The crystal structure of a truncated Aer2, a signal transducer protein from Pseudomonas aeruginosa, consisting of the heme-containing PAS and di-HAMP domains revealed that a distal tryptophan residue ...(Trp283) plays an important role in stabilizing the heme-bound O(2) and intra-molecular signal transduction upon O(2) binding.
Hepatic stellate cells (HSCs) are the primary cell type in liver fibrosis, a significant global health care burden. Cytoglobin (CYGB), a globin family member expressed in HSCs, inhibits HSC ...activation and reduces collagen production. We studied the antifibrotic properties of globin family members hemoglobin (HB), myoglobin (MB), and neuroglobin (NGB) in comparison with CYGB.
We characterized the biological activities of globins in cultured human HSCs (HHSteCs) and their effects on carbon tetrachloride (CCl4)-induced cirrhosis in mice. All globins demonstrated greater antioxidant capacity than glutathione in cell-free systems. Cellular fractionation revealed endocytosis of extracellular MB, NGB, and CYGB, but not HB; endocytosed globins localized to intracellular membranous, cytoplasmic, and cytoskeletal fractions. MB, NGB, and CYGB, but not HB, scavenged reactive oxygen species generated spontaneously or stimulated by H2O2 or transforming growth factor β1 in HHSteCs and reduced collagen 1A1 production via suppressing COL1A1 promoter activity. Disulfide bond-mutant NGB displayed decreased heme and superoxide scavenging activity and reduced collagen inhibitory capacity. RNA sequencing of MB- and NGB-treated HHSteCs revealed downregulation of extracellular matrix–encoding and fibrosis-related genes and HSC deactivation markers. Upregulation of matrix metalloproteinase (MMP)-1 was observed following MB and NGB treatment, and MMP-1 knockdown partially reversed globin-mediated effects on secreted collagen. Importantly, administration of MB, NGB, and CYGB suppressed CCl4-induced mouse liver fibrosis.
These findings revealed unexpected roles for MB and NGB in deactivating HSCs and inhibiting liver fibrosis development, suggesting that globin therapy may represent a new strategy for combating fibrotic liver disease.
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Myoglobin, neuroglobin, and cytoglobin, but not hemoglobin:•Internalize into human hepatic stellate cells via endocytosis pathway.•Scavenge intracellular reactive oxidative species.•Suppress COL1A1 promoter activity and promote matrix metaloproteinase-1 secretion.•Suppress carbon tetrachloride-induced mouse liver fibrosis.
We have studied the structural and enzymatic properties of a diguanylate cyclase from an obligatory anaerobic bacterium Desulfotalea psychrophila, which consists of the N-terminal sensor domain and ...the C-terminal diguanylate cyclase domain. The sensor domain shows an amino acid sequence homology and spectroscopic properties similar to those of the sensor domains of the globin-coupled sensor proteins containing a protoheme. This heme-containing diguanylate cyclase catalyzes the formation of cyclic di-GMP from GTP only when the heme in the sensor domain binds molecular oxygen. When the heme is in the ferric, deoxy, CO-bound, or NO-bound forms, no enzymatic activity is observed. Resonance Raman spectroscopy reveals that Tyr55 forms a hydrogen bond with the heme-bound O2, but not with CO. Instead, Gln81 interacts with the heme-bound CO. These differences of a hydrogen bonding network will play a crucial role for the selective O2 sensing responsible for the regulation of the enzymatic activity.
