Direct Fe2+ Sensing by Iron-responsive Messenger RNA��Repressor Complexes Weakens Binding

Fe²⁺ is now shown to weaken binding between ferritin and mitochondrial aconitase messenger RNA noncoding regulatory structures ((iron-responsive element) (IRE)-RNAs) and the regulatory proteins (IRPs), which adds a direct role of iron to regulation that can complement the well known regulatory protein modification and degradative pathways related to iron-induced mRNA translation. We observe that the K_d value increases 17-fold in 5′-untranslated region IRE-RNA·repressor complexes; Fe²⁺, is studied in the absence of O₂. Other metal ions, Mn²⁺ and Mg²⁺ have similar effects to Fe²⁺ but the required Mg²⁺ concentration is 100 times greater than for Fe²⁺ or Mn²⁺. Metal ions also weaken ethidium bromide binding to IRE-RNA with no effect on IRP fluorescence, using Mn²⁺ as an O₂-resistant surrogate for Fe²⁺, indicating that metal ions bound IRE-RNA but not IRP. Fe²⁺ decreases IRP repressor complex stability of ferritin IRE-RNA 5–10 times compared with 2–5 times for mitochondrial aconitase IRE-RNA, over the same concentration range, suggesting that differences among IRE-RNA structures contribute to the differences in the iron responses observed in vivo. The results show the IRE-RNA·repressor complex literally responds to Fe²⁺, selectively for each IRE-mRNA.Iron (e.g. ferrous sulfate, ferric citrate, and hemin) added to animal cells changes translation rates of messenger RNAs encoding proteins of iron traffic and oxidative metabolism (1–4). To cross cell membranes, iron ions are transported by membrane proteins such as DMT1 or carried on proteins such as transferrin. Inside the cells, iron is mainly in heme, FeS clusters, non-heme iron cofactors of proteins, and iron oxide minerals coated by protein nanocages (ferritins). Iron in transit is thought to be Fe²⁺ in labile “pools” accessible to small molecular weight chelators, and/or bound loosely by chaperones.When iron concentrations in the cells increase, a group of mRNAs with three-dimensional, noncoding structures in the 5′-untranslated region (UTR)³ are derepressed (Fig. 1A), i.e. the fraction of the mRNAs in mRNA·repressor protein complexes, which inhibit ribosome binding, decreases and the fraction of the mRNAs in polyribosomes increases (5–7). The three-dimensional, noncoding mRNA structure, representing a family of related structures, is called the iron-responsive element, or IRE, and the repressors are called iron regulatory proteins (IRPs). Together they are one of the most extensively studied eukaryotic messenger RNA regulatory systems (1–4). In addition to large numbers of cell studies, structures of IRE-RNAs are known from solution NMR (8–12), and the RNA·protein complex from x-ray crystallography (13). Recent data indicate that demetallation of IRP1 and disruption of the [4Fe-4S] cluster that inhibits IRP1 binding to RNA will be enhanced by phosphorylation and low iron concentrations (1, 2, 14–16). Such results can explain the increased IRP1 binding to IRE-mRNAs and increased translational repression when iron concentrations are abnormally low. However, the mechanism to explain dissociation of IRE-RNA·IRP complexes, thereby allowing ribosome assembly and increased proteosomal degradation of IRPs (1, 2, 14, 15) (Fig. 1A), when high iron concentrations are abnormally high, is currently unknown.Open in a separate windowFIGURE 1.IRE-RNA·IRP complexes and a model for depression by excess iron. A, a representative model of iron-induced translation of 5′-UTR IRE-RNAs. This figure is modified from Ref. 7. B, IRE-RNA sites influenced by metal binding related to the crystal structure of the ferritin-IRE-RNA·IRP complex from Ref. 13. The figure was created by T. Tosha using Discovery Studio 1.6 and Protein Data Bank file 2IPY. ■, hydrated Mg²⁺, determined by solution NMR; ▴, Cu¹⁺-1.10-phenanthroline, determined by RNA cleavage in O₂.Metal ion binding changes conformation and function of most RNA classes, e.g. rRNA (17), tRNA (18, 19), ribozymes (20–23), riboswitches (24, 25), possibly hammerhead mRNAs in mammals (26), and proteins. Although the effects of metal ion binding on eukaryotic mRNAs have not been extensively studied, Mg²⁺ is known to cause changes in conformation, shown by changes in radical cleavage sites of