase I (ADAR1) contains the Z-DNA binding domain Zα. Here we report the solution structure of free Zα and map the interaction surface with Z-DNA, confirming roles previously assigned to residues by mutagenesis. Comparison with the crystal structure of the (Zα)2/Z-DNA complex shows that most Z-DNA contacting residues in free Zα are prepositioned to bind Z-DNA, thus minimizing the entropic cost of binding. Comparison with homologous (α+β)helix–turn–helix/B-DNA complexes suggests that binding of Zα to B-DNA is disfavored by steric hindrance, but does not eliminate the possibility that related domains may bind to both B- and Z-DNA.

The importin/karyopherin Kap114 mediates the nuclear import of TATA-binding protein

Two high copy suppressors of temperature-sensitive TATA-binding protein (TBP) mutants were isolated. One suppressor was TIF51A, which encodes eukaryotic translation initiation factor 5A. The other high copy suppressor, YGL241W, also known as KAP114, is one of 14 importin/karyopherin proteins in yeast. These proteins mediate the transport of specific macromolecules into and out of the nucleus. Cells lacking Kap114 partially mislocalize TBP to the cytoplasm. Kap114 binds TBP in vitro, and binding is disrupted in the presence of GTPγS. Therefore, Kap114 is an importer of TBP into the nucleus, but alternative import pathways must also exist.

In vitro selection of a 7-methyl-guanosine binding RNA that inhibits translation of capped mRNA molecules

Using systematic evolution of ligands by exponential enrichment (SELEX), an RNA molecule was isolated that displays a 1,000-fold higher affinity for guanosine residues that carry an N-7 methyl group than for nonmethylated guanosine residues. The methylated guanosine residue closely resembles the 5′ terminal cap structure present on all eukaryotic mRNA molecules. The cap-binding RNA specifically inhibited the translation of capped but not uncapped mRNA molecules in cell-free lysates prepared from either human HeLa cells or from Saccharomyces cerevisiae. These findings indicate that the cap-binding RNA will also be useful in studies of other cap-dependent processes such as pre-mRNA splicing and nucleocytoplasmic mRNA transport.

A protein encoded by a group I intron in Aspergillus nidulans directly assists RNA splicing and is a DNA endonuclease

Some group I introns self-splice in vitro, but almost all are thought to be assisted by proteins in vivo. Mutational analysis has shown that the splicing of certain group I introns depends upon a maturase protein encoded by the intron itself. However the effect of a protein on splicing can be indirect. We now provide evidence that a mitochondrial intron-encoded protein from Aspergillus nidulans directly facilitates splicing in vitro. This demonstrates that a maturase is an RNA splicing protein. The protein-assisted reaction is as fast as that of any other known group I intron. Interestingly the protein is also a DNA endonuclease, an activity required for intron mobilization. Mobile elements frequently encode proteins that promote their propagation. Intron-encoded proteins that also assist RNA splicing would facilitate both the transposition and horizontal transmission of introns.

mRNA binding protein mrnp 41 localizes to both nucleus and cytoplasm

We have identified and molecularly characterized a human protein with a Mr of 40,880 Da. After UV irradiation of HeLa cells, this protein was cross-linked to poly(A)-containing mRNA and was therefore designated mrnp 41 (for mRNA binding protein of 41 kDa). Cell fractionation and immunoblotting showed mrnp 41 in both the cytoplasm and the nucleus and particularly in the nuclear envelope. Immunofluorescence microscopy localized mrnp 41 to distinct foci in the nucleoplasm, to the nuclear rim, and to meshwork-like structures throughout the cytoplasm. The cytoplasmic meshwork staining was disrupted by prior treatment of cells with the actin filament- or microtubule-disrupting drugs cytochalasin or nocodazole, respectively, suggesting association of mrnp 41 with the cytoskeleton. Double immunofluorescence with antibodies against mrnp 41 and the cytoplasmic poly(A) binding protein showed colocalization to the cytoplasmic meshwork. Immunogold electronmicroscopy confirmed mrnp 41’s cytoplasmic and nucleoplasmic localization and revealed a striking labeling of nuclear pore complexes. Together these data suggest that mrnp 41 may function in nuclear export of mRNPs and/or in cytoplasmic transport on, or attachment to, the cytoskeleton. Consistent with a role of mrnp 41 in nuclear export are previous reports that mutations in homologs of mrnp 41 in Schizosaccharomyces pombe, designated Rae1p, or in Saccharomyces cerevisiae, designated Gle2p, result in mRNA accumulation in the nucleus although it is presently not known whether these homologs are mRNA binding proteins as well.