Cytoglobin (Cgb), a newly discovered member of the vertebrate globin family, binds O2 reversibly via its heme, as is the case for other mammalian globins (hemoglobin (Hb), myoglobin (Mb) and ...neuroglobin (Ngb)). While Cgb is expressed in various tissues, its physiological role is not clearly understood. Here, the X-ray crystal structure of wild type human Cgb in the ferric state at 2.4Å resolution is reported. In the crystal structure, ferric Cgb is dimerized through two intermolecular disulfide bonds between Cys38(B2) and Cys83(E9), and the dimerization interface is similar to that of lamprey Hb and Ngb. The overall backbone structure of the Cgb monomer exhibits a traditional globin fold with a three-over-three α-helical sandwich, in which the arrangement of helices is basically the same among all globins studied to date. A detailed comparison reveals that the backbone structure of the CD corner to D helix region, the N terminus of the E-helix and the F-helix of Cgb resembles more closely those of pentacoordinated globins (Mb, lamprey Hb), rather than hexacoordinated globins (Ngb, rice Hb). However, the His81(E7) imidazole group coordinates directly to the heme iron as a sixth axial ligand to form a hexcoordinated heme, like Ngb and rice Hb. The position and orientation of the highly conserved residues in the heme pocket (Phe(CD1), Val(E11), distal His(E7) and proximal His(F8)) are similar to those of other globin proteins. Two alternative conformations of the Arg84(E10) guanidium group were observed, suggesting that it participates in ligand binding to Cgb, as is the case for Arg(E10) of Aplysia Mb and Lys(E10) of Ngb. The structural diversities and similarities among globin proteins are discussed with relevance to molecular evolutionary relationships.
Dietary iron absorption is regulated by duodenal cytochrome
(Dcytb), an integral membrane protein that catalyzes reduction of nonheme Fe
by electron transfer from ascorbate across the membrane. This ...step is essential to enable iron uptake by the divalent metal transporter. Here we report the crystallographic structures of human Dcytb and its complex with ascorbate and Zn
. Each monomer of the homodimeric protein possesses cytoplasmic and apical heme groups, as well as cytoplasmic and apical ascorbate-binding sites located adjacent to each heme. Zn
coordinates to two hydroxyl groups of the apical ascorbate and to a histidine residue. Biochemical analysis indicates that Fe
competes with Zn
for this binding site. These results provide a structural basis for the mechanism by which Fe
uptake is promoted by reducing agents and should facilitate structure-based development of improved agents for absorption of orally administered iron.
Cytoglobin (Cgb) represents a fourth member of the globin superfamily in mammals, but its function is unknown. Site-directed mutagenesis, in which six histidine residues were replaced with alanine, ...was carried out, and the results indicate that the imidazoles of His81 (E7) and His113 (F8) bind to the heme iron as axial ligands in the hexacoordinate and the low-spin state. The optical absorption, resonance Raman, and IR spectral results are consistent with this conclusion. The redox potential measurements revealed an E‘ of 20 mV (vs NHE) in the ferric/ferrous couple, indicating that the imidazole ligands of His81 and His113 are electronically neutral. On the basis of the νFe - CO and νC - O values in the resonance Raman and infrared spectra of the ferrous−CO complexes of Cgb and its mutants, it was found that CO binds to the ferrous iron after the His81 imidazole is dissociated, and three conformers are present in the resultant CO coordination structure. Two are in closed conformations of the heme pocket, in which the bound CO ligand interacts with the dissociated His81 imidazole, while the third is in an open conformation. The νFe - O 2 in the resonance Raman spectra of oxy Cgb can be observed at 572 cm-1, suggesting a polar heme environment. These structural properties of the heme pocket of Cgb are discussed with respect to its proposed in vivo oxygen storage function.
