Genetic Analysis of the nuo Locus,Which Encodes the Proton-Translocating NADH Dehydrogenase in Escherichia coli

Complex I (EC 1.6.99.3) of the bacterium Escherichia coli is considered to be the minimal form of the type I NADH dehydrogenase, the first enzyme complex in the respiratory chain. Because of its small size and relative simplicity, the E. coli enzyme has become a model used to identify and characterize the mechanism(s) by which cells regulate the synthesis and assembly of this large respiratory complex. To begin dissecting the processes by which E. coli cells regulate the expression of nuo and the assembly of complex I, we undertook a genetic analysis of the nuo locus, which encodes the 14 Nuo subunits comprising E. coli complex I. Here we present the results of studies, performed on an isogenic collection of nuo mutants, that focus on the physiological, biochemical, and molecular consequences caused by the lack of or defects in several Nuo subunits. In particular, we present evidence that NuoG, a peripheral subunit, is essential for complex I function and that it plays a role in the regulation of nuo expression and/or the assembly of complex I.

Complex I (NADH:ubiquinone oxidoreductase; EC 1.6.99.3), a type I NADH dehydrogenase that couples the oxidation of NADH to the generation of a proton motive force, is the first enzyme complex of the respiratory chain (2, 35, 47). The Escherichia coli enzyme, considered to be the minimal form of complex I, consists of 14 subunits instead of the 40 to 50 subunits associated with the homologous eukaryotic mitochondrial enzyme (17, 29, 30, 48–50). E. coli also possesses a second NADH dehydrogenase, NDH-II, which does not generate a proton motive force (31). E. coli complex I resembles eukaryotic complex I in many ways (16, 17, 30, 49): it performs the same enzymatic reaction and is sensitive to a number of the same inhibitors, it consists of subunits homologous to those found in all proton-translocating NADH:ubiquinone oxidoreductases studied thus far, it is comprised of a large number of subunits relative to the number that comprise other respiratory enzymes, and it contains flavin mononucleotide and FeS center prosthetic groups. Additionally, it possesses an L-shaped topology (14, 22) like that of its Neurospora crassa homolog (27), and it consists of distinct fragments or subcomplexes. Whereas eukaryotic complex I can be dissected into a peripheral arm and a membrane arm, the E. coli enzyme consists of three subcomplexes referred to as the peripheral, connecting, and membrane fragments (29) (Fig. ​(Fig.1A).1A). The subunit composition of these three fragments correlates approximately with the organization of the 14 structural genes (nuoA to nuoN) (49) of the nuo (for NADH:ubiquinone oxidoreductase) locus (Fig. ​(Fig.1B),1B), an organization that is conserved in several other bacteria, including Salmonella typhimurium (3), Paracoccus denitrificans (53), Rhodobacter capsulatus (12), and Thermus thermophilus (54). The 5′ half of the locus contains a promoter (nuoP), previously identified and located upstream of nuoA (8, 49), and the majority of genes that encode subunits homologous to the nucleus-encoded subunits of eukaryotic complex I and to subunits of the Alcaligenes eutrophus NAD-reducing hydrogenase (17, 29, 30, 49). In contrast, the 3′ half contains the majority of the genes that encode subunits homologous to the mitochondrion-encoded subunits of eukaryotic complex I and to subunits of the E. coli formate-hydrogen lyase complex (17, 29, 30, 49). Whereas the nuclear homologs NuoE, NuoF, and NuoG constitute the peripheral fragment (also referred to as the NADH dehydrogenase fragment [NDF]), the nuclear homologs NuoB, NuoC, NuoD, and NuoI constitute the connecting fragment. The mitochondrial homologs NuoA, NuoH, NuoJ, NuoK, NuoL, NuoM, and NuoN constitute the membrane fragment (29). E. coli complex I likely evolved by fusion of preexisting protein assemblies constituting modules for electron transfer and proton translocation (17–19, 30). Open in a separate windowFIG. 1Schematic of E. coli complex I and the nuo locus. Adapted with permission of the publisher (17, 29, 30, 49). (A) E. coli complex I is comprised of three distinct fragments: the peripheral (light gray), connecting (white), and membrane (dark gray) fragments (17, 29). The peripheral fragment (NDF) is comprised of the nuclear homologs NuoE, -F, and -G and exhibits NADH dehydrogenase activity that oxidizes NADH to NAD⁺; the connecting fragment is comprised of the nuclear homologs NuoB, -C, -D, and -I; and the membrane fragment is comprised of the mitochondrial homologs NuoA, -H, and -J to -N and catalyzes ubiquinone (Q) to its reduced form (QH₂). FMN, flavin mononucleotide. (B) The E. coli nuo locus encodes the 14 Nuo subunits that constitute complex I. The 5′ half of the locus contains a previously identified promoter (nuoP) and the majority of genes that encode the peripheral and connecting subunits (light gray and white, respectively). The 3′ half of the locus contains the majority of the genes encoding the membrane subunits (dark gray). The 3′ end of nuoG encodes a C-Terminal region (CTR) of the NuoG subunit (hatched).Because of its smaller size and relative simplicity, researchers recently have begun to utilize complex I of E. coli, and that of its close relative S. typhimurium, to identify and characterize the mechanism(s) by which cells regulate the synthesis and assembly of this large respiratory complex (3, 8, 46) and to investigate the diverse physiological consequences caused by defects in this enzyme (4, 6, 10, 40, 59). Such defects affect the ability of cells to perform chemotaxis (40), to grow on certain carbon sources (4, 6, 10, 40, 57), to survive stationary phase (59), to perform energy-dependent proteolysis (4), to regulate the expression of at least one gene (32), and to maintain virulence (5).To begin dissecting the processes by which E. coli cells regulate the expression of nuo and the assembly of complex I, we undertook a genetic analysis of the nuo locus. Here, we present the results of studies, performed on an isogenic collection of nuo mutants, that focus on the physiological, biochemical, and molecular consequences caused by the lack of or defects in several Nuo subunits. In particular, we present evidence that NuoG, a peripheral subunit, is essential for complex I function and that it plays a role in the regulation of nuo expression and/or the assembly of complex I.     

