Saccharomyces cerevisiae ferrochelatase forms a homodimer

Ferrochelatase, the last enzyme of the heme biosynthetic pathway, has for years been considered to be active as a monomer. The crystal structure of Bacillus subtilis ferrochelatase confirmed its monomeric structure. However, animal ferrochelatase was found to form a functional dimer. Data presented here indicate that ferrochelatase from the yeast Saccharomyces cerevisiae is also dimeric. Following two-hybrid studies that had shown an interaction of two ferrochelatase molecules, we employed several different, complementary approaches, such as chemical crosslinking, affinity chromatography, and complementation analysis, to prove that in the yeast cells ferrochelatase forms an active dimer. We have isolated a double mutant, hem15D246V/Y248F, which is probably dimerization-defective. We propose a structural model of yeast ferrochelatase, based on the known structure of the human enzyme, which helps us to understand the differences in dimerization between the wild-type and mutant proteins.     

NF-kappaB RelB forms an intertwined homodimer

The X-ray structure of the RelB dimerization domain (DD) reveals that the RelBDD assumes an unexpected intertwined fold topology atypical of other NF-kappaB dimers. All typical NF-kappaB dimers are formed by the association of two independently folded immunoglobulin (Ig) domains. In RelBDD, two polypeptides reconstruct both Ig domains in the dimer with an extra beta sheet connecting the two domains. Residues most critical to NF-kappaB dimer formation are invariant in RelB, and Y300 plays a positive role in RelBDD dimer formation. The presence of RelB-specific nonpolar residues at the surface removes several intradomain surface hydrogen bonds that may render the domain fold unstable. Intertwining may stabilize the RelBDD homodimer by forming the extra beta sheet. We show that, as in the crystal, RelB forms an intertwined homodimer in solution. We suggest that the transiently stable RelB homodimer might prevent its rapid degradation, allowing for heterodimer formation with p50 and p52.     

Human joining peptide: a proopiomelanocortin product secreted as a homodimer

The human (h) POMC gene sequence predicts a 30 amino acid joining peptide (JP) separating the N-terminal fragment [POMC(1-76) or hNT] and ACTH within their common precursor. We used an anti-serum directed against the amidated COOH-terminal end of mouse JP to develop a RIA for the predicted hJP molecule. Immunoreactive JP was detected in tissue extracts from human normal pituitary, ACTH-secreting pituitary- and nonpituitary tumors, and in plasma from patients with ACTH hypersecretory syndromes. Its molar concentration was of the same order of magnitude as, and correlated with, that of the other POMC peptides. Gel exclusion chromatography in 1% formic acid and 6 M guanidine-HCl revealed a predominant immunoreactive material with an apparent mol wt of ca. 6000. After reduction with dithiothreitol this material was recovered in an elution volume identical to that of purified hJP and corresponding to a mol wt of ca. 3000. These data show that POMC processing generates a COOH terminally amidated hJP predominantly secreted as a homodimer, probably through disulfide bonding between the single Cys9 residue of two molecules.     

Plasmodium falciparum serine-repeat antigen (SERA) forms a homodimer through disulfide bond

Plasmodium falciparum serine-repeat antigen (SERA) is one potential blood-stage vaccine candidate and is expressed as a protomer that is subsequently processed into four fragments (P47, P50, P6, and P17). Although recent evidence shows that P50 exhibits chymotrypsin-like protease activity, the function of SERA is still largely unknown. Here, we found that apart from cathepsin L-like cysteine protease, P50 showed significant homology to silicatein-α and testin which were shown to bind to cellular components, suggesting that SERA may have similar function. Immunoprecipitation of schizont lysate and molecular assignment of its precipitate by mass spectrometry provided evidence that SERA forms a homodimer through disulfide bond. Moreover, analysis of the fate of SERA using cell-free system revealed that the kinetics of conversion of SERA dimer into monomer is faster than that of processing of SERA monomer into various fragments. These findings may contribute to elucidate a possible function of SERA other than a protease.     

Budding yeast Dsk2 protein forms a homodimer via its C-terminal UBA domain

Budding yeast Dsk2 is a family of UbL-UBA proteins that can interact with both polyubiquitin and the proteasome, and is thereby thought to function as a shuttle protein in the ubiquitin-proteasome pathway. Here we show that Dsk2 can homodimerize via its C-terminal UBA domain in the absence of ubiquitin. Dsk2 mutants defective in the UBA domain do not dimerize and do not bind polyubiquitin. The expression of Dsk2 UBA mutants fails to restore the growth defect caused by DSK2 disruption although that of wild-type Dsk2 can restore the defect. These results suggest that Dsk2 homodimerization via the UBA domain plays a role in regulating polyubiquitin binding in the ubiquitin-proteasome pathway.     

