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Cited by 6Iron Metabolism and DisordersProtein Structure and DynamicsInsect Resistance and Genetics

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Comparison of insect and human cytochrome b561 proteins: Insights into candidate ferric reductases in insects

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Holst, Jessica D., Murphy, Laura G., Gorman, Maureen J., Ragan, Emily J.·2023·PLOS ONE
DOI 10.1371/journal.pone.0291564PMC PMC10691727

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Abstract

Cytochrome b561 (cytb561) proteins comprise a family of transmembrane oxidoreductases that transfer single electrons across a membrane. Most eukaryotic species, including insects, possess multiple cytb561 homologs. To learn more about this protein family in insects, we carried out a bioinformatics-based investigation of cytb561 family members from nine species representing eight insect orders. We performed a phylogenetic analysis to classify insect cytb561 ortholog groups. We then conducted sequence analyses and analyzed protein models to predict structural elements that may impact the biological functions and localization of these proteins, with a focus on possible ferric reductase activity. Our study revealed three ortholog groups, designated CG1275, Nemy, and CG8399, and a fourth group of less-conserved genes. We found that CG1275 and Nemy proteins are similar to a human ferric reductase, duodenal cytochrome b561 (Dcytb), and have many conserved amino acid residues that function in substrate binding in Dcytb. Notably, CG1275 and Nemy proteins contain a conserved histidine and other residues that play a role in ferric ion reduction by Dcytb. Nemy proteins were distinguished by a novel cysteine-rich cytoplasmic loop sequence. CG8399 orthologs are similar to a putative ferric reductase in humans, stromal cell-derived receptor 2. Like other members of the CYBDOM class of cytb561 proteins, these proteins contain reeler, DOMON, and cytb561 domains. Drosophila melanogaster CG8399 is the only insect cytb561 with known ferric reductase activity. Our investigation of the DOMON domain in CG8399 proteins revealed a probable heme-binding site and a possible site for ferric reduction. The fourth group includes a subgroup of proteins with a conserved “KXXXXKXH” non-cytoplasmic loop motif that may be a substrate binding site and is present in a potential ferric reductase, human tumor suppressor cytochrome b561. This study provides a foundation for future investigations of the biological functions of cytb561 genes in insects.

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Fig 1 Phylogenetic tree of insect and human cytb561 proteins. A phylogenetic analysis of cytb561s from nine species of insects (A. gambiae [Ag], A. mellifera [Am], A. pisum [Ap], C. felis [Cf], D. melanogaster [Dm], T. castaneum [Tc], P. humanus corporis [Ph], P. xuthus [Px], and Z. nevadensis [Zn]), humans (Hs), and a species of lancelet (B. floridae) (Bf) was performed with the maximum likelihood method. An outgroup sequence from T. adhaerens (Ta) was used for rooting the tree. Bootstrap values greater than 70% are shown. Three insect ortholog groups (with one-to-one orthologs in most or all insect species) were identified: CG1275, Nemy, and CG8399. The remaining genes formed a fourth group that has no one-to-one orthologs but includes many species-specific gene expansions. The bootstrap values that support groups of interest are marked with red circles. A branch length scale bar indicates one substitution per site. Of the Group 4 sequences, seven are more similar to human TScytb than any D. melanogaster cytb561s and are referred to as subgroup 4A, whereas the remaining Group 4 proteins belong to subgroup 4B. (Note that the 4A and 4B subgroups are based on sequence comparisons rather than phylogenetic relationship.).