Cytoglobin (Cgb) and neuroglobin (Ngb) are the first examples of hexacoordinated globins from humans and other vertebrates in which a histidine (His) residue at the sixth position of the heme iron is ...an endogenous ligand in both the ferric and ferrous forms. Static and time-resolved resonance Raman and FT-IR spectroscopic techniques were applied in examining the structures in the heme environment of these globins. Picosecond time-resolved resonance Raman (ps-TR3) spectroscopy of transient five-coordinate heme species produced by the photolysis of carbon monoxide (CO) adducts of Cgb and Ngb showed Fe−His stretching (νFe - His) bands at 229 and 221 cm-1, respectively. No time-dependent shift in the νFe - His band of Cgb and Ngb was detected in the 20−1000 ps time domain, in contrast to the case of myoglobin (Mb). These spectroscopic data, combined with previously reported crystallographic data, suggest that the structure of the heme pocket in Cgb and Ngb is altered upon CO binding in a manner different from that of Mb and that the scales of the structural alteration are different for Cgb and Ngb. The structural property of the heme distal side of the ligand-bound forms was investigated by observing the sets of (νFe - CO, νC - O, δFe - C - O) and (νFe - NO, νN - O, δFe - N - O) for the CO and nitric oxide (NO) complexes of Cgb and Ngb. A comparison of the spectra of some distal mutants of Cgb (H81A, H81V, R84A, R84K, and R84T) and Ngb (H64A, H64V, K67A, K67R, and K67T) showed that the CO adducts of Cgb and Ngb contained three conformers and that the distal His (His81 in Cgb and His64 in Ngb) mainly contributes to the interconversion of the conformers. These structural characteristics of Cgb and Ngb are discussed in relation to their ligand binding and physiological properties.
Proteins containing DM9 motifs, which were originally identified in the Drosophila melanogaster genome, are widely distributed in various organisms and are assumed to be involved in their innate ...immune response. In this study, we produced a recombinant protein of CG13321 (rCG13321) from D. melanogaster, which consists of four DM9 motifs, in Escherichia coli cells. In affinity chromatography using a mannose-immobilized column, rCG13321 exhibited mannose-binding ability and was separated into high-affinity and low-affinity fractions, named HA and LA, respectively, based on its binding ability to the column. In addition to having a higher affinity for the column, HA exhibited self-oligomerization ability, suggesting slight differences in tertiary structure. Both LA and HA showed hemagglutinating activity and were able to agglutinate an oligomannose-containing dendrimer, indicating that they have multiple carbohydrate-binding sites. Glycan array analysis suggested that rCG13321 primarily recognizes D-mannose and D-rhamnose through hydrogen bonding with the 2-, 3-, and 4-hydroxy groups. Isothermal titration calorimetry demonstrated that rCG13321 has a comparable affinity to typical lectins. These findings suggest that CG13321 functions as a carbohydrate-binding protein or lectin that recognizes mannose and related carbohydrate-containing molecules on the surface of foreign organisms as a pattern recognition molecule.
HemAT-Bs is a heme-based signal transducer protein responsible for aerotaxis. Time-resolved ultraviolet resonance Raman (UVRR) studies of wild-type and Y70F mutant of the full-length HemAT-Bs and the ...truncated sensor domain were performed to determine the site-specific protein dynamics following carbon monoxide (CO) photodissociation. The UVRR spectra indicated two phases of intensity changes for Trp, Tyr, and Phe bands of both full-length and sensor domain proteins. The W16 and W3 Raman bands of Trp, the F8a band of Phe, and the Y8a band of Tyr increased in intensity at hundreds of nanoseconds after CO photodissociation, and this was followed by recovery in ∼50 μs. These changes were assigned to Trp-132 (G-helix), Tyr-70 (B-helix), and Phe-69 (B-helix) and/or Phe-137 (G-helix), suggesting that the change in the heme structure drives the displacement of B- and G-helices. The UVRR difference spectra of the sensor domain displayed a positive peak for amide I in hundreds of nanoseconds after photolysis, which was followed by recovery in ∼50 μs. This difference band was absent in the spectra of the full-length protein, suggesting that the isolated sensor domain undergoes conformational changes of the protein backbone upon CO photolysis and that the changes are restrained by the signaling domain. The time-resolved difference spectrum at 200 μs exhibited a pattern similar to that of the static (reduced − CO) difference spectrum, although the peak intensities were much weaker. Thus, the rearrangements of the protein moiety toward the equilibrium ligand-free structure occur in a time range of hundreds of microseconds.
HemAT is an O2 sensor and functions as a signal transducer for aerotaxis.
The UV resonance Raman spectra indicated two phases of intensity changes for the Trp, Tyr, and Phe bands of proteins.
Changes in the heme structure drive displacements of the B- and G-helices upon CO photolysis.
Understanding the communication pathway from the sensor to signaling domain of HemAT-Bs.
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