Human cyclooxygenase-2 is a sequence homodimer that functions as a conformational heterodimer

Prostaglandin endoperoxide H synthases 1 and 2, also known as cyclooxygenases (COXs) 1 and 2, convert arachidonic acid (AA) to prostaglandin endoperoxide H(2). Prostaglandin endoperoxide H synthases are targets of nonspecific nonsteroidal anti-inflammatory drugs and COX-2-specific inhibitors called coxibs. PGHS-2 is a sequence homodimer. Each monomer has a peroxidase and a COX active site. We find that human PGHS-2 functions as a conformational heterodimer having a catalytic monomer (E(cat)) and an allosteric monomer (E(allo)). Heme binds tightly only to the peroxidase site of E(cat), whereas substrates, as well as certain inhibitors (e.g. celecoxib), bind the COX site of E(cat). E(cat) is regulated by E(allo) in a manner dependent on what ligand is bound to E(allo). Substrate and nonsubstrate fatty acids (FAs) and some COX inhibitors (e.g. naproxen) preferentially bind to the COX site of E(allo). AA can bind to E(cat) and E(allo), but the affinity of AA for E(allo) is 25 times that for E(cat). Palmitic acid, an efficacious stimulator of human PGHS-2, binds only E(allo) in palmitic acid/murine PGHS-2 co-crystals. Nonsubstrate FAs can potentiate or attenuate actions of COX inhibitors depending on the FA and whether the inhibitor binds E(cat) or E(allo). Our studies suggest that the concentration and composition of the free FA pool in the environment in which PGHS-2 functions in cells, the FA tone, is a key factor regulating PGHS-2 activity and its responses to COX inhibitors. We suggest that differences in FA tone occurring with different diets will likely affect both base-line prostanoid synthesis and responses to COX inhibitors.     

Signal peptide peptidase forms a homodimer that is labeled by an active site-directed gamma-secretase inhibitor

Presenilin (PS) is the presumptive catalytic component of the intramembrane aspartyl protease gamma-secretase complex. Recently a family of presenilin homologs was identified. One member of this family, signal peptide peptidase (SPP), has been shown to be a protease, which supports the hypothesis that PS and presenilin homologs are related intramembrane-cleaving aspartyl proteases. SPP has been reported as a glycoprotein of approximately 45 kDa. Our initial characterization of SPP isolated from human brain and cell lines demonstrated that SPP is primarily present as an SDS-stable approximately 95-kDa protein on Western blots. Upon heating or treatment of this approximately 95-kDa SPP band with acid, a approximately 45-kDa band could be resolved. Co-purification of two different epitope-tagged forms of SPP from a stably transfected cell line expressing both tagged versions demonstrated that the approximately 95-kDa band is a homodimer of SPP. Pulse-chase metabolic labeling studies demonstrated that the SPP homodimer assembles rapidly and is metabolically stable. In a glycerol velocity gradient, SPP sedimented from approximately 100-200 kDa. Significantly the SPP homodimer was specifically labeled by an active site-directed photoaffinity probe (III-63) for PS, indicating that the active sites of SPP and PS/gamma-secretase are similar and providing strong evidence that the homodimer is functionally active. Collectively these data suggest that SPP exists in vivo as a functional dimer.     

Characterization of Human gamma-glutamyl hydrolase in solution demonstrates that the enzyme is a non-dissociating homodimer

Human gamma-glutamyl hydrolase (hGH) is a key enzyme in the metabolism of folic acid and in the pharmacology of many antifolate drugs. hGH catalyzes removal of the poly-gamma-glutamate chains of intracellular folic acid and antifolates. hGH crystallized as a homodimer with two putative active sites. However, the quaternary structure and the number of species of the enzyme in solution have not been determined. hGH has now been characterized using analytical ultracentrifugation and dynamic light scattering. HisTag fusion proteins of wild-type hGH, rat GH, and hGH expressed as a glycosylated protein were studied. Analyses of HisTag wild-type hGH were conducted over a range of protein concentrations (1.4-200 microM), ionic strengths (0-1 M NaCl), and pH (4.5-8.5). A single species with a molecular mass consistent with a homodimer was observed. Glycosylated hGH and HisTag rat gamma-glutamyl hydrolase also formed very stable homodimers. The lack of dissociation of the dimer, the large monomer-monomer interface, and the presence of catalytically essential Tyr-36 in the homodimer interface sequences suggest that homodimer formation is required for the hGH monomer to fold into an active conformation. The conservation of hGH monomer-monomer interface sequences in other mammalian and plant gamma-glutamyl hydrolase molecules suggests that they also exist as stable homodimers.     