Mutational Biosynthesis of Novel Rapamycins by a Strain of Streptomyces hygroscopicus NRRL 5491 Disrupted in rapL,Encoding a Putative Lysine Cyclodeaminase

The gene rapL lies within the region of the Streptomyces hygroscopicus chromosome which contains the biosynthetic gene cluster for the immunosuppressant rapamycin. Introduction of a frameshift mutation into rapL by ΦC31 phage-mediated gene replacement gave rise to a mutant which did not produce significant amounts of rapamycin. Growth of this rapL mutant on media containing added l-pipecolate restored wild-type levels of rapamycin production, consistent with a proposal that rapL encodes a specific l-lysine cyclodeaminase important for the production of the l-pipecolate precursor. In the presence of added proline derivatives, rapL mutants synthesized novel rapamycin analogs, indicating a relaxed substrate specificity for the enzyme catalyzing pipecolate incorporation into the macrocycle.Rapamycin is a 31-member macrocyclic polyketide produced by Streptomyces hygroscopicus NRRL 5491 which, like the structurally related compounds FK506 and immunomycin (Fig. ​(Fig.1),1), has potent immunosuppressive properties (24). Such compounds are potentially valuable in the treatment of autoimmune diseases and in preventing the rejection of transplanted tissues (16). The biosynthesis of rapamycin requires a modular polyketide synthase, which uses a shikimate-derived starter unit (11, 20) and which carries out a total of fourteen successive cycles of polyketide chain elongation that resemble the steps in fatty acid biosynthesis (2, 27). l-Pipecolic acid is then incorporated (21) into the chain, followed by closure of the macrocyclic ring, and both these steps are believed to be catalyzed by a pipecolate-incorporating enzyme (PIE) (18), the product of the rapP gene (8, 15). Further site-specific oxidations and O-methylation steps (15) are then required to produce rapamycin. Open in a separate windowFIG. 1Structures of rapamycin, FK506, and immunomycin.The origin of the pipecolic acid inserted into rapamycin has been previously established (21) to be free l-pipecolic acid derived from l-lysine (although the possible role of d-lysine as a precursor must also be borne in mind) (9). Previous work with other systems has suggested several alternative pathways for pipecolate formation from lysine (22), but the results of the incorporation of labelled lysine into the pipecolate moiety of immunomycin (Fig. ​(Fig.1)1) clearly indicate loss of the α-nitrogen atom (3). More recently, the sequencing of the rap gene cluster revealed the presence of the rapL gene (Fig. ​(Fig.2),2), whose deduced gene product bears striking sequence similarity to two isoenzymes of ornithine deaminase from Agrobacterium tumefaciens (25, 26). Ornithine deaminase catalyzes the deaminative cyclization of ornithine to proline, and we have proposed (15) that the rapL gene product catalyzes the analogous conversion of l-lysine to l-pipecolate (Fig. ​(Fig.3).3). Open in a separate windowFIG. 2A portion of the rapamycin biosynthetic gene cluster which contains ancillary (non-polyketide synthase) genes (15, 27). PKS, polyketide synthase.Open in a separate windowFIG. 3(A) The conversion of l-ornithine to l-proline by ornithine cyclodeaminase (17). (B) Proposed conversion of l-lysine to l-pipecolic acid by the rapL gene product.Here, we report the use of ΦC31 phage-mediated gene replacement (10) to introduce a frameshift mutation into rapL and the ability of the mutant to synthesize rapamycins in the absence or presence of added pipecolate or pipecolate analogs.     