IRE-RNA with 1,10-phenanthrolene-iron and proton shifts in the one-dimensional NMR spectrum (12, 27). The Mg²⁺ effects are observed at low magnesium concentrations (0.1–0.5 mm) and low molar stoichiometries (1:1 and 2:1 = Mg:RNA).We hypothesized that Fe²⁺ could directly change the binding of the IRE-mRNA to the iron regulatory protein for several reasons. First, other metal ions influence the IRE-RNA structure (12, 27). Second, in IRE-RNA/IRP cocrystals there are exposed RNA sites in the IRE-RNA/IRP complex that are accessible for interactions (13) (Fig. 1B). Third, regions in the IRE-RNA are hypersensitive to Fe²⁺-EDTA/ascorbate/H₂O₂, suggesting selective interactions with metals and/or solvent (28). We now report that Fe²⁺ weakens IRE-RNA/IRP binding, whereas Mg²⁺ requires 100 times the concentration and Mn²⁺ is comparable with Fe²⁺; the Fe²⁺ effect was diminished in mutant IRE-RNA and IRE-RNA selective in wild type sequences: ferritin IRE-RNA > mt-aconitase IRE-RNA.     

Activation of Heterologous Gene Expression by the Large Isoform of Hepatitis Delta Antigen

Hepatitis delta virus (HDV) encodes two isoforms of its principal gene product, hepatitis delta antigen (HDAg). These two forms play distinctive and complementary roles in viral replication. Here we report that the large (LHDAg), but not the small (SHDAg), isoform of HDAg has the capacity to activate the expression of cotransfected genes driven by a variety of promoters, including the pre-S, S, and C promoters of hepatitis B virus. Mutational analysis of the C-terminal 19 amino acids unique to LHDAg shows that changing prolines to alanines in the two PXXP motifs in this region specifically ablates the activation function without abolishing another activity of LHDAg, namely, its ability to inhibit HDV RNA synthesis. However, C-terminal truncations that also disrupt these PXXP motifs only slightly diminished the activation function, indicating that the proline mutations were not acting by inactivating potential SH3 interactions that could be mediated by these motifs. Mutation of the isoprenylated cysteine to serine decreases but does not abolish the activation activity, and overexpression of SHDAg does not interfere with the transactivation function of LHDAg. Although the mechanism and biological significance of this activity of LHDAg remain unknown, the presence of this activity serves as yet another marker that functionally distinguishes this protein from the closely related isoform SHDAg.Hepatitis delta virus (HDV) is an RNA virus that requires coinfection with hepatitis B virus (HBV) to complete its life cycle. The helper function supplied by HBV is limited to the provision of envelope proteins (hepatitis B surface antigens) for the completion of HDV assembly (28, 29, 31). HDV RNA replication is independent of its HBV helper (19). In fact, the presence of HDV suppresses HBV replication in vivo (30, 39). Nonetheless, clinical studies have shown that HDV infection can be associated with more severe hepatitis than HBV alone and is often implicated in cases of fulminant hepatitis (4, 32).The genome of HDV is a circular, single-stranded RNA of about 1,700 nucleotides (nt), of which approximately 70% are self-complementary (for a review, see references 20 and 21). This self-complemetarity allows the genome to form an unbranched rod-like structure. A unique functional protein, hepatitis delta antigen (HDAg), is encoded by the genome (3, 38), and two isoforms of this protein are produced during infection. The canonical small form of HDAg (SHDAg) is 195 amino acids (aa) long; it harbors an N-terminal coiled-coil domain responsible for oligomerization (37), a central domain responsible for binding to the RNA genome (7, 23), a nuclear localization signal (2, 7), and a C-terminal glycine- and proline-rich region with an uncertain function. This form of HDAg is essential for viral RNA replication, although it is not itself a polymerase. Host RNA polymerase II is thought to supply the polymerase function for replication (15, 26). During viral replication, an RNA editing event occurs at the UAG termination codon of SHDAg, allowing readthrough of another 19 aa (Fig. ​(Fig.1)1) to generate the large isoform of the protein, LHDAg (25). Since LHDAg contains all of the domains of SHDAg, it too can form multimers with itself and with the SHDAg isoform, bind HDV RNA (as a homo- or heteromultimer), and be localized to the nucleus. Open in a separate windowFIG. 