Genome-wide analysis of HIF-2α chromatin binding sites under normoxia in human bronchial epithelial cells (BEAS-2B) suggests its diverse functions.

Constitutive functional HIF-2α was recently identified in cancer and stem cell lines under normoxia. In this study, BEAS-2B, a bronchial epithelial cell line, was shown to constitutively express active HIF-2α under normoxia and exhibit markers of pluripotency including Oct-4, Nanog, and sphere formation. Oct-4 expression was reduced after knockdown of HIF-2α under normoxia. Global enrichment analysis of HIF-2α demonstrated the diverse functions of HIF-2α under normoxia. Bioinformatics analysis of the enriched loci revealed an enhancer role of HIF-2α binding sites, involvement of HIF-2α interacting proteins, and enriched de novo motifs which suggest the diverse role of HIF-2α in pseudohypoxia. The low ratio of the discovered loci overlapping with those revealed in cancer cell lines 786-O (16.1%) and MCF-7 (15.9%) under hypoxia indicated a prevailing non-canonical mechanism. Hypoxia had positive, marginal or adverse effects on the enrichment of the selected loci in ChIP-PCR assays. Deletion of the N-terminal activation domain (N-TAD) of HIF-2α disrupted the reporting activity of two of the loci annotated to ELN and ANKRD31. Hypoxia incurring abundance variation of HIF-2α may misrepresent the N-TAD functions as canonical hypoxia inducible features via C-TAD activation. Elucidation of the pseudohypoxia functions of constitutive HIF-2α is useful for resolving its role in malignancy and pluripotency.

Lys-315 at the Interfaces of Diagonal Subunits of δ-Crystallin Plays a Critical Role in the Reversibility of Folding and Subunit Assembly.

δ-Crystallin is the major structural protein in avian eye lenses and is homologous to the urea cycle enzyme argininosuccinate lyase. This protein is structurally assembled as double dimers. Lys-315 is the only residue which is arranged symmetrically at the diagonal subunit interfaces to interact with each other. This study found that wild-type protein had both dimers and monomers present in 2-4 M urea whilst only monomers of the K315A mutant were observed under the same conditions, as judged by sedimentation velocity analysis. The assembly of monomeric K315A mutant was reversible in contrast to wild-type protein. Molecular dynamics simulations showed that the dissociation of primary dimers is prior to the diagonal dimers in wild-type protein. These results suggest the critical role of Lys-315 in stabilization of the diagonal dimer structure. Guanidinium hydrochloride (GdmCl) denatured wild-type or K315A mutant protein did not fold into functional protein. However, the urea dissociated monomers of K315A mutant protein in GdmCl were reversible folding through a multiple steps mechanism as measured by tryptophan and ANS fluorescence. Two partly unfolded intermediates were detected in the pathway. Refolding of the intermediates resulted in a conformation with greater amounts of hydrophobic regions exposed which was prone to the formation of protein aggregates. The formation of aggregates was not prevented by the addition of α-crystallin. These results highlight that the conformational status of the monomers is critical for determining whether reversible oligomerization or aggregate formation occurs.

The C Terminus of the Core ß-Ladder Domain in Japanese Encephalitis Virus Nonstructural Protein 1 Is Flexible for Accommodation of Heterologous Epitope Fusion.