Ceramide Synthesis Is Modulated by the Sphingosine Analog FTY720 via a Mixture of Uncompetitive and Noncompetitive Inhibition in an Acyl-CoA Chain Length-de pend ent Manner

FTY720, a sphingosine analog, is in clinical trials as an immunomodulator. The biological effects of FTY720 are believed to occur after its metabolism to FTY720 phosphate. However, very little is known about whether FTY720 can interact with and modulate the activity of other enzymes of sphingolipid metabolism. We examined the ability of FTY720 to modulate de novo ceramide synthesis. In mammals, ceramide is synthesized by a family of six ceramide synthases, each of which utilizes a restricted subset of acyl-CoAs. We show that FTY720 inhibits ceramide synthase activity in vitro by noncompetitive inhibition toward acyl-CoA and uncompetitive inhibition toward sphinganine; surprisingly, the efficacy of inhibition depends on the acyl-CoA chain length. In cultured cells, FTY720 has a more complex effect, with ceramide synthesis inhibited at high (500 nm to 5 μm) but not low (<200 nm) sphinganine concentrations, consistent with FTY720 acting as an uncompetitive inhibitor toward sphinganine. Finally, electrospray ionization-tandem mass spectrometry demonstrated, unexpectedly, elevated levels of ceramide, sphingomyelin, and hexosylceramides after incubation with FTY720. Our data suggest a novel mechanism by which FTY720 might mediate some of its biological effects, which may be of mechanistic significance for understanding its mode of action.FTY720 (2-amino-(2-2-[4-octylphenyl]ethyl)propane 1,3-diol hydrochloride), also known as Fingolimod, is an immunosuppressant drug currently being tested in clinical trials for organ transplantation and autoimmune diseases such as multiple sclerosis (1). FTY720 is a structural analog of sphingosine, a key biosynthetic intermediate in sphingolipid (SL)² metabolism (see Fig. 1). In vivo, FTY720 is rapidly phosphorylated by sphingosine kinase 2 (2, 3) to form FTY720 phosphate (FTY720-P), an analog of sphingosine 1-phosphate (S1P) (see Fig. 1A). FTY720-P binds to S1P receptors (S1PRs) (4, 5) and thereby induces a variety of phenomena such as T-lymphocyte migration from lymphoid organs (6–9); accordingly, FTY720 treatment results in lymphopenia as lymphocytes (especially T-cells) become sequestered inside lymphoid organs (10–12). The ability of FTY720 to sequester lymphocytes has stimulated its use in treatment of allograft rejection and autoimmune diseases (13), and FTY720 is currently under phase III clinical trials for treatment of relapsing-remitting multiple sclerosis (14).Open in a separate windowFIGURE 1.SL structure and metabolism. A, structures of SLs and SL analogs used in this study. B, metabolic inter-relationships between SLs and the metabolism of FTY720. The enzymes are denoted in italics. LPP3, lipid phosphate phosphatase 3; LPP1α, lipid phosphate phosphatase 1α.Apart from the binding of FTY720-P to S1PRs, the ability of FTY720 to inhibit S1P lyase (15) (see Fig. 1B), and its inhibitory effect on cytosolic phospholipase A2 (16), whose activity can be modulated by ceramide 1-phosphate (17), little is known about whether FTY720 or FTY720-P can modulate the activity of other enzymes of SL metabolism. Because FTY720 is an analog of sphingosine, one of the two substrates of ceramide synthase (CerS) (see Fig. 1), we now examine whether FTY720 can modulate CerS activity. CerS utilizes fatty acyl-CoAs to N-acylate sphingoid long chain bases. Six CerS exist in mammals, each of which uses a restricted subset of acyl-CoAs (18–23). We demonstrate that FTY720 inhibits CerS activity and that the extent of inhibition varies according to the acyl chain length of the acyl-CoA substrate. Surprisingly, FTY720 inhibits CerS activity toward acyl-CoA via noncompetitive inhibition and toward sphinganine via uncompetitive inhibition. Finally, the mode of interaction of FTY720 with CerS in cultured cells depends on the amount of available sphinganine. Together, we show that FTY720 modulates ceramide synthesis, which may be of relevance for understanding its biological effects in vivo and its role in immunomodulation.     

The Nutrigenetics of Hyperhomocysteinemia: QUANTITATIVE PROTEOMICS REVEALS DIFFERENCES IN THE METHIONINE CYCLE ENZYMES OF GENE-INDUCED VERSUS DIET-INDUCED HYPERHOMOCYSTEINEMIA*

