Histidine-rich Motif Research Articles

Crystal Structure of Imidazole Glycerol-phosphate Dehydratase: DUPLICATION OF AN UNUSUAL FOLD

Imidazole glycerol-phosphate dehydratase (IGPD) catalyzes the sixth step of histidine biosynthesis. The enzyme is of fundamental biochemical interest, because it catalyzes removal of a non-acidic hydrogen atom in the dehydration reaction. It is also a potential target for development of herbicides. IGPD is a metalloenzyme in which transition metals induce aggregation and are required for catalysis. Addition of 1 equivalent of Mn(2+)/subunit is shown by analytical ultracentrifugation to induce the formation of 24-mers from trimeric IGPD. Two histidine-rich motifs may participate in metal binding and aggregation. The 2.3-A crystal structure of metal-free trimeric IGPD from the fungus Filobasidiella neoformans reveals a novel fold containing an internal repeat, apparently the result of gene duplication. The 95-residue alpha/beta half-domain occurs in a few other proteins, including the GHMP kinase superfamily (galacto-homoserine-mevalonate-phosphomevalonate), but duplication to form a compact domain has not been seen elsewhere. Conserved residues cluster at two types of sites in the trimer, each site containing a conserved histidine-rich motif. A model is proposed for the intact, active 24-mer in which all highly conserved residues, including the histidine-rich motifs in both the N- and C-terminal halves of the polypeptide, cluster at a common site between trimers. This site is a candidate for the active site and also for metal binding leading to aggregation of trimers. The structure provides a basis for further studies of enzyme function and mechanism and for development of more potent and specific herbicides.

The variant ‘his-box’ of the C18-Δ9-PUFA-specific elongase IgASE1 from Isochrysis galbana is essential for optimum enzyme activity

IgASE1, a C18-Δ9-polyunsaturated fatty acid-specific fatty acid elongase component from Isochrysis galbana, contains a variant histidine box (his-box) with glutamine replacing the first histidine of the conserved histidine-rich motif present in all other known equivalent proteins. The importance of glutamine and other variant amino acid residues in the his-box of IgASE1 was determined by site-directed mutagenesis. Results showed that all the variation in amino acid sequence between this motif in IgASE1 and the consensus sequences of other elongase components was required for optimum enzyme activity. The substrate specificity was shown to be unaffected by these changes suggesting that components of the his-box are not directly responsible for substrate specificity.

Merging of multiple signals regulating delta9 fatty acid desaturase gene transcription in Saccharomyces cerevisiae.

Fatty acid desaturation, which requires molecular oxygen (O2) as an electron acceptor, is catalyzed by delta9 fatty acid desaturase, which is encoded by OLE1 in Saccharomyces cerevisiae. Transcription of the OLE1 gene is repressed by unsaturated fatty acids (UFAs) and activated by hypoxia and low temperatures via the endoplasmic reticulum membrane protein Mga2p. We previously reported the isolation of the nfo3-1 (negative factor for OLE1) mutant, which exhibits enhanced expression of OLE1 in the presence of UFA and under aerobic conditions. In this work, we demonstrated that the NFO3 gene is identical to OLE1 and that the nfo3-1 mutation (renamed ole1-101) alters arginine-346, in the vicinity of the conserved histidine-rich motif essential for the catalytic function of the Ole1 protein, to lysine. The ratio of UFAs to total fatty acids in the ole1-101 mutant was 60%, compared to 75% in the wild type, suggesting that the reduction in relative levels of intracellular UFAs activates OLE1 transcription. However, in ole1-101 cells grown in the presence of oleic acid, the level of OLE1 expression remained high, although the relative amount of UFAs in the ole1-101 mutant cells was almost the same as that in wild-type cells growing under the same conditions. By contrast, when cells were grown with linoleic acid, which has a lower melting point than oleic acid, the elevation of the OLE1 expression level due to the ole1-101 mutation was almost completely suppressed. These observations suggest that the ole1-101 cells activate OLE1 transcription by sensing not only the intracellular UFA level, but also membrane fluidity or the nature of the UFA species itself. Furthermore, we found that not only the fatty acid- regulated (FAR) element but also the O2- regulated (O2R) element in the OLE1 promoter was involved in the activation of OLE1 transcription by the ole1-101 mutation, and that the effects of the low-oxygen signal and the ole1-101-generated signal on OLE1 expression were not additive. Taken together, these findings suggest that signals associated with hypoxia, low temperatures and intracellular UFA depletion activate OLE1 transcription by a common pathway.