Sterol regulatory element binding protein-1c is a major mediator of insulin action on the hepatic expression of glucokinase and lipogenesis-related genes

Hepatic glucokinase plays a key role in glucose metabolism as underlined by the anomalies associated with glucokinase mutations and the consequences of tissue-specific knock-out. In the liver, glucokinase transcription is absolutely dependent on the presence of insulin. The cis-elements and trans-acting factors that mediate the insulin effect are presently unknown; this is also the case for most insulin-responsive genes. We have shown previously that the hepatic expression of the transcription factor sterol regulatory element binding protein-1c (SREBP-1c) is activated by insulin. We show here in primary cultures of hepatocytes that the adenovirus-mediated transduction of a dominant negative form of SREBP-1c inhibits the insulin effect on endogenous glucokinase expression. Conversely, in the absence of insulin, the adenovirus-mediated transduction of a dominant positive form of SREBP-1c overcomes the insulin dependency of glucokinase expression. Hepatic fatty acid synthase and Spot-14 are insulin/glucose-dependent genes. For this latter class of genes, the dominant positive form of SREBP-1c obviates the necessity for the presence of insulin, whereas glucose potentiates the effect of SREBP-1c on their expression. In addition, the insulin dependency of lipid accumulation in cultured hepatocytes is overcome by the dominant positive form of SREBP-1c. We propose that SREBP-1c is a major mediator of insulin action on hepatic gene expression and a key regulator of hepatic glucose/lipid metabolism.

CCAAT/enhancer binding protein (C/EBP) sites are required for HIV-1 replication in primary macrophages but not CD4+ T cells

The importance of CCAAT/enhancer binding proteins (C/EBPs) and binding sites for HIV-1 replication in primary macrophages, T cell lines and primary CD4+ T cells was examined. When lines overexpressing the C/EBP dominant-negative protein LIP were infected with HIV-1, replication occurred in Jurkat T cells but not in U937 promonocytes, demonstrating a requirement for C/EBP activators by HIV-1 only in promonocytes. Primary macrophages did not support the replication of HIV-1 harboring mutant C/EBP binding sites in the long terminal repeat but Jurkat, H9 and primary CD4+ T cells supported replication of wild-type and mutant HIV-1 equally well. Thus the requirement for C/EBP sites is also confined to monocyte/macrophages. The requirement for C/EBP proteins and sites identifies the first uniquely macrophage-specific regulatory mechanism for HIV-1 replication.

Direct interaction of Gβγ with a C-terminal Gβγ-binding domain of the Ca2+ channel α1 subunit is responsible for channel inhibition by G protein-coupled receptors

Several classes of voltage-gated Ca2+ channels (VGCCs) are inhibited by G proteins activated by receptors for neurotransmitters and neuromodulatory peptides. Evidence has accumulated to indicate that for non-L-type Ca2+ channels the executing arm of the activated G protein is its βγ dimer (Gβγ). We report below the existence of two Gβγ-binding sites on the A-, B-, and E-type α1 subunits that form non-L-type Ca2+ channels. One, reported previously, is in loop 1 connecting transmembrane domains I and II. The second is located approximately in the middle of the ca. 600-aa-long C-terminal tails. Both Gβγ-binding regions also bind the Ca2+ channel β subunit (CCβ), which, when overexpressed, interferes with inhibition by activated G proteins. Replacement in α1E of loop 1 with that of the G protein-insensitive and Gβγ-binding-negative loop 1 of α1C did not abolish inhibition by G proteins, but the exchange of the α1E C terminus with that of α1C did. This and properties of α1E C-terminal truncations indicated that the Gβγ-binding site mediating the inhibition of Ca2+ channel activity is the one in the C terminus. Binding of Gβγ to this site was inhibited by an α1-binding domain of CCβ, thus providing an explanation for the functional antagonism existing between CCβ and G protein inhibition. The data do not support proposals that Gβγ inhibits α1 function by interacting with the site located in the loop I–II linker. These results define the molecular mechanism by which presynaptic G protein-coupled receptors inhibit neurotransmission.

Multiple pathways to bypass the enhancer requirement of sigma 54 RNA polymerase: Roles for DNA and protein determinants

Sigma 54 is a required factor for bacterial RNA polymerase to respond to enhancers and directs a mechanism that is a hybrid between bacterial and eukaryotic transcription. Three pathways were found that bypass the enhancer requirement in vitro. These rely on either deletion of the sigma 54 N terminus or destruction of the DNA consensus −12 promoter recognition element or altering solution conditions to favor transient DNA melting. Each of these allows unstable heparin-sensitive pre-initiation complexes to form that can be driven to transcribe in the absence of both enhancer protein and ATP β–γ hydrolysis. These disparate pathways are proposed to have a common basis in that multiple N-terminal contacts may mediate the interactions between the polymerase and the DNA region where melting originates. The results raise possibilities for common features of open complex formation by different RNA polymerases.