Hyperhomocysteinemia has long been associated with atherosclerosis and thrombosis and is an independent risk factor for cardiovascular disease. Its causes include both genetic and environmental factors. Although homocysteine is produced in every cell as an intermediate of the methionine cycle, the liver contributes the major portion found in circulation, and fatty liver is a common finding in homocystinuric patients. To understand the spectrum of proteins and associated pathways affected by hyperhomocysteinemia, we analyzed the mouse liver proteome of gene-induced (cystathionine β-synthase (CBS)) and diet-induced (high methionine) hyperhomocysteinemic mice using two-dimensional difference gel electrophoresis and Ingenuity Pathway Analysis. Nine proteins were identified whose expression was significantly changed by 2-fold (p ≤ 0.05) as a result of genotype, 27 proteins were changed as a result of diet, and 14 proteins were changed in response to genotype and diet. Importantly, three enzymes of the methionine cycle were up-regulated. S-Adenosylhomocysteine hydrolase increased in response to genotype and/or diet, whereas glycine N-methyltransferase and betaine-homocysteine methyltransferase only increased in response to diet. The antioxidant proteins peroxiredoxins 1 and 2 increased in wild-type mice fed the high methionine diet but not in the CBS mutants, suggesting a dysregulation in the antioxidant capacity of those animals. Furthermore, thioredoxin 1 decreased in both wild-type and CBS mutants on the diet but not in the mutants fed a control diet. Several urea cycle proteins increased in both diet groups; however, arginase 1 decreased in the CBS^+/− mice fed the control diet. Pathway analysis identified the retinoid X receptor signaling pathway as the top ranked network associated with the CBS^+/− genotype, whereas xenobiotic metabolism and the NRF2-mediated oxidative stress response were associated with the high methionine diet. Our results show that hyperhomocysteinemia, whether caused by a genetic mutation or diet, alters the abundance of several liver proteins involved in homocysteine/methionine metabolism, the urea cycle, and antioxidant defense.Homocysteine (Hcy)1 is a thiol-containing amino acid that is produced in every cell of the body as an intermediate of the methionine cycle (Fig. 1, Reactions 1–5) (1). Once formed, the catabolism of homocysteine occurs via three enzymatic pathways. 1) Hcy is remethylated back to methionine using vitamin B₁₂-dependent methionine synthase (Fig. 1, Reaction 4) and/or 2) betaine-homocysteine methyltransferase (BHMT) (Fig. 1, Reaction 5), and 3) Hcy is converted to cysteine via the transsulfuration pathway using CBS and γ-cystathionase (Fig. 1, Reactions 6 and 7). Under normal conditions ∼40–50% of the Hcy that is produced in the liver is remethylated, ∼40–50% is converted to cysteine, and a small amount is exported (1–3). However, when Hcy production is increased (i.e. increased dietary methionine/protein intake) or when Hcy catabolism is decreased (i.e. CBS deficiency or B vitamin deficiencies), excess Hcy is exported into the extracellular space, resulting in hyperhomocysteinemia (1–5).Open in a separate windowFig. 1.Homocysteine metabolism in liver and kidney. In classical homocystinuria, the initial enzyme of the transsulfuration pathway, CBS (Reaction 6), is deficient. MTHF, methylenetetrahydrofolate; THF, tetrahydrofolate; DHF, dihydrofolate; MeCbl, methylcobalamin; DMG, dimethylglycine; PLP, pyridoxal 5′-phosphate.Homocystinuria was first described in the 1960s by Carson et al. (6): they observed 10 pediatric patients with severely elevated levels of Hcy in the urine and hypermethioninemia. Normal concentrations of plasma total homocysteine (tHcy) range from 5 to 12 μm (7); however, in homocystinuria, tHcy levels can exceed 100 μm. Homocystinuric patients present with mental retardation, abnormal bone growth, fine hair, malar flush, and dislocation of the lens of the eye, and most die from premature cardiovascular disease (6, 8). Autopsy findings indicate widespread thromboembolism, arteriosclerosis, and fatty livers (6, 8). Mudd et al. (9, 10) identified the cause of homocystinuria as a defect in the enzyme cystathionine β-synthase. A recent study of newborn infants in Denmark estimated the birth prevalence for CBS heterozygosity to be about 1:20,000 (11).Plasma tHcy concentrations are also directly correlated with dietary methionine/protein intake (12, 13). Guttormsen et al. (13) demonstrated that a protein-rich meal affected tHcy for at least 8–24 h. When normal subjects were fed a low protein-containing breakfast (12–15 g), plasma methionine levels increased slightly after 2 h (22.5–27.5 μm), but tHcy levels did not change significantly. However, when these same subjects were fed a high protein meal (52 g), plasma methionine levels peaked after 4 h (38 μm), and tHcy rose steadily until a maximum level was reached 8 h postmeal (7.6 versus 8.5 μm) (13). Thus, the following questions can be raised. How does the hepatic proteome respond to a hyperhomocysteinemic diet, and are the changes that accompany such a diet the same as or different from those that may be observed in gene-induced hyperhomocysteinemia?Because hyperhomocysteinemia is a strong independent risk factor for cardiovascular, cerebrovascular, and peripheral vascular disease, most of the current research has focused on the mechanisms involved in Hcy-induced endothelial dysfunction (14–24). The results of those studies have concluded that Hcy induces intracellular oxidative stress by generating ROS, which in turn lead to decreased bioavailable nitric oxide (NO), altered gene expression, increased endoplasmic reticulum stress, and activation of cholesterol biosynthesis. Also, several studies have examined the association between hyperhomocysteinemia and alcoholic liver disease, but few have looked at the effect of Hcy on the non-alcoholic liver even though fatty liver is a constant finding in homocystinuria (6, 8), and the liver is the major source of circulating Hcy (4, 5, 10). We hypothesize that 1) the liver proteome will respond to hyperhomocysteinemia by altering the expression of proteins involved in methionine/homocysteine metabolism and antioxidant defense and that 2) the set of proteins that are expressed when hyperhomocysteinemia is induced by CBS deficiency will differ from those expressed as a result of a high methionine diet. In the present study, we use a well established mouse model of CBS deficiency to study the early changes in the liver proteome that accompany hyperhomocysteinemia (25).     