Specificity of interactions of galectin-3 with Chrp, a cysteine- and histidine-rich cytoplasmic protein

Earlier work described the cloning of a gene from murine 3T3 cells encoding a cytoplasmic protein Chrp containing a cysteine- and histidine-rich motif characteristic of Zn-finger proteins. The interaction of Chrp with murine galectin-3 first became evident in a yeast two-hybrid screen, but it was also observed in co-precipitation experiments from 3T3 cell lysates. Here, the formation of equimolar complexes by murine Chrp and hamster galectin-3 is shown. Moreover, we found that Chrp binds to the carbohydrate-recognition domain (CRD) of hamster galectin-3 and not to the N-terminal domain carrying the proline- and glycine-rich repeats characteristic of galectin-3 and absent in other galectins. However, galectin-1 does not bind to Chrp, although its CRD is homologous to the galectin-3 CRD. Finally, we report that galectin-3, in a complex with Chrp, binds to laminin in surface plasmon resonance experiments with similar kinetics and affinity as it does in the free state. The formation of higher-order complexes containing these proteins and additional binding partners may be relevant to cytoplasmic functions involving galectin-3.

Production of cobalamin and sirohaem in Bacillus megaterium: an investigation into the role of the branchpoint chelatases sirohydrochlorin ferrochelatase (SirB) and sirohydrochlorin cobalt chelatase (CbiX).

One of the four operons required for cobalamin biosynthesis in Bacillus megaterium is also associated with sirohaem synthesis, and contains three genes, sirA, sirB and sirC. By undertaking functional complementation experiments and in vitro assays using recombinantly produced enzymes, we have been able to demonstrate that (1) SirA acts as a uroporphyrinogen III methyltransferase, transforming uroporphyrinogen III into precorrin-2, (2) SirC acts as an NAD(+) dehydrogenase, responsible for the oxidation of precorrin-2 into sirohydrochlorin, and (3) SirB acts as a ferrochelatase, responsible for the insertion of a ferrous ion into sirohydrochlorin to give sirohaem. Comparative sequence analysis reveals that the primary structure of SirB is highly similar to that of the cobalt chelatase involved in cobalamin biosynthesis in Bacillus megaterium, CbiX, with the exception that CbiX contains a C-terminal histidine-rich motif. Surprisingly, CbiX has been shown (using EPR) to contain a 4Fe-4S centre, a redox centre that is absent from SirB.

Δ6-Desaturase of Mucor rouxii with High Similarity to Plant Δ6-Desaturase and Its Heterologous Expression in Saccharomyces cerevisiae

Gamma-linolenic acid (GLA, γ-C18:3) is an essential fatty acid that plays a vital role in biological structures and cellular functions. Based on available sequence information and using polymerase chain reaction (PCR) technique, we cloned from the fungus Mucor rouxii the entire coding sequence of a Δ6-desaturase enzyme, which is responsible for the transformation of linoleic acid into GLA. The deduced amino acid sequence of M. rouxii gene showed the highest homology with the plant Δ6-desaturase. It comprises the characteristics of membrane-bound desaturases, including histidine-rich motifs and hydrophobic regions. A cytochrome b5-like domain was observed at the N-terminus. In addition to three conserved histidine-rich motifs, we found an additional histidine-rich motif, HKHHSH, downstream of the cytochrome b5-like domain, which is not present in previously cloned Δ6-desaturase genes. Heterologous expression of the M. rouxii cDNA in Saccharomyces cerevisiae resulted in the synthesis and accumulation of GLA.