UNLABELLED: NS1 is the only nonstructural protein that enters the lumen of the endoplasmic reticulum (ER), where NS1 is glycosylated, forms a dimer, and is subsequently secreted during flavivirus replication as dimers or hexamers, which appear to be highly immunogenic to the infected host, as protective immunity can be elicited against homologous flavivirus infections. Here, by using a trans-complementation assay, we identified the C-terminal end of NS1 derived from Japanese encephalitis virus (JEV), which was more flexible than other regions in terms of housing foreign epitopes without a significant impact on virus replication. This mapped flexible region is located in the conserved tip of the core ß-ladder domain of the multimeric NS1 structure and is also known to contain certain linear epitopes, readily triggering specific antibody responses from the host. Despite becoming attenuated, recombinant JEV with insertion of a neutralizing epitope derived from enterovirus 71 (EV71) into the C-terminal end of NS1 not only could be normally released from infected cells, but also induced dual protective immunity for the host to counteract lethal challenge with either JEV or EV71 in neonatal mice. These results indicated that the secreted multimeric NS1 of flaviviruses may serve as a natural protein carrier to render epitopes of interest more immunogenic in the C terminus of the core ß-ladder domain. IMPORTANCE: The positive-sense RNA genomes of mosquito-borne flaviviruses appear to be flexible in terms of accommodating extra insertions of short heterologous antigens into their virus genes. Here, we illustrate that the newly identified C terminus of the core ß-ladder domain in NS1 could be readily inserted into entities such as EV71 epitopes, and the resulting NS1-epitope fusion proteins appeared to maintain normal virus replication, secretion ability, and multimeric formation from infected cells. Nonetheless, such an insertion attenuated the recombinant JEV in mice, despite having retained the brain replication ability observed in wild-type JEV. Mother dams immunized with recombinant JEV expressing EV71 epitope-NS1 fused proteins elicited neutralizing antibodies that protected the newborn mice against lethal EV71 challenge. Together, our results implied a potential application of JEV NS1 as a viral carrier protein to express a heterologous epitope to stimulate dual/multiple protective immunity concurrently against several pathogens.

Characterization of a new mouse p53 variant: loss-of-function and gain-of-function.

BACKGROUND: p53 is a major tumor suppressor that is inactivated in over 50% of human cancer types through either mutation or inactivating interactions with viral or cellular proteins. The uncertainties around the link between p53 status, therapeutic response, and outcome in cancer suggest that additional factors may be involved. p53 isoforms that are generated via the alternative splicing pathway may be promising candidates for further investigation. RESULT: In this study, we report one new p53 protein with two internally deleted regions, resulting in one deleted amino acid fragment (from amino acid residues 42 to 89) and one reading frame-shift region (from amino acid residues 90-120) compared to wild-type p53. The functional status of the new p53 protein, which has a defect in its proline-rich and N-terminal DNA-binding domains, was characterized as possessing an intact conformation, exhibiting no transactivation activity, exerting a dominant-negative effect and an interacting with a coactivator with an arginine methyltransferase activity. CONCLUSION: Taken together, our findings provide valuable information about the structure and function of p53 for the regulation of transactivation activity and cellular protein-protein interactions. Furthermore, natural p53 isoforms will help us understand the functional roles of the p53 family and potential therapeutics for p53-dependent cancers.

Caffeine induces tumor cytotoxicity via the regulation of alternative splicing in subsets of cancer-associated genes.

Caffeine causes a diverse range of pharmacological effects that are time- and concentration-dependent and reversible. The detailed mechanisms of caffeine in tumor suppression via tumor suppressor protein p53 remain unclear. The isoforms of p53 are physiological proteins that are expressed in normal cells and generated via alternative promoters, splicing sites and/or translational initiation sites. In this study, we investigated how caffeine modulated cell cycle arrest and apoptosis via the expression of various alternatively spliced p53 isoforms. Caffeine reduced p53α expression and induced the expression of p53ß, which contains an alternatively spliced p53 C-terminus. In HeLa cells, the expression levels of many serine/arginine-rich splicing factors, including serine/arginine-rich splicing factors 2 and 3, were altered by caffeine. Serine/arginine-rich splicing factor 3 was a promising candidate for the serine/arginine-rich splicing factors responsible for the alternative splicing of p53 in response to caffeine treatment. In addition to p53-dependent functions, multiple target genes of serine/arginine-rich splicing factor 3 suggest that caffeine can regulate epithelial-mesenchymal-transition and hypoxic conditions to inhibit the survival of tumor cells. In summary, our data provide a new pathway of caffeine-modulated tumor suppression via the alternative splicing of the target genes of serine/arginine-rich splicing factor 3.

Ganoderma tsugae Extract Inhibits Growth of HER2-Overexpressing Cancer Cells via Modulation of HER2/PI3K/Akt Signaling Pathway.