Fig 1 Phylogenetic tree of insect and human cytb561 proteins. A phylogenetic analysis of cytb561s from nine species of insects (A. gambiae [Ag], A. mellifera [Am], A. pisum [Ap], C. felis [Cf], D. melanogaster [Dm], T. castaneum [Tc], P. humanus corporis [Ph], P. xuthus [Px], and Z. nevadensis [Zn]), humans (Hs), and a species of lancelet (B. floridae) (Bf) was performed with the maximum likelihood method. An outgroup sequence from T. adhaerens (Ta) was used for rooting the tree. Bootstrap values greater than 70% are shown. Three insect ortholog groups (with one-to-one orthologs in most or all insect species) were identified: CG1275, Nemy, and CG8399. The remaining genes formed a fourth group that has no one-to-one orthologs but includes many species-specific gene expansions. The bootstrap values that support groups of interest are marked with red circles. A branch length scale bar indicates one substitution per site. Of the Group 4 sequences, seven are more similar to human TScytb than any D. melanogaster cytb561s and are referred to as subgroup 4A, whereas the remaining Group 4 proteins belong to subgroup 4B. (Note that the 4A and 4B subgroups are based on sequence comparisons rather than phylogenetic relationship.).CC BY · PubMed Central
Fig 2 Group consensus logos for the cytb561 core domain. Group-specific alignments were used to create group consensus sequences in Jalview. The four conserved histidines are labeled H1-H4 with yellow highlighting for the non-cytoplasmic histidines and blue highlighting for the cytoplasmic histidines. The positions of amino acid residues of interest are labeled relative to the nearest conserved histidine. If the corresponding residue in Dcytb was predicted to contact a heme or ascorbate, that position is indicated by a symbol: non-cytoplasmic heme (square), non-cytoplasmic ascorbate (upside-down triangle), cytoplasmic heme (red diamond), or cytoplasmic ascorbate (red triangle). The Jalview consensus rows show the most common residue at that position listed below the logo; a + is used where there are two or more residues with the highest percentage and a–is used to show gaps.
Fig 2 Group consensus logos for the cytb561 core domain. Group-specific alignments were used to create group consensus sequences in Jalview. The four conserved histidines are labeled H1-H4 with yellow highlighting for the non-cytoplasmic histidines and blue highlighting for the cytoplasmic histidines. The positions of amino acid residues of interest are labeled relative to the nearest conserved histidine. If the corresponding residue in Dcytb was predicted to contact a heme or ascorbate, that position is indicated by a symbol: non-cytoplasmic heme (square), non-cytoplasmic ascorbate (upside-down triangle), cytoplasmic heme (red diamond), or cytoplasmic ascorbate (red triangle). The Jalview consensus rows show the most common residue at that position listed below the logo; a + is used where there are two or more residues with the highest percentage and a–is used to show gaps.CC BY · PubMed Central
Fig 3 Insect cytb561 models with sequence alignment overlay demonstrate residue conservation. AlphaFold models of mature proteins are shown. Sequence conservation was determined by ChimeraX using AL2CO conservation scores. The model is gradient colored with high conservation (+1.5 score) in magenta and low conservation (-1.5 score) in cyan; sequence gaps are shown in tan. Conserved histidines H1-H4 (not all visible in all panels) are shown in stick form with nitrogen in blue. The models are oriented with the cytoplasmic side of the protein at the bottom. Note that the amino- and carboxyl- termini for all models except the CG8399 model are near the bottom of the panels. Locations of H1-4 are indicated. Scale bars depict 10 Å. A) D. melanogaster CG1275 (Lys95-Glu340) aligned with the CG1275 sequences. B) D. melanogaster Nemy (Lys40-Ile290) aligned with the Nemy sequences. C) D. melanogaster CG8399 (Ser402-Gly645) aligned with the multi-domain CG8399 sequences. The reeler and DOMON domains are omitted. D) A. gambiae XP_320673.4 (all residues) aligned with the Group 4A sequences. E) D. melanogaster CG13077 (all residues) aligned with the Group 4B sequences.