The effect of Zn 2+ on the secondary structure of a histidine-rich fusogenic peptide and its interaction with lipid membranes

Membrane fusion between uncharged lipid vesicles can be triggered by the peptide sequence ‘B18’ from the fertilization protein ‘bindin’, but it only proceeds efficiently in the presence of Zn 2+ ions. We studied (i) the interaction of Zn 2+ with the fusogenic peptide B18, (ii) the binding of B18 to 1-palmitoyl-2-oleoylglycero-3-phosphocholine (POPC), and (iii) the ternary system POPC/B18/Zn 2+. The complex formation of Zn 2+ with the central histidine-rich motif of B18 appears to shift the secondary structure away from a β-sheet towards an α-helical conformation. Here we observe for the first time an essentially α-helical structure of the peptide when immersed in POPC bilayers which appears to represent its functional fusogenic state. Infrared linear dichroism suggests a peripheral, oblique insertion mode of B18, mediated by the hydrophobic patches along one side of the amphipathic peptide. Furthermore, the hydration level of the peptide is reduced, suggesting that the hydrophobic region of the bilayer is involved in the lipid/peptide interactions. The hydration capacity of the POPC/B18/Zn 2+ system is distinctly smaller than that of POPC/Zn 2+ without peptide. The accompanying decrease in the number of tightly bound water molecules per lipid can be interpreted as a reduction in the repulsive ‘hydration’ forces, which usually prevent the spontaneous fusion of lipid vesicles. Binding of the B18 peptide in the presence of Zn 2+ effectively renders the membrane surface more hydrophobic, thus allowing fusion to proceed.

Structure analysis of a fusogenic peptide sequence from the sea urchin fertilization protein bindin.

The structure of "B18", an 18-residue fusogenic peptide from the sea urchin fertilization protein bindin, was investigated in several membrane-mimicking environments with circular dichroism and nuclear magnetic resonance spectroscopy. The fully conserved peptide sequence represents the minimal functional part of the 24 kDa protein, which can bind to membranes and induce fusion of lipid vesicles. The B18 peptide undergoes a coil-helix transition in the presence of TFE, showing a transient tendency to self-associate. Its NMR structure in 30% TFE exhibits two helical regions at either side, connected by a flexible loop. In DPC and SDS detergent micelles, this loop becomes distinctly bent, presumably due to the high degree of curvature of the micelles. The loop contains a histidine-rich motif for binding zinc, which is required for the fusogenic function of the peptide. Therefore, we monitored the structural response of B18 and of recombinant bindin toward this ion. Like TFE, and in a mutually cooperative manner, zinc induces a partially helical structure in both the peptide and the protein. Complex formation via the histidine residues rigidifies the flexible loop and is accompanied by self-association of the molecules. The data suggest that the zinc-bound functional state is a continuous amphipathic alpha-helix, bearing some resemblance to a leucine zipper. Two hydrophobic patches on one face could favorably penetrate into a membrane, while two arginines on the other face could interact with lipid phosphate groups. The three-dimensional model of the B18 sequence thus contributes to a better understanding of peptide-induced vesicle fusion in general, and of the lipid-protein interactions of sperm bindin in particular.

Membrane Fusion Is Induced by a Distinct Peptide Sequence of the Sea Urchin Fertilization Protein Bindin

Fertilization in the sea urchin is mediated by the membrane-associated acrosomal protein bindin, which plays a key role in the adhesion and fusion between sperm and egg. We have investigated the structure/function relationship of an 18-amino acid peptide fragment "B18," which represents the minimal membrane binding motif of the protein and resembles a putative fusion peptide. The peptide was found to mimic the behavior of its parent protein bindin with respect to (a) its high affinity for lipid bilayers, (b) the ability to aggregate and fuse vesicles, (c) the binding of Zn2+ by a histidine-rich motif, (d) the tendency to self-assemble, and (e), as indicated earlier, the adhesion to cell surface polysaccharides. Fluorescence and light scattering assays were used here to monitor peptide-induced lipid mixing, leakage, and aggregation of large unilamellar sphingomyelin/cholesterol vesicles. For these activities, B18 requires the presence of Zn2+ ions, with which it forms oligomeric complexes and assumes a partially alpha-helical conformation, as observed by circular dichroism. We conclude that aggregation and fusion involves a "trans-complex" between peptides on apposing vesicles that are connected by Zn2+ bridges.