Ganoderma, also known as Lingzhi or Reishi, has been used for medicinal purposes in Asian countries for centuries. It is a medicinal fungus with a variety of biological properties including immunomodulatory and antitumor activities. In this study, we investigated the molecular mechanisms by which Ganoderma tsugae (GT), one of the most common species of Ganoderma, inhibits the proliferation of HER2-overexpressing cancer cells. Here, we show that a quality assured extract of GT (GTE) inhibited the growth of HER2-overexpressing cancer cells in vitro and in vivo and enhanced the growth-inhibitory effect of antitumor drugs (e.g., taxol and cisplatin) in these cells. We also demonstrate that GTE induced cell cycle arrest by interfering with the HER2/PI3K/Akt signaling pathway. Furthermore, GTE curtailed the expression of the HER2 protein by modulating the transcriptional activity of the HER2 gene and the stability/degradation of the HER2 protein. In conclusion, this study suggests that GTE may be a useful adjuvant therapeutic agent in the treatment of cancer cells that highly express HER2.

A non-covalent interaction between small ubiquitin-like modifier-1 and Zac1 regulates Zac1 cellular functions.

Zac1, a zinc-finger protein that regulates apoptosis and cell cycle arrest 1, such as p53, can induce cell-cycle arrest and apoptosis. The transactivation and coactivation functions of Zac1 may occur at non-promyelocytic leukemia nuclear body (PML-NB) sites in the presence of other PML-NB components, including ubiquitin-conjugating 9 (Ubc9). It is unclear whether post-translational modification of Zac1 by the small ubiquitin-like modifier SUMO plays a role in the coactivation functions of Zac1 for the regulation of the p21 gene. Mutagenesis experiments revealed that the two SUMO-binding lysine residues of Zac1, K237 and K424, repress the transactivation activity of Zac1. Studies using a SUMO-1 C-terminal di-glycine motif mutant that is deficient in the ability to form covalent bonds with lysines, SUMO-1 (GA), and a dominant-negative Ubc9 construct (C93S) indicated that SUMO-1 might regulate Zac1 transactivation and coactivation via a non-covalent interaction. Unlike the wild-type Zac1, which induced apoptosis, the Zac1 (K237/424R) double mutant had the ability to induce autophagy. The functional role of p21 remains to be investigated. SUMO-1 selectively suppressed the induction of the p21 gene and protein by wild-type Zac1 but not by the Zac1 (K237/424R) double mutant. Moreover, wild-type Ubc9 but not Ubc9 (C93S) further potentiated the suppression of SUMO-1 in all Zac1-induced p21 promoter activities. Our data reveal that p21 may be an important factor for the prevention of Zac1-induced apoptosis without affecting autophagosome formation. This work indicates that Zac1 functions are regulated, at least in part, via non-covalent interactions with SUMO-1 for the induction of p21, which is important for the modulation of apoptosis.

α-Crystallin protects human arginosuccinate lyase activity under freeze-thaw conditions.

Argininosuccinate lyase (ASL) catalyzes the conversion of argininosuccinate into arginine and fumarate, a key step in the biosynthesis of urea and arginine. ASL is a tetrameric enzyme but it dissociates into inactive dimers under low temperature conditions. This study investigates the inactivation process under low temperature conditions. Inactivation was caused by dissociation of tetrameric ASL into dimers, with increased exposure of hydrophobic areas without disturbance of the secondary structure or the microenvironment surrounding the key tryptophan residues. Most activity was retained when temperatures were changed at a rate of >1 °C/min, whilst freezing or thawing more slowly resulted in greater loss of activity. Inactivation was reduced by inclusion of α-crystallin, a structural protein found in ocular lenses and a member of the small heat-shock protein family, by stabilization of the ASL quaternary structure. In addition, α-crystallin was able to restore the function of ASL that had been inactivated by slow freezing and thawing. The effect of α-crystallin was similar to that of bovine serum albumin, suggesting that both proteins exerted their effects by hydrophobic interactions. α-Crystallin therefore acts as a cryo-preservative that protects ASL activity during freezing and thawing.

Distinct interactions of αA-crystallin with homologous substrate proteins, δ-crystallin and argininosuccinate lyase, under thermal stress.