1Sequence of the 19 aa unique to the C terminus of LHDAg. The PXXP motifs are underlined. Below are shown the amino acid changes present in the mutants employed in this study. The positions of the termination codons introduced into the truncation mutants are indicated by asterisks.Despite these similarities, the two HDAgs have very distinct functions (22) and play complementary roles in HDV replication, which takes place largely in the nuclei of infected cells (34). While SHDAg activates HDV RNA replication, LHDAg is a trans-dominant inhibitor of this process (8). By contrast, LHDAg, but not SHDAg, is capable of interacting with the HBV envelope proteins to mediate envelopment of the HDV ribonucleoprotein in viral assembly (6). This interaction has been shown to require farnesylation of a cysteine residue found in the C-terminal 19 aa unique to LHDAg (27, 16). Furthermore, it has been shown recently that only LHDAg is phosphorylated in cells (1).In this report, we describe yet another activity of LHDAg that further differentiates it from the related isoform SHDAg, i.e., the ability to activate gene expression in trans.     

Poliovirus 2C Protein Forms Homo-oligomeric Structures Required for ATPase Activity

The poliovirus protein 2C plays an essential role in viral RNA replication, although its precise biochemical activities or structural requirements have not been elucidated. The protein has several distinctive properties, including ATPase activity and membrane and RNA binding, that are conserved among orthologs of many positive-strand RNA viruses. Sequence alignments have placed these proteins in the SF3 helicase family, a subset of the AAA+ ATPase superfamily. A feature common to AAA+ proteins is the formation of oligomeric rings that are essential for their catalytic functions. Here we show that a recombinant protein, MBP-2C, in which maltose-binding protein was fused to 2C, formed soluble oligomers and that ATPase activity was restricted to oligomer-containing fractions from gel-filtration chromatography. The active fraction was visualized by negative-staining electron microscopy as ring-like particles composed of 5–8 protomers. This conclusion was confirmed by mass measurements obtained by scanning transmission electron microscopy. Mutation of amino acid residues in the 2C nucleotide-binding domain demonstrated that loss of the ability to bind or hydrolyze ATP did not affect oligomerization. Co-expression of active MBP-2C and inactive mutant proteins generated mixed oligomers that exhibited little ATPase activity, suggesting that incorporation of inactive subunits eliminates the function of the entire particle. Finally, deletion of the N-terminal 38 amino acids blocked oligomerization of the fusion protein and eliminated ATPase activity, despite retention of an unaltered nucleotide-binding domain.Poliovirus is the prototype member of the Picornaviridae family. The 7.5-kb positive sense RNA genome encodes both capsid and noncapsid proteins that are necessary for virus replication. Translation of the viral genome into a single polyprotein yields both functionally distinct precursors and final products that are required for productive viral replication via an orchestrated series of co- and post-translational cleavage events catalyzed by viral proteinases. Replication of the viral RNA occurs in the cytoplasm, localized on the surfaces of newly formed membranous structures that develop after infection. Viral and host proteins involved in viral RNA replication form a poorly characterized, nuclease-resistant replication complex associated with the remodeled membrane structures. Numerous studies have demonstrated that viral protein 2C and its precursor 2BC play key roles in viral RNA replication, yet their actual biochemical functions in this complex reaction remain undefined (1–3). Protein 2C has been shown to interact with other elements of the viral replication apparatus, including 3AB (4), 3C proteinase (5), and the cloverleaf structure at the 5′-end of the viral genome (6). More recently it was shown that reticulon-3, a cellular protein involved in membrane trafficking and endoplasmic reticulum