Comparative Studies of Class IIa Bacteriocins of Lactic Acid Bacteria

Four class IIa bacteriocins (pediocin PA-1, enterocin A, sakacin P, and curvacin A) were purified to homogeneity and tested for activity toward a variety of indicator strains. Pediocin PA-1 and enterocin A inhibited more strains and had generally lower MICs than sakacin P and curvacin A. The antagonistic activity of pediocin-PA1 and enterocin A was much more sensitive to reduction of disulfide bonds than the antagonistic activity of sakacin P and curvacin A, suggesting that an extra disulfide bond that is present in the former two may contribute to their high levels of activity. The food pathogen Listeria monocytogenes was among the most sensitive indicator strains for all four bacteriocins. Enterocin A was most effective in inhibiting Listeria, having MICs in the range of 0.1 to 1 ng/ml. Sakacin P had the interesting property of being very active toward Listeria but not having concomitant high levels of activity toward lactic acid bacteria. Strains producing class IIa bacteriocins displayed various degrees of resistance toward noncognate class IIa bacteriocins; for the sakacin P producer, it was shown that this resistance is correlated with the expression of immunity genes. It is hypothesized that variation in the presence and/or expression of such immunity genes accounts in part for the remarkably large variation in bacteriocin sensitivity displayed by lactic acid bacteria.Many lactic acid bacteria (LAB), including members of the genera Lactococcus, Lactobacillus, Carnobacterium, Enterococcus, and Pediococcus, are known to secrete small, ribosomally synthesized antimicrobial peptides called bacteriocins (26, 29, 34). Some of these peptides undergo posttranslational modifications (class I bacteriocins), whereas others are not modified (class II bacteriocins) (29, 34). Class II bacteriocins contain between 30 and 60 residues and are usually positively charged at a neutral pH. Studies of a large number of class II bacteriocins have led to subgrouping of these compounds (29, 34). One of the subgroups, class IIa, contains bacteriocins that are characterized by the presence of YGNG and CXXXXCXV sequence motifs in their N-terminal halves as well as by their strong inhibitory effect on Listeria (e.g., 3, 4, 22, 23, 27, 28, 31, 38, 45) (Fig. ​(Fig.1).1). Because of their effectiveness against the food pathogen Listeria, class IIa bacteriocins have potential as antimicrobial agents in food and feed. Open in a separate windowFIG. 1Sequence alignment of class IIa bacteriocins. Residue numbering is according to the sequence of pediocin PA-1. Cysteine residues are printed in boldface; the two known class IIa bacteriocins with four cysteine residues are in the upper group. No attempt was made to optimize the alignment in the C-terminal halves of the peptides. Piscicolin 126 is identical to piscicocin V1a (4). Carnobacteriocin BM1 most probably is identical to piscicocin V1b (4). Sakacin P most probably is identical to bavaricin A (30). Curvacin A is identical to sakacin A (2). The consensus sequence includes residues conserved in at least 8 of the 12 sequences shown; 100% conserved residues are underlined.Class IIa bacteriocins act by permeabilizing the membrane of their target cells (1, 5, 6, 9, 10, 26, 28). The most recent studies on the mode of action of these bacteriocins indicate that antimicrobial activity does not require a specific receptor and is enhanced by (but not fully dependent on) a membrane potential (9, 28). Little is known about bacteriocin structure, and unravelling the relationships between structure and function is one of the great challenges in current bacteriocin research. A logical starting point for structure-function studies is a thorough study of the differences in activity and target cell specificity between naturally occurring homologous bacteriocins. A few such studies have been described, but these suffer from either a very limited number of tested indicator strains or the use of culture supernatants instead of purified bacteriocins (3, 4, 17, 45). The use of purified bacteriocins for comparative analyses is absolutely essential, since it is becoming increasingly evident that bacteriocin producers produce more than one bacteriocin (4, 8, 38, 48; this study).In the present study, the activities of four pure class IIa bacteriocins (pediocin PA-1, enterocin A, curvacin A, and sakacin P) (Fig. ​(Fig.1)1) were tested against a large number of LAB as well as several strains of the food pathogen Listeria monocytogenes. The bacteriocins were purified from their respective producer strains by use of an optimized purification protocol yielding highly pure samples. The contribution of disulfide formation was assessed and found to be important for activity. The effects of the purified bacteriocins on (noncognate) class IIa bacteriocin-producing strains are described, and the implications of our findings for immunity and resistance are discussed.     

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.     