δ-Crystallin is a taxon-specific eye lens protein that was recruited from argininosuccinate lyase (ASL) through gene sharing. ASL is a metabolic enzyme that catalyzes the reversible conversion of argininosuccinate into arginine and fumarate and shares about 70% sequence identity and similar overall topology with δ-crystallin. ASL has a lower thermal stability than δ-crystallin. In this study, we show that the small heat shock protein, αA-crystallin, functions as a molecular chaperone, and enhanced thermal stability of both δ-crystallin and ASL. The stoichiometry for efficient protection of the two substrate proteins by αA-crystallin was determined by slowly increasing the temperature. N- or C-terminal truncated mutants of δ-crystallin co-incubated with αA-crystallin showed higher thermal stability than wild-type enzyme, and the stoichiometry for efficient protection was the same. Thermal unfolding of δ-crystallin or ASL in the presence of αA-crystallin followed a similar three-state model, as determined by circular dichroism analyses. A stable intermediate which retained about 30% α-helical structure was observed. Protection from thermal denaturation by αA-crystallin was by interaction with partly unfolded ASL or δ-crystallin to form high molecular weight heteroligomers, as judged by size-exclusive chromatography and SDS-PAGE analyses. Aggregate formation of ASL was significantly reduced in the presence of αA-crystallin. The extent of protection of ASL and δ-crystallin at different ratios of αA-crystallin were described by hyperbolic and sigmoidal curves, respectively. These results suggest the preferential recognition of partly unfolded ASL by αA-crystallin. In contrast, unstable δ-crystallin might trigger a cooperative interaction by higher stoichiometries of αA-crystallin leading to fuller protection. The different interactions of αA-crystallin with the two homologous but functionally different substrate proteins show its behavior as a chaperone is variable.

Substitution of residues at the double dimer interface affects the stability and oligomerization of goose delta-crystallin.

Delta-crystallin is the major structural protein in avian and reptilian eye lenses, and confers special refractive properties. The protein is a homotetramer arranged as a dimer of dimers. In the present study, the roles of the side chains of Glu267, Lys315, and Glu327, which provide hydrogen bonds at the double dimer interface, were investigated. Hydrophobic side chain substitution led to all mutant proteins having an unstable dimer interface. The E267L/E327L mutant had the greatest sensitivity to temperature, urea and guanidinium hydrochloride denaturation, and the most extensive exposure of hydrophobic patches, as judged by 1-anilinonaphthalene-8-sulfonic acid fluorescence, CD, and tryptophan fluorescence. In contrast, the E267L/K315L/E327L mutant showed higher stability than the E267L/E327L mutant. Some level of the dissociated dimeric form was observed in the K315L mutant, but it was not observed for the K315A and E267L/K315L mutants. The E327L mutant was partially in the dissociated dimeric form, whereas the E267/E327L mutant was predominantly dissociated into dimers. In contrast, the triple mutant of E267L/K315L/E327L retained a tetrameric structure. In the presence of urea, a stable monomeric intermediate with higher stability than the wild type was identified for the K315A mutant. Disruption of interfacial interactions at Glu267 led to polymerization of partly unfolded intermediates in the presence of 3 m urea. However, these polymeric forms were not observed with combinations of the E267L mutation with other mutations. These results indicate that these hydrogen bonds, which are present at different contact surfaces in the dimer-dimer interface, perform distinct functions in double dimer assembly. The coordination of these interactions is critical for the stability and tetramer formation of delta-crystallin.

Physical and functional interactions between hnRNP K and PRMT family proteins.

The mechanism underlying the protein-protein interaction of hnRNP K and PRMT family proteins is unclear. We examined and confirmed the arginine methylation of hnRNP K protein by PRMT1, not CARM1, via their direct binding. We also studied hnRNP K protein complexes containing CARM1, as well as PRMT1, using co-immunoprecipitation analysis. PRMT family proteins might be involved in the regulation of hnRNP K functions in nuclear receptor coactivator, transactivation, and p21 gene and protein expressions. We believe these observations will help provide insights into the regulation of hnRNP K protein functions via the recruitment of its associated proteins, including its arginine methylation-modifying proteins.

The roles of Tyr(91) and Lys(162) in general acid-base catalysis in the pigeon NADP+-dependent malic enzyme.