Fig 3 Insect cytb561 models with sequence alignment overlay demonstrate residue conservation. AlphaFold models of mature proteins are shown. Sequence conservation was determined by ChimeraX using AL2CO conservation scores. The model is gradient colored with high conservation (+1.5 score) in magenta and low conservation (-1.5 score) in cyan; sequence gaps are shown in tan. Conserved histidines H1-H4 (not all visible in all panels) are shown in stick form with nitrogen in blue. The models are oriented with the cytoplasmic side of the protein at the bottom. Note that the amino- and carboxyl- termini for all models except the CG8399 model are near the bottom of the panels. Locations of H1-4 are indicated. Scale bars depict 10 Å. A) D. melanogaster CG1275 (Lys95-Glu340) aligned with the CG1275 sequences. B) D. melanogaster Nemy (Lys40-Ile290) aligned with the Nemy sequences. C) D. melanogaster CG8399 (Ser402-Gly645) aligned with the multi-domain CG8399 sequences. The reeler and DOMON domains are omitted. D) A. gambiae XP_320673.4 (all residues) aligned with the Group 4A sequences. E) D. melanogaster CG13077 (all residues) aligned with the Group 4B sequences.CC BY · PubMed Central
Fig 4 Alignment of Dcytb, AtCytb561-B, Nemy, and CG1275 protein sequences. The two cytb561 sequences with known structure, human Dcytb (PDB 5ZLG) and A. thaliana AtCytb561-B (PDB 4O7G), were aligned with CG1275 and Nemy sequences from A. gambiae (Ag), A. mellifera (Am), A. pisum (Ap), C. felis (Cf), D. melanogaster (Dm), T. castaneum (Tc), P. humanus corporis (Ph), P. xuthus (Px), and Z. nevadensis (Zn). (Note that only the A. pisum CG1275 sequence most similar to Dcytb was included.) Alpha helices from crystal structures (Dcytb, AtCytb561-B) or predicted in AlphaFold models (Nemy, CG1275) are shown in boxes: helix 1 carboxyl end (gray), helix 2 (red), helix 3 (yellow), helix 4 (green), and helix 5 (blue). Conserved residues are noted under the alignment (* indicates completely conserved, the four strictly conserved histidines are labeled with their number rather than *, and ^ indicates highly conserved with a Jalview conservation score of 8 or 9). Predicted Dcytb heme-contacting residues are labeled with a square (non-cytoplasmic heme) or diamond (cytoplasmic heme), and ascorbate-contacting residues are labeled with an upside-down triangle (non-cytoplasmic ascorbate) or triangle (cytoplasmic ascorbate). A blue vertical line indicates the position of 51 amino acids found only in A. pisum XP_001949276.1, which were omitted from the figure. Coloring by amino acid residue: basic residues in blue (Arg) or cyan (Lys); acidic residues (Asp and Glu) in dark pink; amide residues (Asn and Gln) in light pink; hydroxylic residues (Ser and Thr) in pale purple; aromatic residues in light peach (Phe and Tyr) or dark peach (Trp); sulfur-containing residues in dark green (Cys) or light green (Met); His in orange; Pro in yellow; Ile, Leu, and Val in pale yellow; and Ala and Gly in white.