Novel Inhibitors Complexed with Glutamate Dehydrogenase: ALLOSTERIC REGULATION BY CONTROL OF PROTEIN DYNAMICS*

Mammalian glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of l-glutamate to 2-oxoglutarate using NAD(P)⁺ as coenzyme. Unlike its counterparts from other animal kingdoms, mammalian GDH is regulated by a host of ligands. The recently discovered hyperinsulinism/hyperammonemia disorder showed that the loss of allosteric inhibition of GDH by GTP causes excessive secretion of insulin. Subsequent studies demonstrated that wild-type and hyperinsulinemia/hyperammonemia forms of GDH are inhibited by the green tea polyphenols, epigallocatechin gallate and epicatechin gallate. This was followed by high throughput studies that identified more stable inhibitors, including hexachlorophene, GW5074, and bithionol. Shown here are the structures of GDH complexed with these three compounds. Hexachlorophene forms a ring around the internal cavity in GDH through aromatic stacking interactions between the drug and GDH as well as between the drug molecules themselves. In contrast, GW5074 and bithionol both bind as pairs of stacked compounds at hexameric 2-fold axes between the dimers of subunits. The internal core of GDH contracts when the catalytic cleft closes during enzymatic turnover. None of the drugs cause conformational changes in the contact residues, but all bind to key interfaces involved in this contraction process. Therefore, it seems likely that the drugs inhibit enzymatic turnover by inhibiting this transition. Indeed, this expansion/contraction process may play a major role in the inter-subunit communication and allosteric regulation observed in GDH.Glutamate dehydrogenase (GDH)² is found in all living organisms and catalyzes the reversible oxidative deamination of l-glutamate to 2-oxoglutarate using NAD(P)⁺ as coenzyme (1). In eukaryotic organisms, GDH resides within the inner mitochondrial matrix where it catabolizes glutamate to feed 2-oxoglutarate to the Krebs cycle. Although there is some debate as to the directionality of the reaction, the high K_m for ammonium in the reductive amination reaction seems to prohibit the reverse reaction under normal conditions in most organisms (2). GDH from animals, but not other kingdoms (3), is allosterically regulated by a wide array of ligands (3–9). GTP (9–11), and with ∼100-fold lower affinity, ATP (3), is a potent inhibitor of the reaction and acts by increasing the binding affinity for the product, thereby slowing down enzymatic turnover (11). Hydrophobic compounds such as palmitoyl-CoA (12), steroid hormones (13), and steroid hormone analogs such as diethylstilbestrol (5) are also potent inhibitors. ADP is an activator of GDH (3, 6, 10, 11, 14) that acts in an opposite manner to GTP by facilitating product release. Leucine is a poor substrate for GDH but is also an allosteric activator for the enzyme (8). Its activation is akin to ADP but acts at site distinct from ADP (15).The crystal structures of the bacterial (16–18) and animal forms (19, 20) of GDH have shown that the general architecture and the locations of the catalytically important residues have remained unchanged throughout evolution. The structure of GDH (Fig. 1) is essentially two trimers of subunits stacked directly on top of each other with each subunit being composed of at least three domains (19–22). The bottom domain makes extensive contacts with a subunit from the other trimer. Resting on top of this domain is the “NAD binding domain” that has the conserved nucleotide binding motif. Animal GDH has a long protrusion, “antenna,” rising above the NAD binding domain that is not found in bacteria, plants, fungi, and the vast majority of protists. The antenna from each subunit lies immediately behind the adjacent, counterclockwise neighbor within the trimer. Because these intertwined antennae are only found in the forms of GDH that are allosterically regulated by numerous ligands, it is reasonable to speculate that it plays a role in regulation.Open in a separate windowFIGURE 1.Conformational transitions and locations of ligand binding sites in bovine glutamate dehydrogenase. A, a ribbon diagram of apo-bovine glutamate dehydrogenase with each of the identical subunits represented by different colors. The subunit arrangement is that of a trimer of dimers where anti-parallel β-strands form extensive interactions between the subunits stacked on top of each other. This pairing is represented by different shades of the same color. The conformational changes that during substrate binding are shown by the numbered arrows. As substrate binds, the NAD⁺ binding domain closes (1). The ascending helix of the antenna moves toward the pivot helix of the adjacent subunit (2). The short helix of the descending strand of the antenna becomes extended and distorted at the carboxyl end (3). Finally, the internal cavity of the helix compresses, bringing the three pairs closer together (4). B shows the structure of ADP (green spheres) bound to the apo-form of GDH and the location of Arg-463 (mauve spheres) that is involved in ADP activation (22). C shows the location of the inhibitor, GTP (mauve spheres), bound to the NADH (gray spheres), and glutamate (orange spheres) abortive complex. The green arrow notes the approximate location of one of the two sites (Lys-420) modified by 5′-FSBA (48). Comparing B and C, the closing of the catalytic cleft and the movement of the pivot helix is evident.From the structures GDH alone and complexed with various ligands, it is clear that GDH undergoes large conformational changes during each catalytic cycle (19–22) (the locations of these changes are summarized in Fig. 1). Substrate binds to the deep recesses of the cleft between the coenzyme binding domain and the lower domain. Coenzyme binds along the coenzyme binding domain surface of the cleft. Upon binding, the coenzyme binding domain rotates by ∼18° to firmly close down upon the substrate and coenzyme (Fig. 1, arrow 1). As the catalytic cleft closes, the base of each of the long ascending helices in the antenna appears to rotate out in a counterclockwise manner to push against the “pivot” helix of the adjacent subunit (Fig. 1, arrow 2). There is a short helix in the descending loop of the antenna that becomes distended and shorter as the mouth closes in a manner akin to an extending spring (Fig. 1, arrow 3). The pivot helix rotates in a counterclockwise manner along the helical axes as well as rotating counterclockwise around the trimer 3-fold axis. Finally, the entire hexamer seems to compress as the mouth closes (Fig. 1, arrow 4). The three pairs of subunits that sit on top of each other move as a rigid units toward each other, compressing the cavity at the core of the hexamer. This last conformational change will be further examined in this work. Allosteric regulation is likely exacted by controlling some or all of these conformational changes.The reason for complex animal regulation came from studies that linked GDH regulation with insulin and ammonia homeostasis. The connection between GDH and insulin regulation was initially established using a non-metabolizable analog of leucine (7, 23), BCH (β-2-aminobicycle[2.2.1]-heptane-2-carboxylic acid). These studies demonstrated that activation of GDH was tightly correlated with increased glutaminolysis and release of insulin. In addition, it has also been noted that factors that regulate GDH also affect insulin secretion (24). The in vivo importance of GDH in glucose homeostasis was demonstrated by the discovery that a genetic hypoglycemic disorder, the hyperinsulinemia/hyperammonemia (HHS) syndrome, is caused by loss of GTP regulation of GDH (25–27). Children with HHS have increased β-cell responsiveness to leucine and susceptibility to hypoglycemia following high protein meals (28). This is likely due to uncontrolled catabolism of amino acids yielding high ATP levels that stimulate insulin secretion as well as high serum ammonium levels. During glucose-stimulated insulin secretion in normal individuals, it has been proposed that the generation of high energy phosphates inhibits GDH and promotes conversion of glutamate to glutamine, which, alone or combined, might amplify the release of insulin (29, 30). Further support for this contention came from studies on the inhibitory effects of the polyphenolic compounds from green tea on BCH-stimulated insulin secretion (31). This not only lent support for the contention that GDH plays a significant role in insulin homeostasis, but also suggests that the HHS disorder might be directly controlled pharmaceutically. The role of GDH in insulin homeostasis is summarized in Fig. 2.Open in a separate windowFIGURE 2.Link between GDH and insulin homeostasis. This figure shows the role of GDH in BCH stimulated insulin secretion and how GDH inhibitors affect this process (29, 30). In energy-depleted β-cells, a BCH ramp stimulates insulin secretion. Here, the major energy source is glutaminolysis via phosphate-dependent glutaminase and GDH, because the concentration of GDH inhibitors (ATP/GTP) have been depleted and the phosphate-dependent glutaminase activator P_i (inorganic phosphate) has been increased. BCH stimulates glutamine utilization via GDH activation, thus providing the ATP signal necessary for insulin secretion. GDH inhibitors block this process by inhibiting GDH activity.To both find a more stable pharmaceutical agent to control HHS and to better understand the allosteric regulation of GDH, a high throughput screen was performed to identify new GDH inhibitors (32). Of the ∼30,000 compounds tested, ∼20 demonstrated significant activity. Three of the most active compounds, hexachlorophene, GW5074, and bithionol, were chosen for further analysis in this study. As shown here, all three compounds exhibit essentially non-competitive inhibition of the reaction and therefore do not compete with either substrate or coenzyme. Structural studies are presented here that demonstrate that six hexachlorophene (HCP) molecules bind to the inner core of the GDH hexamer, forming an internal ring via aromatic interactions. In contrast, bithionol and GW5074 bind as pairs between dimers of GDH subunits further away from the core of the enzyme. None of these compounds induce significant conformational changes in their immediate vicinity, and the mechanism of action is not clear from the location of their binding sites. However, detailed analysis of the various GDH complexes shows all of the drugs are binding to contact areas in the core of the hexamer that appear to be expanding and contracting during catalytic turnover. Therefore, inhibition is likely due interference with this “breathing” process.     