The role of general acid-base catalysis in the enzymatic mechanism of NADP+-dependent malic enzyme was examined by detailed steady-state kinetic studies through site-directed mutagenesis of the Tyr(91) and Lys(162) residues in the putative catalytic site of the enzyme. Y91F and K162A mutants showed approx. 200- and 27000-fold decreases in k(cat) values respectively, which could be partially recovered with ammonium chloride. Neither mutant had an effect on the partial dehydrogenase activity of the enzyme. However, both Y91F and K162A mutants caused decreases in the k(cat) values of the partial decarboxylase activity of the enzyme by approx. 14- and 3250-fold respectively. The pH-log(k(cat)) profile of K162A was found to be different from the bell-shaped profile pattern of wild-type enzyme as it lacked a basic pK(a) value. Oxaloacetate, in the presence of NADPH, can be converted by malic enzyme into L-malate by reduction and into enolpyruvate by decarboxylation activities. Compared with wild-type, the K162A mutant preferred oxaloacetate reduction to decarboxylation. These results are consistent with the function of Lys(162) as a general acid that protonates the C-3 of enolpyruvate to form pyruvate. The Tyr(91) residue could form a hydrogen bond with Lys(162) to act as a catalytic dyad that contributes a proton to complete the enol-keto tautomerization.

Human Spot 14 protein is a p53-dependent transcriptional coactivator via the recruitment of thyroid receptor and Zac1.

Spot 14 is an acidic homodimeric protein with no sequence similarity to other mammalian gene products. Its biochemical function remains unclear. Recent studies have shown that the human Spot 14 locus is in the chromosomal region 11q13 and is frequently amplified in breast cancers, suggesting that it plays a role in the gene regulation involved in cell growth during tumorigenesis. Our previous work has demonstrated that human Spot 14 protein physically and functionally interacts with thyroid receptor in the regulation of malic enzyme gene expression. In this study, we investigated the subcellular distribution of human Spot 14 protein using enhanced green fluorescence protein and infer that its localization might be affected by thyroid hormone. Our results also demonstrate that the potential transactivation activity of human Spot 14 protein is regulated by its C-terminal region. Human Spot 14 protein is involved both in the regulation of malic enzyme promoter activity, and in the regulation of p53-dependent transactivation. It does not interact directly with p53, whereas it is able to directly interact with thyroid receptor and Zac1 (zinc finger protein which regulates apoptosis and cell cycle arrest 1) using glutathione-S-transferase pull-down assay. Hence, human Spot 14 protein might regulate the p53 target gene, p21(WAF1/Cip1), via its direct interaction with the thyroid receptor or other p53 coactivators, such as Zac1. These findings provide a molecular rationale for the role of human Spot 14 protein in the p53-dependent transcriptional activation of specific genes via diverse pathways in cells.

Human spot 14 protein interacts physically and functionally with the thyroid receptor.

Spot 14 (S14) is a small acidic protein with no sequence similarity to other mammalian gene products. Its biochemical function is elusive. Recent studies have shown that, in some cancers, human S14 (hS14) localizes to the nucleus and is amplified, suggesting that it plays a role in the regulation of lipogenic enzymes during tumorigenesis. In this study, we purified untagged hS14 protein and then demonstrated, using various biochemical methods, including analytic ultracentrifugation, that hS14 might form a homodimer. We also found several lines of evidence to suggest physical and functional interactions between hS14 and the thyroid hormone receptor (TR). The ubiquitous expression of hS14 in various cell lines and its cell-type-dependent functions demonstrated in this study suggest that it acts as a positive or negative cofactor of the TR to regulate malic enzyme gene expression. These findings provide a molecular rationale for the role of hS14 in TR-dependent transcriptional activation of the expression of specific genes.

Critical roles of conserved carboxylic acid residues in pigeon cytosolic NADP+-dependent malic enzyme.