Fig 4 Alignment of Dcytb, AtCytb561-B, Nemy, and CG1275 protein sequences. The two cytb561 sequences with known structure, human Dcytb (PDB 5ZLG) and A. thaliana AtCytb561-B (PDB 4O7G), were aligned with CG1275 and Nemy sequences from A. gambiae (Ag), A. mellifera (Am), A. pisum (Ap), C. felis (Cf), D. melanogaster (Dm), T. castaneum (Tc), P. humanus corporis (Ph), P. xuthus (Px), and Z. nevadensis (Zn). (Note that only the A. pisum CG1275 sequence most similar to Dcytb was included.) Alpha helices from crystal structures (Dcytb, AtCytb561-B) or predicted in AlphaFold models (Nemy, CG1275) are shown in boxes: helix 1 carboxyl end (gray), helix 2 (red), helix 3 (yellow), helix 4 (green), and helix 5 (blue). Conserved residues are noted under the alignment (* indicates completely conserved, the four strictly conserved histidines are labeled with their number rather than *, and ^ indicates highly conserved with a Jalview conservation score of 8 or 9). Predicted Dcytb heme-contacting residues are labeled with a square (non-cytoplasmic heme) or diamond (cytoplasmic heme), and ascorbate-contacting residues are labeled with an upside-down triangle (non-cytoplasmic ascorbate) or triangle (cytoplasmic ascorbate). A blue vertical line indicates the position of 51 amino acids found only in A. pisum XP_001949276.1, which were omitted from the figure. Coloring by amino acid residue: basic residues in blue (Arg) or cyan (Lys); acidic residues (Asp and Glu) in dark pink; amide residues (Asn and Gln) in light pink; hydroxylic residues (Ser and Thr) in pale purple; aromatic residues in light peach (Phe and Tyr) or dark peach (Trp); sulfur-containing residues in dark green (Cys) or light green (Met); His in orange; Pro in yellow; Ile, Leu, and Val in pale yellow; and Ala and Gly in white.CC BY · PubMed Central
Fig 5 D. melanogaster CG1275 and Nemy models overlayed on Dcytb heme- and ascorbate-bound structure. AlphaFold structures of D. melanogaster CG1275 and Nemy were overlayed with Dcytb (PDB 5ZLG) using Chimera Matchmaker. Transmembrane helices are labeled (TM1-TM6), with Dcytb in tan, CG1275 in pale pink, and Nemy in pale blue. The four conserved histidines are shown in stick form; in the Dcytb structure, H1 is in red, H2 in yellow, H3 in green, and H4 in dark blue. Ascorbate (ASC) in black stick, heme in tan stick (with iron shown as orange spheres), and zinc (Zn2+) ion coordination in purple are from the 5ZLG structure. Stick side chains, ascorbate, and heme have oxygen in red and nitrogen in blue. The alpha carbon RMSD for CG1275 with 5ZLG is 0.679 Å for the 208 pruned atom pairs and 1.458 Å across all 222 pairs. The alpha carbon RMSD for Nemy with 5ZLG is 0.990 Å for the 188 pruned atom pairs and 2.657 Å across all 222 pairs. A) Overlay of D. melanogaster CG1275 and Nemy with Dcytb. B) Non-cytoplasmic ascorbate binding, zinc coordination, and heme coordination with H1 (red) and H3 (green) shown in stick form. C) Cytoplasmic ascorbate binding and heme coordination with H2 (yellow) and H4 (dark blue) shown in stick form.
Fig 5 D. melanogaster CG1275 and Nemy models overlayed on Dcytb heme- and ascorbate-bound structure. AlphaFold structures of D. melanogaster CG1275 and Nemy were overlayed with Dcytb (PDB 5ZLG) using Chimera Matchmaker. Transmembrane helices are labeled (TM1-TM6), with Dcytb in tan, CG1275 in pale pink, and Nemy in pale blue. The four conserved histidines are shown in stick form; in the Dcytb structure, H1 is in red, H2 in yellow, H3 in green, and H4 in dark blue. Ascorbate (ASC) in black stick, heme in tan stick (with iron shown as orange spheres), and zinc (Zn2+) ion coordination in purple are from the 5ZLG structure. Stick side chains, ascorbate, and heme have oxygen in red and nitrogen in blue. The alpha carbon RMSD for CG1275 with 5ZLG is 0.679 Å for the 208 pruned atom pairs and 1.458 Å across all 222 pairs. The alpha carbon RMSD for Nemy with 5ZLG is 0.990 Å for the 188 pruned atom pairs and 2.657 Å across all 222 pairs. A) Overlay of D. melanogaster CG1275 and Nemy with Dcytb. B) Non-cytoplasmic ascorbate binding, zinc coordination, and heme coordination with H1 (red) and H3 (green) shown in stick form. C) Cytoplasmic ascorbate binding and heme coordination with H2 (yellow) and H4 (dark blue) shown in stick form.CC BY · PubMed Central
