Characterization of DrosophilaHemoglobin

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Hemoglobins at high concentration have been isolated long ago from some insect larvae living in hypoxic environments. Conversely, a monomeric hemoglobin has been discovered recently in the fruit fly Drosophila melanogaster as intracellular protein expressed both in larvae and in the adult fly. Such a finding indicates that the oxygen supply in insects may be more complex than previously thought, relying not only on O 2 diffusion through the tubular tracheal system, but also on carriermediated transport and storage. We present here the crystal structure of recombinant D. melanogaster hemoglobin at 1.20 Å resolution. Spectroscopic data show that the protein displays a hexacoordinated heme, whose axial ligands are the proximal and distal His residues. Such bis-His ligation of the heme has sizable effects on the protein local structure. Three protein matrix cavities, comparable in size but not in topological locations with those of sperm whale myoglobin, are spread through the protein matrix; one of these can host a xenon atom. Additionally, D. melanogaster hemoglobin binds one molecule of 3-(cyclohexylamino)propanesulfonic acid (CAPS) buffer at a surface pocket, next to the EF hinge. Despite the high resolution achieved, no sequence/structure features specifically supporting the heme hexa-to pentacoordination transition required for diatomic ligand binding could be recognized.

Section: Or Hemocyanins (Hcs) (2-4)mentioning

confidence: 99%

Bishistidyl Heme Hexacoordination, a Key Structural Property in Drosophila melanogaster Hemoglobin

Sanctis¹,

Dewilde

Vonrhein

et al. 2005

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“…Similar hexacoordinate globins have also been isolated from bacteria and unicellular eukaryotes (truncated hemoglobins (15,16)) as well as from plants (nonsymbiotic hemoglobins (17)(18)(19)) and insects (20). The exogenous ligand replaces the endogenous one upon binding at the sixth coordination, and therefore, hexacoordination has been considered as a novel mechanism for regulating ligand binding affinity to the heme (13).…”

mentioning

confidence: 94%

Structural Dynamics in the Active Site of Murine Neuroglobin and Its Effects on Ligand Binding

Nienhaus

Kriegl

Nienhaus

2004

132

We have examined the effects of active site residues on ligand binding to the heme iron of mouse neuroglobin using steady-state and time-resolved visible spectroscopy. Absorption spectra of the native protein, mutants H64L and K67L and double mutant H64L/K67L were recorded for the ferric and ferrous states over a wide pH range (pH 4 -11), which allowed us to identify a number of different species with different ligands at the sixth coordination, to characterize their spectroscopic properties, and to determine the pK values of active site residues. In flash photolysis experiments on CO-ligated samples, reaction intermediates and the competition of ligands for the sixth coordination were studied. These data provide insights into structural changes in the active site and the role of the key residues His 64 and Lys 67 . His 64 interferes with exogenous ligand access to the heme iron. Lys 67 sequesters the distal pocket from the solvent. The heme iron is very reactive, as inferred from the fast ligand binding kinetics and the ability to bind water or hydroxyl ligands to the ferrous heme. Fast bond formation favors geminate rebinding; yet the large fraction of bimolecular rebinding observed in the kinetics implies that ligand escape from the distal pocket is highly efficient. Even slight pH variations cause pronounced changes in the association rate of exogenous ligands near physiological pH, which may be important in functional processes.

“…As a first step toward this goal, we conducted a genome-wide computational survey for potential HIF target genes in both Drosophila and C. elegans as characterized by the presence of reportedly functional HRE sequences within 600 nucleotides upstream from start codons and downstream from stop codons (72). One of the genes we retrieved as a multi-HREflanked ORF was glob1, the recently discovered (73) singlecopy gene for Drosophila globin that generates a high O 2 affinity (P 50 ϳ 0.1-0.2 torr) hemoglobin in association with the tracheal system and the fat body of fly embryos, larvae, and adults (74). Fig.…”

Section: Resultsmentioning

confidence: 99%

“…3a shows a diagram of the globin gene, including TATA box-like transcription start sites and potential HIFbinding HREs in 5Ј-and 3Ј-flanks as well as the coding region. At least four different globin transcripts from two different promoters are produced in vivo (74). All four mRNAs share the same coding region (exons 1-3) (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Regulation of Drosophila Hypoxia-inducible Factor (HIF) Activity in SL2 Cells

Gorr

Tomita

Wappner

et al. 2004

Although hypoxia-inducible factor-␣ (HIF␣) subunitspecific hydroxylation and proteolytic breakdown explain the binary switch between the presence (hypoxia) and absence (normoxia) of HIFs, little is known of the mechanisms that fine-tune HIF activity under constant, rather than changing, oxygen tensions. Here, we report that the Drosophila HIF␣ homolog, the basic helix-loophelix/PAS protein Sima (Similar), in hypoxic cultures of SL2 cells is expressed in full-length (fl) and splice variant (sv) isoforms. The following evidence supports the role of flSima as functional HIF␣ and the role of SL2 HIF as a transcriptional activator or suppressor. The pO 2 dependence of Sima abundance matched that of HIF activity. HIF-dependent changes in candidate target gene expression were detected through variously effective stimuli: hypoxia (strong) > iron chelation, e.g. desferrioxamine (moderate) > > transition metals, e.g. cobalt Ӎ normoxia (ineffective). Sima overexpression augmented hypoxic induction or suppression of different targets. In addition to the full-length exon 1-12 transcript yielding the 1510-amino acid HIF␣ homolog, the sima gene also expressed, specifically under hypoxia, an exon 1-7/12 splice variant, which translated into a 426-amino acid Sima truncation termed svSima. svSima contains basic helix-loop-helix and PAS sequences identical to those of flSima, but, because of deletion of exons 8 -11, lacks the oxygen-dependent degradation domain and nuclear localization signals. Overexpressed svSima failed to transactivate reporter genes. However, it attenuated HIF (Sima⅐Tango)-stimulated reporter expression in a dose-dependent manner. Thus, svSima has the potential to regulate Drosophila HIF function under steady and hypoxic pO 2 by creating a cytosolic sink for the Sima partner protein Tango.