Heterogeneous nuclear ribonucleoprotein A1 binds to the transcription-regulatory region of mouse hepatitis virus RNA

A cellular protein, previously described as p35/38, binds to the complementary (−)-strand of the leader RNA and intergenic (IG) sequence of mouse hepatitis virus (MHV) RNA. The extent of the binding of this protein to IG sites correlates with the efficiency of the subgenomic mRNA transcription from that IG site, suggesting that it is a requisite transcription factor. We have purified this protein and determined by partial peptide sequencing that it is heterogeneous nuclear ribonucleoprotein (hnRNP) A1, an abundant, primarily nuclear protein. hnRNP A1 shuttles between the nucleus and cytoplasm and plays a role in the regulation of alternative RNA splicing. The MHV(−)-strand leader and IG sequences conform to the consensus binding motifs of hnRNP A1. Recombinant hnRNP A1 bound to these two RNA regions in vitro in a sequence-specific manner. During MHV infection, hnRNP A1 relocalizes from the nucleus to the cytoplasm, where viral replication occurs. These data suggest that hnRNP A1 is a cellular factor that regulates the RNA-dependent RNA transcription of the virus.

The activation domain of the enhancer binding protein p45NF-E2 interacts with TAFII130 and mediates long-range activation of the α- and β-globin gene loci in an erythroid cell line

We have used the interaction between the erythroid-specific enhancer in hypersensitivity site 2 of the human β-globin locus control region and the globin gene promoters as a paradigm to examine the mechanisms governing promoter/enhancer interactions in this locus. We have demonstrated that enhancer-dependent activation of the globin promoters is dependent on the presence of both a TATA box in the proximal promoter and the binding site for the erythroid-specific heteromeric transcription factor NF-E2 in the enhancer. Mutational analysis of the transcriptionally active component of NF-E2, p45NF-E2, localizes the critical region for this function to a proline-rich transcriptional activation domain in the NH2-terminal 80 amino acids of the protein. In contrast to the wild-type protein, expression of p45 NF-E2 lacking this activation domain in an NF-E2 null cell line fails to support enhancer-dependent transcription in transient assays. More significantly, the mutated protein also fails to reactivate expression of the endogenous β- or α-globin loci in this cell line. Protein-protein interaction studies reveal that this domain of p45 NF-E2 binds specifically to a component of the transcription initiation complex, TATA binding protein associated factor TAFII130. These findings suggest one potential mechanism for direct recruitment of distal regulatory regions of the globin loci to the individual promoters.

Spinophilin, a novel protein phosphatase 1 binding protein localized to dendritic spines

Dendritic spines receive the vast majority of excitatory synaptic contacts in the mammalian brain and are presumed to contain machinery for the integration of various signal transduction pathways. Protein phosphatase 1 (PP1) is greatly enriched in dendritic spines and has been implicated in both the regulation of ionic conductances and long-term synaptic plasticity. The molecular mechanism whereby PP1 is localized to spines is unknown. We have now characterized a novel protein that forms a complex with the catalytic subunit of PP1 and is a potent modulator of PP1 enzymatic activity in vitro. Within the brain this protein displays a remarkably distinct localization to the heads of dendritic spines and has therefore been named spinophilin. Spinophilin has the properties expected of a scaffolding protein localized to the cell membrane and contains a single consensus sequence in PSD95/DLG/zo-1, which implies cross-linking of PP1 to transmembrane protein complexes. We propose that spinophilin represents a novel targeting subunit for PP1, which directs the enzyme to those substrates in the dendritic spine compartment, e.g., neurotransmitter receptors, which mediate the regulation of synaptic function by PP1.

Structural mimicry of a native protein by a minimized binding domain

The affinity between molecules depends both on the nature and presentation of the contacts. Here, we observe coupling of functional and structural elements when a protein binding domain is evolved to a smaller functional mimic. Previously, a 38-residue form of the 59-residue B-domain of protein A, termed Z38, was selected by phage display. Z38 contains 13 mutations and binds IgG only 10-fold weaker than the native B-domain. We present the solution structure of Z38 and show that it adopts a tertiary structure remarkably similar to that observed for the first two helices of B-domain in the B-domain/Fc complex [Deisenhofer, J. (1981) Biochemistry 20, 2361–2370], although it is significantly less stable. Based on this structure, we have improved on Z38 by designing a 34-residue disulfide-bonded variant (Z34C) that has dramatically enhanced stability and binds IgG with 9-fold higher affinity. The improved stability of Z34C led to NMR spectra with much greater chemical shift dispersion, resulting in a more precisely determined structure. Z34C, like Z38, has a structure virtually identical to the equivalent region from native protein A domains. The well-defined hydrophobic core of Z34C reveals key structural features that have evolved in this small, functional domain. Thus, the stabilized two-helix peptide, about half the size and having one-third of the remaining residues altered, accurately mimics both the structure and function of the native domain.