Spectroscopic characterization of metal binding by Klebsiella aerogenes UreE urease accessory protein

The urease accessory protein encoded by ureE from Klebsiella aerogenes is proposed to function in Ni(II) delivery to the urease apoprotein. Wild-type UreE contains a histidine-rich region at its carboxyl terminus and binds 5–6 Ni per dimer, whereas the functionally active but truncated H144*UreE lacks the histidine-rich motif and binds only two Ni per dimer [Brayman TG, Hausinger RP (1996) J Bacteriol 178 : 5410-5416]. For both proteins, Cu(II), Co(II), and Zn(II) ions compete for the Ni-binding sites. In order to characterize the coordination environments of bound metals, especially features that are unique to Ni, the Ni-, Cu-, and Co-bound forms of H144*UreE were studied by a combination of EPR, ESEEM, hyperfine-shifted 1H-NMR, XAS, and RR spectroscopic methods. For each metal ion, the two binding sites per homodimer were spectroscopically distinguishable. For example, the two Ni-binding sites each have pseudo-octahedral geometry in an N/O coordination environment, but differ in their number of histidine donors. The two Cu-binding sites have tetragonal geometry with two histidine donors each; however, the second Cu ion is bound by at least one cysteine donor in addition to the N/O-type donors found for the first Cu ion. Two Co ions are bound to H144*UreE in pseudo-octahedral geometry with N/O coordination, but the sites differ in the number of histidine donors that can be observed by NMR. The differences in coordination for each type of metal ion are relevant to the proposed function of UreE to selectively facilitate Ni insertion into urease in vivo.

A novel phloem-specific gene is expressed preferentially in aerial portions of Vicia faba

We have isolated a gene from bean (Vicia faba L.), called Vein1, that encodes a novel protein. The Vein1 cDNA was isolated as a result of a differential screen for genes that are expressed in leaves but not in the most common cell type, the mesophyll cell. Northern blot analysis revealed that Vein 1 transcripts are differentially expressed in the plant with expression in leaves, stems and sepals but not in petals, mesophyll cells or roots. In situ hybridization studies of stem and leaf sections indicate that the expression of Vein1 is localized to the phloem tissue. Interestingly, Vein1 was differentially expressed in stem tissue with the highest expression in the oldest internodes. The deduced Vein1 protein sequence does not share homology with any known protein sequences. The 17 kDa Vein1 protein is highly hydrophilic and contains a histidine-rich motif, where six out of seven amino acids are histidines. The function of Vein1 is unknown, although the expression patterns suggests that it may play a role in mature phloem tissue in the aerial parts of the plant.

Characterisation of potato ( Solanum tuberosum L.) extensins: a novel extensin-like cDNA from dormant tubers

A cDNA library from dormant potato tubers was screened for extensin (Ext)-like cDNAs (PTEL). The cDNAs recovered were partially sequenced and could be divided into two classes. One class, containing the repeating amino acid (aa) motifs, SPPPPSPKYVYK and SPPPPSPSPPPPYYYK, constitutes the potato counterpart of the tomato Ext-encoding gene previously isolated by Zhou et al. [Plant Mol. Biol. 20 (1992) 5–17]. A full-length cDNA from the second class was fully sequenced. The product of this cDNA has the repetitive sequence and proline-rich composition typical of Ext. Features worthy of note are the signal peptide sequence, which shares close homology with signal sequences of a number of Ext of diverse origin, and the presence of a novel, histidine-rich motif (SPPPHHHHPVYK) repeated within the sequence. Wounding of the potato tuber causes a marked increase in Ext-like mRNAs, although the species induced are not those present in dormant tissue.