structure, binds polioviral as well as other picornaviral 2C proteins, and this plays an essential albeit undefined role in the process of virus replication (7).Poliovirus 2C is tightly associated with intracellular membranes and can be cross-linked to actively replicating viral RNA isolated from infected cells (8). In addition, individual expression of 2C or 2BC in mammalian cells induces the formation of reorganized membrane structures from the endoplasmic reticulum, and thus these proteins have been implicated in the formation of virus-induced replication complexes (9, 10).Poliovirus 2C is a 329-amino acid protein that is relatively highly conserved among members of the Picornaviridae family and is predicted to contain at least three domains (see Fig. 3) (11, 12). A centrally located nucleotide-binding domain (NBD)² is the most highly conserved region of the protein. Flanking the NBD, at the N terminus of poliovirus 2C, is a region (amino acids 1–54) that contains a predicted amphipathic helix (amino acids 19–36) (13, 14) that specifies localization of 2C to the membrane (15). The C-terminal portion of the protein contains a small Cys-rich region that binds zinc (16), and a region that is thought to be involved in RNA binding and also may interact with membranes (12, 17). Biochemical studies of purified poliovirus 2C fusion proteins have demonstrated that the protein manifests an ATPase and a much weaker GTPase activity (18, 19). Mutations in NBD signature sequences result in impairment or abrogation of viral RNA replication when introduced into full-length or replicon viral RNAs (2, 20). The sensitivity of poliovirus RNA replication to millimolar concentrations of guanidine-HCl has been attributed to 2C: guanidine inhibits the ATPase activity of purified 2C protein in vitro, and viral mutants that are resistant or dependent for growth in the presence of guanidine harbor alterations in the NBD (21, 22). In other domains of the protein, mutagenesis by insertion of sequences in the region C-terminal to the NBD causes temperature-dependent packaging defects (23), whereas gross changes to the upstream sequence in the region encompassing the predicted amphipathic helix also affect viral RNA replication (13). Taken together, these data imply that 2C is a multifunctional protein.Open in a separate windowFIGURE 3.Schematic diagram of domains predicted for poliovirus protein 2C. The locations of the amphipathic helix, Walker A, B, and C (WA, WB, and C) motifs, a conserved arginine residue (R), and a zinc-binding domain are indicated. Amino acid residues are identified by number below the box, and the WA and WB mutations used in this study are indicated.Comparative sequence alignments of the central NBD of picornaviral 2C proteins have grouped these proteins within the SF3 helicase family (24), although there is no evidence that poliovirus 2C protein has helicase activity or that such activity is necessary for virus replication (19, 25). The SF3 helicase family was originally identified in and appears to be limited to the genomes of DNA and small RNA viruses (26, 27). The NBDs of SF3 family members include the characteristic Walker A and B motifs along with a distinguishing C-motif (sensor) within a 100- to 120-amino acid region. The Walker A motif is specified by a GXXXGK(T/S) signature, which interacts with the phosphates of ATP; the Walker B signature is defined by MDD, where the Asp (or Glu) residues interact with Mg²⁺ or water and contribute to nucleotide hydrolysis activity. Motif C consists of an Asn residue preceded by a run of hydrophobic amino acids located C-terminal to the Walker B motif (27).Although few biochemical or structural data are available for the picornaviral 2C proteins or other SF3 ATPases from RNA viruses, some SF3 helicase members encoded by DNA viruses have been quite well characterized (28, 29). These data indicate that DNA virus SF3 family proteins belong to the AAA+ superfamily, a functionally diverse group of proteins whose biological activities include protein folding, cytoskeletal regulation, and DNA replication (30). A common feature is the formation of higher order oligomers (predominantly hexamers and heptamers) that are essential for function. Indeed, structures of SV40 and papillomavirus E1 proteins resolved by x-ray