Fig 6 Heme binding by Dcytb and D. melanogaster CG1275 and Nemy. AlphaFold structures of D. melanogaster CG1275 and Nemy were overlayed with Dcytb (5ZLG) using Chimera Matchmaker. Dcytb loops are in tan and helices are shown in red (helix 2), yellow (helix 3), green (helix 4), dark blue (helix 5), and purple (helix 6). CG1275 is in pale pink, and Nemy is in pale blue. Ascorbate (ASC) in black stick, heme in tan stick (with iron shown as orange spheres), and zinc (Zn2+) ion coordination in purple are from the 5ZLG structure. Select side chains are shown in stick form and are labeled by position relative to conserved heme-coordinating histidines with Dcytb amino acid numbering in parentheses. Key glycines are shown in black ribbon. Stick side chains, ascorbate, and heme have oxygen in red and nitrogen in blue. Hydrogen bonds shown as cyan dashed lines, including to the A and D heme-propionates (labeled as in [18]). Water molecules shown as red spheres. A) Cytoplasmic heme region. B) Non-cytoplasmic heme region. Hydrogen bonds predicted by ChimeraX or observed in Dcytb structure (Asn115 to D heme-propionate; Glu180 to A heme-propionate, [18]).
Fig 6 Heme binding by Dcytb and D. melanogaster CG1275 and Nemy. AlphaFold structures of D. melanogaster CG1275 and Nemy were overlayed with Dcytb (5ZLG) using Chimera Matchmaker. Dcytb loops are in tan and helices are shown in red (helix 2), yellow (helix 3), green (helix 4), dark blue (helix 5), and purple (helix 6). CG1275 is in pale pink, and Nemy is in pale blue. Ascorbate (ASC) in black stick, heme in tan stick (with iron shown as orange spheres), and zinc (Zn2+) ion coordination in purple are from the 5ZLG structure. Select side chains are shown in stick form and are labeled by position relative to conserved heme-coordinating histidines with Dcytb amino acid numbering in parentheses. Key glycines are shown in black ribbon. Stick side chains, ascorbate, and heme have oxygen in red and nitrogen in blue. Hydrogen bonds shown as cyan dashed lines, including to the A and D heme-propionates (labeled as in [18]). Water molecules shown as red spheres. A) Cytoplasmic heme region. B) Non-cytoplasmic heme region. Hydrogen bonds predicted by ChimeraX or observed in Dcytb structure (Asn115 to D heme-propionate; Glu180 to A heme-propionate, [18]).CC BY · PubMed Central
Fig 7 Ascorbate binding in Dcytb, CG1275, and Nemy. AlphaFold structures of D. melanogaster CG1275 and Nemy were overlayed with Dcytb (5ZLG) using Chimera Matchmaker. Dcytb loops are in tan and helices are shown in yellow (helix 3), green (helix 4), and dark blue (helix 5). CG1275 is in pale pink, and Nemy is in pale blue. Ascorbate (ASC) in black stick, heme in tan stick (with iron shown as orange spheres), and metal ion coordination in purple are from the 5ZLG structure. Select side chains are shown in stick form and are labeled by position relative to conserved heme-coordinating histidines with Dcytb amino acid numbering in parentheses (unless otherwise noted as Nemy numbering). Stick side chains, ascorbate, and heme have oxygen in red and nitrogen in blue. Hydrogen bonds shown as cyan dashed lines. A) Cytoplasmic ascorbate region. Ascorbate oxygens involved in bonding are labeled 1–3 and 6. B) Non-cytoplasmic ascorbate region with predicted hydrogen bonds to Dcytb, CG1275, and Nemy shown in cyan; an additional predicted hydrogen bond between Nemy Lys161 and Glu236 is also shown. Zinc coordination bonds are shown as orange dashed lines.