A Quantitative Chemical Proteomics Approach to Profile the Specific Cellular Targets of Andrographolide,a Promising Anticancer Agent That Suppresses Tumor Metastasis

Drug target identification is a critical step toward understanding the mechanism of action of a drug, which can help one improve the drug''s current therapeutic regime and expand the drug''s therapeutic potential. However, current in vitro affinity-chromatography-based and in vivo activity-based protein profiling approaches generally face difficulties in discriminating specific drug targets from nonspecific ones. Here we describe a novel approach combining isobaric tags for relative and absolute quantitation with clickable activity-based protein profiling to specifically and comprehensively identify the protein targets of andrographolide (Andro), a natural product with known anti-inflammation and anti-cancer effects, in live cancer cells. We identified a spectrum of specific targets of Andro, which furthered our understanding of the mechanism of action of the drug. Our findings, validated through cell migration and invasion assays, showed that Andro has a potential novel application as a tumor metastasis inhibitor. Moreover, we have unveiled the target binding mechanism of Andro with a combination of drug analog synthesis, protein engineering, and mass-spectrometry-based approaches and determined the drug-binding sites of two protein targets, NF-κB and actin.As most drugs exert pharmacological effects by interacting with their target proteins, the identification of these target proteins is a critical step in unraveling the mechanisms of drug action. It is also imperative for our understanding of the pharmacodynamics of a known drug, suggesting potentially unrevealed actions and thus refining future clinical applications of the substance. Traditional approaches used to identify protein targets of a drug typically utilize immobilized drug affinity chromatography coupled with mass spectrometry (MS)¹ (1, 2). These methods can be applied to cell lysates, but not in an in vivo setting, because of the requirement of a solid support. In vitro target profiling might not accurately reflect the drug''s actions in the in vivo physiological environment. To overcome this limitation, several groups have used activity-based protein profiling (ABPP) combined with bio-orthogonal click chemistry to identify drug targets both in vitro and in vivo (supplemental Fig. S1) (3–15). ABPP probes exert their functions via covalent reactions with the target proteins or photoaffinity-based labeling via the incorporation of photoreactive groups. With the increasing sensitivity of modern MS platforms, low-abundance protein targets can be successfully identified. Although both conventional affinity chromatography and recent ABPP-based methods allow us to detect a set of candidate protein targets for a drug, it remains difficult to discriminate specific interactions from nonspecific ones. Thus, more time and effort are needed for subsequent validation because of the presence of a large number of nonspecific binders. Therefore, there is an urgent need to develop comprehensive unbiased methods for specific target identification. Quantitative proteomics has been used to profile enriched kinases using cell-lysate-based kinobead pull-down. However, these types of experiments are mainly suitable for studying kinase inhibitors (16). Recently, proteomics methods based on stable isotope labeling of amino acids in cell culture (SILAC) have been applied to determine the specific binders of small molecules or proteins with certain post-translational modifications (17–19). These studies have shed light on how quantitative proteomics can improve the specificity of target protein identification. Nevertheless, because of the inherent limitations of SILAC, the complete incorporation of isotopic amino acids via such an approach takes a long time. Furthermore, it is also extremely difficult to apply the SILAC approach to tissue and body fluid samples, which are of particular relevance to biomedical research.Here we introduce a clickable activity-based protein profiling (ICABPP) approach based on the use of isobaric tags for relative and absolute quantitation (iTRAQ) for the unbiased specific and comprehensive identification of target proteins in live cells. iTRAQ is a stable isotope labeling approach for multiplexed quantitative proteome profiling (20). An overview of the technique is illustrated in Fig. 1A. In this assay, cells are first incubated with a clickable probe or with DMSO, which serves as a negative control. After the probe permeates the cell, and covalently binds to its dedicated in situ targets, the washed cells are lysed, clicked with biotin-N₃ tag, and enriched through avidin pull-down in parallel. The beads are washed thoroughly, and the bond proteins are directly digested on the beads with trypsin. The resulting peptides are labeled with their respective iTRAQ reagents, pooled together for further identification and quantification via LC-MS/MS. This technique enabled us to discriminate specific protein targets from nonspecific, and endogenously biotinylated proteins. Biological replicates of probe- or DMSO-treated samples are included to overcome experimental variations. As shown in Fig. 1B, nonspecific binding proteins'' iTRAQ reporters have equal or similar intensities, whereas specific target proteins enriched by the probe show highly differential intensities relative to the DMSO-treated control samples (as illustrated by the significantly higher reporter intensities of 116 and 117 versus 113 and 114 shown in Fig. 1B). The multiplexing nature of the iTRAQ-based chemical proteomics method allows replicated enrichments to be compared within a single LC-MS/MS analysis, thereby increasing the accuracy of specific target identifications, and minimizing experimental errors.Open in a separate windowFig. 1.Identifying specific drug targets using ICABPP approach in live cells. A, live cells were treated with DMSO, and clickable probe in duplicate. Cells were lysed and tagged with biotin-N3 using click chemistry in parallel. The biotin-bearing target proteins were enriched through avidin pull-down, and directly digested on beads. The resulting peptides of the two biological replicates of control pulled-down samples were labeled with 113 and 114, respectively, and two probe pulled-down samples were labeled with 116 and 117, respectively. The labeled peptides were combined in order to be identified, and quantified via LC-MS/MS. B, for the nonspecific targets, the iTRAQ reporters showed similar intensities, whereas for the specific targets, the reporters showed highly differential intensities.In this context, the ICABPP approach was applied to identify protein targets of andrographolide (Andro) (Fig. 2), a natural product with known anti-inflammation, and anti-cancer effects (21–25), in live cancer cells. A spectrum of 75 potential Andro targets was identified with high confidence, which suggested that Andro may exert anti-cancer effects by acting on multiple targets to interfere with several cellular signaling pathways. Two targets, NF-κB and β-actin, were validated by in vitro binding assay, and direct binding site mapping. Furthermore, our data revealed a novel mechanism of Andro in suppressing tumor metastasis.Open in a separate windowFig. 2.Chemical structures of Andro, reduced Andro analog RA, and Andro-based clickable ABPP probes P1 and P2.     

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.