crystallography revealed hexameric rings whose formation is dependent on the presence of ATP and magnesium (31, 32). These analyses showed that elements of two adjoining subunits are used to form the nucleotide binding site in the cleft between them. The nucleotide thus facilitates the formation and stability of a functional oligomer (33). Structure-based sequence alignment of these DNA SF3 helicases highlighted additional conserved residues that contribute to the nucleotide binding site in an adjacent protomer (29).Poliovirus 2C differs significantly from SF3 DNA helicases, both in terms of overall size and properties such as membrane association. The low but significant homology among members of the SF3 family may indicate that there is commonality in biophysical properties such as oligomerization. The accumulated biochemical and genetic data for 2C oligomerization are contradictory. Yeast two-hybrid analysis of 2C proteins from polio (34) and related coxsackievirus (35) and teschovirus (36) failed to demonstrate a strong propensity for oligomerization of 2C, whereas biochemical studies (34) and a mammalian two-hybrid study (37) of poliovirus proteins suggested stable 2C-2C interactions may occur. Furthermore, genetic studies examining the mechanism of virus sensitivity or resistance to guanidine were interpreted as indicating that poliovirus 2C functioned as an oligomer (22). As part of our effort to understand the contribution(s) of 2C in the poliovirus replication cycle, we sought to determine the oligomerization status of the 2C protein and potential commonality with other related SF3 DNA helicases.     

Dissecting an Allosteric Switch in Caspase-7 Using Chemical and Mutational Probes

Apoptotic caspases, such as caspase-7, are stored as inactive protease zymogens, and when activated, lead to a fate-determining switch to induce cell death. We previously discovered small molecule thiol-containing inhibitors that when tethered revealed an allosteric site and trapped a conformation similar to the zymogen form of the enzyme. We noted three structural transitions that the compounds induced: (i) breaking of an interaction between Tyr-223 and Arg-187 in the allosteric site, which prevents proper ordering of the catalytic cysteine; (ii) pinning the L2′ loop over the allosteric site, which blocks critical interactions for proper ordering of the substrate-binding groove; and (iii) a hinge-like rotation at Gly-188 positioned after the catalytic Cys-186 and Arg-187. Here we report a systematic mutational analysis of these regions to dissect their functional importance to mediate the allosteric transition induced by these compounds. Mutating the hinge Gly-188 to the restrictive proline causes a massive ∼6000-fold reduction in catalytic efficiency. Mutations in the Arg-187–Tyr-223 couple have a far less dramatic effect (3–20-fold reductions). Interestingly, although the allosteric couple mutants still allow binding and allosteric inhibition, they partially relieve the mutual exclusivity of binding between inhibitors at the active and allosteric sites. These data highlight a small set of residues critical for mediating the transition from active to inactive zymogen-like states.Caspases are a family of dimeric cysteine proteases whose members control the ultimate steps for apoptosis (programmed cell death) or innate inflammation among others (for reviews, see Refs. 1 and 2). During apoptosis, the upstream initiator caspases (caspase-8 and -9) activate the downstream executioner caspases (caspase-3, -6, and-7) via zymogen maturation (3). The activated executioner caspases then cleave upwards of 500 key proteins (4–6) and DNA, leading to cell death. Due to their pivotal role in apoptosis, the caspases are involved both in embryonic development and in dysfunction in diseases including cancer and stroke (7). The 11 human caspases share a common active site cysteine-histidine dyad (8), and derive their name, cysteine aspartate proteases, from their exquisite specificity for cleaving substrate proteins after specific aspartate residues (9–13). Thus, it has been difficult to develop active site-directed inhibitors with significant specificity for one caspase over the others (14). Despite difficulties in obtaining specificity, there has been a long-standing correlation between