Fig 7 Ascorbate binding in Dcytb, CG1275, and Nemy. AlphaFold structures of D. melanogaster CG1275 and Nemy were overlayed with Dcytb (5ZLG) using Chimera Matchmaker. Dcytb loops are in tan and helices are shown in yellow (helix 3), green (helix 4), and dark blue (helix 5). CG1275 is in pale pink, and Nemy is in pale blue. Ascorbate (ASC) in black stick, heme in tan stick (with iron shown as orange spheres), and metal ion coordination in purple are from the 5ZLG structure. Select side chains are shown in stick form and are labeled by position relative to conserved heme-coordinating histidines with Dcytb amino acid numbering in parentheses (unless otherwise noted as Nemy numbering). Stick side chains, ascorbate, and heme have oxygen in red and nitrogen in blue. Hydrogen bonds shown as cyan dashed lines. A) Cytoplasmic ascorbate region. Ascorbate oxygens involved in bonding are labeled 1–3 and 6. B) Non-cytoplasmic ascorbate region with predicted hydrogen bonds to Dcytb, CG1275, and Nemy shown in cyan; an additional predicted hydrogen bond between Nemy Lys161 and Glu236 is also shown. Zinc coordination bonds are shown as orange dashed lines.CC BY · PubMed Central
Fig 8 Cytoplasmic side of D. melanogaster CG8399 model. AlphaFold structure of D. melanogaster CG8399 overlayed with multi-domain CG8399 consensus sequence alignment. The model is gradient colored with high conservation (1.68 score) in magenta to low conservation (-1.89 score) in cyan. Select side chains are shown in stick form with oxygen in red and nitrogen in blue and are labeled by position relative to conserved heme-coordinating histidines with D. melanogaster CG8399 amino acid numbering in parentheses. Ascorbate binding on the cytoplasmic surface of CG8399 may be facilitated by a cluster of aromatic and basic residues: Lys432, Trp446, Phe447, Arg501, and Arg509. Also shown are conserved H2 (His450) and H4 (His516), which coordinate the cytoplasmic heme iron (not present in model). Two well-conserved prolines at positions H3+22 and H3+24 stabilize the loop between helices 3 and 4. Other cytoplasmic side positions with well conserved residues include H2-7, which typically has a lysine but is Thr443 in D. melanogaster, and H4-3 (Asp513). Scale bar depicts 10 Å.
Fig 8 Cytoplasmic side of D. melanogaster CG8399 model. AlphaFold structure of D. melanogaster CG8399 overlayed with multi-domain CG8399 consensus sequence alignment. The model is gradient colored with high conservation (1.68 score) in magenta to low conservation (-1.89 score) in cyan. Select side chains are shown in stick form with oxygen in red and nitrogen in blue and are labeled by position relative to conserved heme-coordinating histidines with D. melanogaster CG8399 amino acid numbering in parentheses. Ascorbate binding on the cytoplasmic surface of CG8399 may be facilitated by a cluster of aromatic and basic residues: Lys432, Trp446, Phe447, Arg501, and Arg509. Also shown are conserved H2 (His450) and H4 (His516), which coordinate the cytoplasmic heme iron (not present in model). Two well-conserved prolines at positions H3+22 and H3+24 stabilize the loop between helices 3 and 4. Other cytoplasmic side positions with well conserved residues include H2-7, which typically has a lysine but is Thr443 in D. melanogaster, and H4-3 (Asp513). Scale bar depicts 10 Å.CC BY · PubMed Central
Fig 9 A conserved “KXXXXKXH” motif in Group 4A and related proteins. A) Truncated alignment of animal TScytb and Group 4A sequences shows a conserved “KXXXXKXH” motif upstream of the conserved non-cytoplasmic heme-coordinating H3. Coloring by amino acid residue as in Fig 4. Abbreviations: Cf, C. felis; Aten, A. tenebrosa; Hrob, H. robusta; Drer, D. rerio; Xlae, X. laevis; Pmon, P. montanus; Mmus, M. musculus; Hsap, H. sapiens; Apla, A. planci; Osin, O. sinensis; Am, A. mellifera; Tc, T. castaneum; Zn, Z. nevadensis; Px, P. xuthus; Ag, A. gambiae. B) Full alignment of animal Tscytb and Group 4A sequences overlayed on AlphaFold model of A. gambiae XP_3200673.4 (AF-Q7PZD0-F1-model_v4). H1-H4 shown in stick form with nitrogen in blue. C) Non-cytoplasmic side of the protein contains completely conserved lysines (H2+22, H3-7), a histidine (H3-5), a tryptophan (H4+23), and a phenylalanine (H1-3), in addition to H1 and H3; these residues are shown in stick with nitrogen atoms in blue. B-C) Amino acid conservation is shown by a gradient of color as in Fig 3. Scale bars show 10 Å.