The C-terminal domain of the MutL homolog from Neisseria gonorrhoeae forms an inverted homodimer

The mismatch repair (MMR) pathway serves to maintain the integrity of the genome by removing mispaired bases from the newly synthesized strand. In E. coli, MutS, MutL and MutH coordinate to discriminate the daughter strand through a mechanism involving lack of methylation on the new strand. This facilitates the creation of a nick by MutH in the daughter strand to initiate mismatch repair. Many bacteria and eukaryotes, including humans, do not possess a homolog of MutH. Although the exact strategy for strand discrimination in these organisms is yet to be ascertained, the required nicking endonuclease activity is resident in the C-terminal domain of MutL. This activity is dependent on the integrity of a conserved metal binding motif. Unlike their eukaryotic counterparts, MutL in bacteria like Neisseria exist in the form of a homodimer. Even though this homodimer would possess two active sites, it still acts a nicking endonuclease. Here, we present the crystal structure of the C-terminal domain (CTD) of the MutL homolog of Neisseria gonorrhoeae (NgoL) determined to a resolution of 2.4 Å. The structure shows that the metal binding motif exists in a helical configuration and that four of the six conserved motifs in the MutL family, including the metal binding site, localize together to form a composite active site. NgoL-CTD exists in the form of an elongated inverted homodimer stabilized by a hydrophobic interface rich in leucines. The inverted arrangement places the two composite active sites in each subunit on opposite lateral sides of the homodimer. Such an arrangement raises the possibility that one of the active sites is occluded due to interaction of NgoL with other protein factors involved in MMR. The presentation of only one active site to substrate DNA will ensure that nicking of only one strand occurs to prevent inadvertent and deleterious double stranded cleavage.     

The fourth transmembrane segment forms the interface of the dopamine D2 receptor homodimer

Considerable evidence suggests that G-protein-coupled receptors form homomeric and heteromeric dimers in vivo. Unraveling the structural mechanism for cross-talk between receptors in a dimeric complex must start with the identification of the presently unknown dimer interface. Here, by using cysteine cross-linking, we identify the fourth transmembrane segment (TM4) as a symmetrical dimer interface in the dopamine D2 receptor. Cross-linking is unaffected by ligand binding, and ligand binding and receptor activation are unaffected by cross-linking, suggesting that the receptor is a constitutive dimer. The accessibility of adjacent residues in TM4, however, is affected by ligand binding, implying that the interface has functional significance.     

The Aer protein of Escherichia coli forms a homodimer independent of the signaling domain and flavin adenine dinucleotide binding

In vivo cross-linking between native cysteines in the Aer receptor of Escherichia coli showed dimer formation at the membrane anchor and in the putative HAMP domain. Dimers also formed in mutants that did not bind flavin adenine dinucleotide and in truncated peptides without a signaling domain and part of the HAMP domain.     

The purified activated glucocorticoid receptor is a homodimer

The structure of purified preparations of activated (DNA-binding) glucocorticoid receptor (GR) was analyzed in the presence or absence of DNA. A 35-base pair DNA fragment harboring a strong GR-binding site from the mouse mammary tumor virus promoter (-189/-166) was used for stoichiometric analysis of the GR.DNA complex. Glycerol gradient centrifugation was utilized in order to separate the 6 S GR.DNA complex from the 4 S GR and the 3 S DNA fragment. Synthetic glucocorticoid [3H]triamcinolone acetonide bound to GR and 32P-5'-end-labeled DNA fragment were used as probes for quantitation of each component. Such experiments demonstrated that two hormone molecules (two 87.5-kDa GR peptides) are associated with each cognate DNA site. Quantitative DNase I footprinting confirmed this result. The formation of the GR.DNA complex was ligand-dependent, but once formed the complex remained stable after ligand dissociation. Incubation of GR with 0.01-0.1% (w/v) glutaraldehyde resulted in a shift in its sedimentation rate from 4 to 6 S. Gel filtration chromatography of glutaraldehyde-treated GR resulted in a complex of slightly larger size than the gamma-globulin standard (158 kDa). Gel filtration of GR without glutaraldehyde treatment gave the identical result. This suggests that a GR multimer, probably a homodimer, is stable during gel filtration chromatography but needs to be stabilized by glutaraldehyde cross-linking or DNA during glycerol gradient centrifugation. We conclude that the activated GR exists as a homodimer when unbound as well as when bound to DNA.     