efficacy of caspase inhibitors in vitro and their ability to inhibit caspases and apoptosis in vivo (for review, see Ref. 31). Thus, a clear understanding of in vitro inhibitor function is central to the ability control caspase function in vivo.Caspase-7 has been a paradigm for understanding the structure and dynamics of the executioner caspases (15–21). The substrate-binding site is composed of four loops; L2, L3, and L4 are contributed from one-half of the caspase dimer, and L2′ is contributed from the other half of the caspase dimer (Fig. 1). These loops appear highly dynamic as they are only observed in x-ray structures when bound to substrate or substrate analogs in the catalytically competent conformation (17–19, 22) (Fig. 1B).Open in a separate windowFIGURE 1.Allosteric site and dimeric structure in caspase-7. A, the surface of active site-bound caspase-7 shows a large open allosteric (yellow) site at the dimer interface. This cavity is distinct from the active sites, which are bound with the active site inhibitor DEVD (green sticks). B, large subunits of caspase-7 dimers (dark green and dark purple) contain the active site cysteine-histidine dyad. The small subunits (light green and light purple) contain the allosteric site cysteine 290. The conformation of the substrate-binding loops (L2, L2′, L3, and L4) in active caspase-7 (Protein Data Bank (PDB) number 1f1j) is depicted. The L2′ loop (spheres) from one-half of the dimer interacts with the L2 loop from the other half of the dimer. C, binding of allosteric inhibitors influences the conformation of the L2′ loop (spheres), which folds over the allosteric cavity (PDB number 1shj). Subunit rendering is as in panel A. Panels A, B, and C are in the same orientation.A potential alternative to active site inhibitors are allosteric inhibitors that have been seeded by the discovery of selective cysteine-tethered allosteric inhibitors for either apoptotic executioner caspase-3 or apoptotic executioner caspase-7 (23) as well as the inflammatory caspase-1 (24). These thiol-containing compounds bind to a putative allosteric site through disulfide bond formation with a thiol in the cavity at the dimer interface (Fig. 1A) (23, 24). X-ray structures of caspase-7 bound to allosteric inhibitors FICA³ and DICA (Fig. 2) show that these compounds trigger conformational rearrangements that stabilize the inactive zymogen-like conformation over the substrate-bound, active conformation. The ability of small molecules to hold mature caspase-7 in a conformation that mimics the naturally occurring, inactive zymogen state underscores the utility and biological relevance of the allosteric mechanism of inhibition. Several structural changes are evident between these allosterically inhibited and active states. (i) The allosteric inhibitors directly disrupt an interaction between Arg-187 (next to the catalytic Cys-186) and Tyr-223 that springs the Arg-187 into the active site (Fig. 3), (ii) this conformational change appears to be facilitated by a hinge-like movement about Gly-188, and (iii) the L2′ loop folds down to cover the allosteric inhibitor and assumes a zymogen-like conformation (Fig. 1C) (23).Open in a separate windowFIGURE 2.Structure of allosteric inhibitors DICA and FICA. DICA and FICA are hydrophobic small molecules that bind to an allosteric site at the dimer interface of caspase-7. Binding of DICA/FICA is mediated by a disulfide between the compound thiol and Cys-290 in caspase-7.Open in a separate windowFIGURE 3.Movement of L2′ blocking arm. The region of caspase-7 encompassing the allosteric couple Arg-187 and Tyr-223 is boxed. The inset shows the down orientation of Arg-187 and Tyr-223 in the active conformation with DEVD substrate mimic (orange spheres) in the active site. In the allosteric/zymogen conformation, Arg-187 and Tyr-223 are pushed up by DICA (blue spheres).Here, using mutational analysis and small molecule inhibitors, we assess the importance of these three structural units to modulate both the inhibition of the enzyme and the coupling between allosteric and active site labeling. Our data suggest that the hinge movement and pinning of the L2-L2′ are most critical for transitioning between the active and inactive forms of the enzyme.     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.