Fig 9 A conserved “KXXXXKXH” motif in Group 4A and related proteins. A) Truncated alignment of animal TScytb and Group 4A sequences shows a conserved “KXXXXKXH” motif upstream of the conserved non-cytoplasmic heme-coordinating H3. Coloring by amino acid residue as in Fig 4. Abbreviations: Cf, C. felis; Aten, A. tenebrosa; Hrob, H. robusta; Drer, D. rerio; Xlae, X. laevis; Pmon, P. montanus; Mmus, M. musculus; Hsap, H. sapiens; Apla, A. planci; Osin, O. sinensis; Am, A. mellifera; Tc, T. castaneum; Zn, Z. nevadensis; Px, P. xuthus; Ag, A. gambiae. B) Full alignment of animal Tscytb and Group 4A sequences overlayed on AlphaFold model of A. gambiae XP_3200673.4 (AF-Q7PZD0-F1-model_v4). H1-H4 shown in stick form with nitrogen in blue. C) Non-cytoplasmic side of the protein contains completely conserved lysines (H2+22, H3-7), a histidine (H3-5), a tryptophan (H4+23), and a phenylalanine (H1-3), in addition to H1 and H3; these residues are shown in stick with nitrogen atoms in blue. B-C) Amino acid conservation is shown by a gradient of color as in Fig 3. Scale bars show 10 Å.CC BY · PubMed Central
Fig 10 Model of D. melanogaster CG8399 demonstrating interactions between domains, and a putative iron binding site. A-C) Models were colored by ChimeraX based on amino acid conservation in the nine multidomain CG8399 sequences. Color gradient as in Fig 3. Residues in stick form show oxygen in red, nitrogen in blue and sulfur in yellow. A-D) Scale bars depict 10 Å. A) CG8399 has three distinct domains: reeler (top left), DOMON (middle right), and cytb561 (bottom). Residues expected to be heme-coordinating are shown in stick (some not visible). B) Selected sidechains at the interface of the DOMON (top) and cytb561 (bottom) domains. Predicted heme-coordinating residues (Met286 and His379) in the DOMON domain are positioned at the bottom of the domain where it is predicted to interact with the cytb561 domain. Interdomain hydrogen bonds were predicted between the sidechain of Lys285 (DOMON) and the backbone of Leu472 (cytb561), as well as the sidechain of Arg383 (DOMON) and the backbone of Pro538 (cytb561). Potential salt bridge interactions may form in the cytb561 domain loop regions between Glu471 and Lys539, as well as Arg403 and Glu541. C) A putative metal binding site in a highly conserved loop with two aspartates and a serine residue. D) Space filling model of the same part of the structure depicted in C, with Coulombic coloring calculated by ChimeraX, red for negative charge, white for neutral, and dark blue for positive charge.