Malic enzyme catalyses the reduction of NADP+ to NADPH and the decarboxylation of L-malate to pyruvate through a general acid/base mechanism. Previous kinetic and structural studies differ in their interpretation of the amino acids responsible for the general acid/base mechanism. To resolve this discrepancy, we used site-directed mutagenesis and kinetic analysis to study four conserved carboxylic amino acids. With the D257A mutant, the Km for Mn2+ and the kcat decreased relative to those of the wild-type by sevenfold and 28-fold, respectively. With the E234A mutant, the Km for Mg2+ and L-malate increased relative to those of the wild-type by 87-fold and 49-fold, respectively, and the kcat remained unaltered, which suggests that the E234 residue plays a critical role in bivalent metal ion binding. The kcat for the D235A and D258A mutants decreased relative to that of the wild-type by 7800-fold and 5200-fold, respectively, for the overall reaction, by 800-fold and 570-fold, respectively, for the pyruvate reduction partial reaction, and by 371-fold and 151-fold, respectively, for the oxaloacetate decarboxylation. The activities of the overall reaction and the pyruvate reduction partial reaction of the D258A mutant were rescued by the presence of 50 mM sodium azide. In contrast, small free acids did not have a rescue effect on the activities of the E234A, D235A, and D257A mutants. These data suggest that D258 may act as a general base to extract the hydrogen of the C2 hydroxy group of L-malate with the aid of D235-chelated Mn2+ to polarize the hydroxyl group.

Modulation of glucocorticoid receptor-interacting protein 1 (GRIP1) transactivation and co-activation activities through its C-terminal repression and self-association domains.

Glucocorticoid receptor-interacting protein 1 (GRIP1), a p160 family nuclear receptor co-activator, possesses at least two autonomous activation domains (AD1 and AD2) in the C-terminal region. AD1 activity appears to be mediated by CBP/p300, whereas AD2 activity is apparently mediated through co-activator-associated arginine methyltransferase 1 (CARM1). The mechanisms responsible for regulating the activities of AD1 and AD2 are not well understood. We provide evidence that the GRIP1 C-terminal region may be involved in regulating its own transactivation and nuclear receptor co-activation activities through primary self-association and a repression domain. We also compared the effects of the GRIP1 C terminus with those of other factors that functionally interact with the GRIP1 C terminus, such as CARM1. Based on our results, we propose a regulatory mechanism involving conformational changes to GRIP1 mediated through its intramolecular and intermolecular interactions, and through modulation of the effects of co-repressors on its repression domains. These are the first results to indicate that the structural components of GRIP1, especially those of the C terminus, might functionally modulate its putative transactivation activities and nuclear receptor co-activator functions.

Nitric oxide induces prion protein via MEK and p38 MAPK signaling.

The prion diseases or transmissible spongiform encephalopathy, such as human Creutzfeldt-Jakob disease (CJD) and so-called mad cow disease, are attributed to the causative agent, the scrapie variant of prion protein (PrP(Sc)) which causes fatal neurodegeneration. To investigate if stresses such as nitric oxide (NO) induced the cellular isoform of prion protein (PrP(C)), lipopolysaccharide, and sodium nitroprusside were used to treat N2a and NT2 cells, which resulted in elevated levels of the PRNP mRNA and prion protein. The signaling pathway for the NO-induced PrP(C) production involved guanylyl cyclase, MEK, and p38 MAPK as shown by the effect of specific pharmacological inhibitors ODQ, PD98059, and SB203580, respectively. Knowing the PrP induction by the biologically existing stimulus, this study provides useful information about the possible cellular mechanism and strategies for the treatment of CJD.

Characterization of the interactions between Asp141 and Phe236 in the Mn2+-l-malate binding of pigeon liver malic enzyme.