The thyroid hormone-inactivating deiodinase functions as a homodimer

The type 3 deiodinase (D3) inactivates thyroid hormone action by catalyzing tissue-specific inner ring deiodination, predominantly during embryonic development. D3 has gained much attention as a player in the euthyroid sick syndrome, given its robust reactivation during injury and/or illness. Whereas much of the structure biology of the deiodinases is derived from studies with D2, a dimeric endoplasmic reticulum obligatory activating deiodinase, little is known about the holostructure of the plasma membrane resident D3, the deiodinase capable of thyroid hormone inactivation. Here we used fluorescence resonance energy transfer in live cells to demonstrate that D3 exists as homodimer. While D3 homodimerized in its native state, minor heterodimerization was also observed between D3:D1 and D3:D2 in intact cells, the significance of which remains elusive. Incubation with 0.5-1.2 m urea resulted in loss of D3 homodimerization as assessed by bioluminescence resonance energy transfer and a proportional loss of enzyme activity, to a maximum of approximately 50%. Protein modeling using a D2-based scaffold identified potential dimerization surfaces in the transmembrane and globular domains. Truncation of the transmembrane domain (DeltaD3) abrogated dimerization and deiodinase activity except when coexpressed with full-length catalytically inactive deiodinase, thus assembled as DeltaD3:D3 dimer; thus the D3 globular domain also exhibits dimerization surfaces. In conclusion, the inactivating deiodinase D3 exists as homo- or heterodimer in living intact cells, a feature that is critical for their catalytic activities.     

Characterization of wild-type and mutant forms of human tryptophan hydroxylase 2

Tryptophan hydroxylase (TPH) catalyses the rate-limiting step in the biosynthesis of serotonin. In vertebrates, the homologous genes tph1 and tph2 encode two different enzymes with distinct patterns of expression, enzyme kinetics and regulation. Variants of TPH2 have recently reported to be associated with reduced serotonin production and behavioural alterations in man and mice. We have produced the human forms of these enzymes in Esherichia coli and in human embryonic kidney cell lines (HEK293) and examined the effects of mutations on their heterologous expression levels, solubility, thermal stability, secondary structure, and catalytic properties. Pure human TPH2 P449R (corresponds to mouse P447R) had comparable catalytic activity (V(max)) and solubility relative to the wild type, but had decreased thermal stability; whereas human TPH2 R441H had decreased activity, solubility and stability. Thus, we consider the variations in kinetic values between wild-type and TPH2 mutants to be of secondary importance to their effects on protein stability and solubility. These findings provide potential molecular explanations for disorders related to the central serotonergic system, such as depression or suicidal behaviour.     

Human macrophages degrade tryptophan upon induction by interferon-gamma

Human peripheral blood mononuclear cells, monocytes-macrophages and T-cells were stimulated with human recombinant interferon-gamma, interferon-alpha and phytohemagglutinin. The culture supernatants were analyzed for tryptophan, kynurenine, 3-hydroxyanthranilic acid, anthranilic acid and neopterin by high performance liquid chromatography. Tryptophan was decreased and the four other compounds were increased in supernatants of peripheral blood mononuclear cells activated by interferon-gamma (250 U/ml), interferon-alpha (10.000 U/ml) and phytohemagglutinin (1 microgram/ml). After splitting of peripheral blood mononuclear cells by adherence, the monocytes and macrophages but not the T-cells degraded tryptophan upon stimulation by interferon-gamma in a dose dependent manner. Supernatants of phytohemagglutinin stimulated but not of resting T-cells were found to induce tryptophan degradation by macrophages, the active principle being neutralized by an antiserum for interferon-gamma. Thus phytohemagglutinin acts by activating T-cells to release interferon-gamma which in turn induces macrophages to degrade tryptophan. In all experiments the appearance of neopterin in the culture media was correlated to the observed tryptophan degradation.     

Human plasma lipofuscin melanins formed from tryptophan metabolites

Incubation of human plasma with the tryptophan metabolites 3-hydroxy-DL-kynurenine (3HK) or 3-hydroxyanthranilic acid (3HAA) yields soluble brown pigments with the fluorescence spectrophotometric and paper chromatographic properties of plasma lipofuscin (PL). An alternative to the recognized tyrosine pathway of melanogenesis is therefore demonstrated in human plasma.