Fig 10 Model of D. melanogaster CG8399 demonstrating interactions between domains, and a putative iron binding site. A-C) Models were colored by ChimeraX based on amino acid conservation in the nine multidomain CG8399 sequences. Color gradient as in Fig 3. Residues in stick form show oxygen in red, nitrogen in blue and sulfur in yellow. A-D) Scale bars depict 10 Å. A) CG8399 has three distinct domains: reeler (top left), DOMON (middle right), and cytb561 (bottom). Residues expected to be heme-coordinating are shown in stick (some not visible). B) Selected sidechains at the interface of the DOMON (top) and cytb561 (bottom) domains. Predicted heme-coordinating residues (Met286 and His379) in the DOMON domain are positioned at the bottom of the domain where it is predicted to interact with the cytb561 domain. Interdomain hydrogen bonds were predicted between the sidechain of Lys285 (DOMON) and the backbone of Leu472 (cytb561), as well as the sidechain of Arg383 (DOMON) and the backbone of Pro538 (cytb561). Potential salt bridge interactions may form in the cytb561 domain loop regions between Glu471 and Lys539, as well as Arg403 and Glu541. C) A putative metal binding site in a highly conserved loop with two aspartates and a serine residue. D) Space filling model of the same part of the structure depicted in C, with Coulombic coloring calculated by ChimeraX, red for negative charge, white for neutral, and dark blue for positive charge.CC BY · PubMed Central
Fig 11 Lysosomal targeting signal in CG1275. An alignment of the C-terminal portion of CG1275 proteins. Helix predictions from the AlphaFold model of D. melanogaster CG1275 shown in gray (helix 6) and an additional helical region is in black. The EDXXLL motif is boxed in blue in the consensus logo.
Fig 11 Lysosomal targeting signal in CG1275. An alignment of the C-terminal portion of CG1275 proteins. Helix predictions from the AlphaFold model of D. melanogaster CG1275 shown in gray (helix 6) and an additional helical region is in black. The EDXXLL motif is boxed in blue in the consensus logo.CC BY · PubMed Central

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Metadata

Authors
Holst, J. D., Murphy, L. G., Gorman, M. J., & Ragan, E. J.
Affiliations
  • Metropolitan State University of Denver (US)
  • Kansas State University (US)
Corresponding author
Ragan, Emily J.
ORCID
Venue
PLOS ONE
Publisher
Public Library of Science (PLoS)
Published
2023-12-01
Volume
18
Issue
12
Pages
e0291564
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0291564
PMC
PMC10691727
OpenAlex
W4389234010
Cited by
6
References
71
Citations by year
2025: 4 · 2024: 2
FWCI
1.23
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Division of Integrative Organismal Systems (NSF IOS-1656388, NSF IOS-1656407)
  • Foundation for the National Institutes of Health (NIH R35GM141859, R01-GM129325, 5U41HG000739)
Topics
  • Health Sciences › Medicine › Hematology › Iron Metabolism and Disorders
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Insect Resistance and Genetics
Keywords
cytochrome b561, Dcytb, heme binding site, substrate binding site, Drosophila melanogaster
MeSH terms
Animals, Cytochrome b Group, Drosophila melanogaster, Humans, Iron, Oxidoreductases, Phylogeny, FMN Reductase

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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How to cite

Cite CoChat for the checks on this page, and the paper for its own findings.

This page
CoChat. (2026). Citation check: Comparison of insect and human cytochrome b561 proteins: Insights into candidate ferric reductases in insects. https://app.cochat.ai/papers/10.1371/journal.pone.0291564
The paper
Holst, J. D., Murphy, L. G., Gorman, M. J., & Ragan, E. J. (2023). Comparison of insect and human cytochrome b561 proteins: Insights into candidate ferric reductases in insects. PLOS ONE. https://doi.org/10.1371/journal.pone.0291564

Data sources

Bibliographic data: Semantic Scholar, OpenAlex, Crossref.

Contains information from the Semantic Scholar Academic Graph Datasets, made available under the ODC Attribution License (ODC-BY 1.0).

Semantic Scholar’s data is described in: Kinney, R., et al. (2023). The Semantic Scholar Open Data Platform. arXiv:2301.10140.