The cytosolic malic enzyme from pigeon liver is very sensitive to the metal-catalysed oxidation systems. Our previous studies using the Cu2+-ascorbate as the oxidation system showed that the enzyme was oxidized and cleaved at several positions, including Asp141. The recently resolved crystal structure of pigeon liver malic enzyme revealed that Asp141 was near to the metal-binding site, but was not a direct metal ligand. However, Asp141 is located next to Phe236, which directly follows the metal ligands Glu234 and Asp235. Mutation at Asp141 caused a drastic effect on the metal-binding affinity of the enzyme. Since Asp141 and Phe236 are highly conserved in most species of malic enzyme, we used a double-mutant cycle to study the possible interactions between these two residues. Four single mutants [D141A (Asp141-->Ala), D141N, F236A and F236L] and four double mutants (D141A/F236A, D141N/F236A, D141A/F236L and D141N/F236L), plus the wild-type enzyme were successfully cloned, expressed and purified to homogeneity. The secondary, tertiary and quaternary structures of these mutants, as assessed by CD, fluorescence and analytical ultracentrifuge techniques, were similar to that of the wild-type enzyme. Initial velocity experiments were performed to derive the various kinetic parameters, which were used to analyse further the free energy change and the coupling energy (DeltaDeltaG(int)) between any two residues. The dissociation constants for Mn2+ ( K (d,Mn)) of the D141A and F236A mutants were increased by approx. 6- and 65-fold respectively, compared with that of the wild-type enzyme. However, the K (d,Mn) for the double mutant D141A/F236A was only increased by 150-fold. A coupling energy of -2.12 kcal/mol was obtained for Asp141 and Phe236. We suggest that Asp141 is involved in the second sphere of the metal-binding network of the enzyme.

Structural studies of the pigeon cytosolic NADP(+)-dependent malic enzyme.

Malic enzymes are widely distributed in nature, and have important biological functions. They catalyze the oxidative decarboxylation of malate to produce pyruvate and CO(2) in the presence of divalent cations (Mg(2+), Mn(2+)). Most malic enzymes have a clear selectivity for the dinucleotide cofactor, being able to use either NAD(+) or NADP(+), but not both. Structural studies of the human mitochondrial NAD(+)-dependent malic enzyme established that malic enzymes belong to a new class of oxidative decarboxylases. Here we report the crystal structure of the pigeon cytosolic NADP(+)-dependent malic enzyme, in a closed form, in a quaternary complex with NADP(+), Mn(2+), and oxalate. This represents the first structural information on an NADP(+)-dependent malic enzyme. Despite the sequence conservation, there are large differences in several regions of the pigeon enzyme structure compared to the human enzyme. One region of such differences is at the binding site for the 2'-phosphate group of the NADP(+) cofactor, which helps define the cofactor selectivity of the enzymes. Specifically, the structural information suggests Lys362 may have an important role in the NADP(+) selectivity of the pigeon enzyme, confirming our earlier kinetic observations on the K362A mutant. Our structural studies also revealed differences in the organization of the tetramer between the pigeon and the human enzymes, although the pigeon enzyme still obeys 222 symmetry.

Effect of metal binding on the structural stability of pigeon liver malic enzyme.

The cytosolic malic enzyme from the pigeon liver is sensitive to chemical denaturant urea. When monitored by protein intrinsic fluorescence or circular dichroism spectral changes, an unfolding of the enzyme in urea at 25 degrees C and pH 7.4 revealed a biphasic phenomenon with an intermediate state detected at 4-5 m urea. The enzyme activity was activated by urea up to 1 m but was completely lost before the intermediate state was detected. This suggests that the active site region of the enzyme was more sensitive to chemical denaturant than other structural scaffolds. In the presence of 4 mm Mn(2+), the urea denaturation pattern of malic enzyme changed to monophasic. Mn(2+) helped the enzyme to resist phase I urea denaturation. The [urea](0.5) for the enzyme inactivation shifted from 2.2 to 3.8 m. Molecular weight determined by the analytical ultracentrifuge indicated that the tetrameric enzyme was dissociated to dimers in the early stage of phase I denaturation. In the intermediate state at 4-5 m urea, the enzyme showed polymerization. However, the polymer forms were dissociated to unfolded monomers at a urea concentration greater than 6 m. Mn(2+) retarded the polymerization of the malic enzyme. Three mutants of the enzyme with a defective metal ligand (E234Q, D235N, E234Q/D235N) were cloned and purified to homogeneity. These mutant malic enzymes showed a biphasic urea denaturation pattern in the absence or presence of Mn(2+). These results indicate that the Mn(2+) has dual roles in the malic enzyme. The metal ion not only plays a catalytic role in stabilization of the reaction intermediate, enol-pyruvate, but also stabilizes the overall tetrameric protein architecture.