Funktionelle und strukturelle Untersuchungen von Häm-Peptid/Protein-Komplexen: ein Beitrag zur Aufklärung der Häm-vermittelten Regulation von Proteinen im Organismus

Dem Biomolekül Häm werden in der Natur in Verbindung mit Proteinen zahlreiche essentielle Funktionen in Bezug auf Gastransport, Elektronentransfer und Substratoxidation zugeschrieben. Im Vergleich dazu ist über die Funktion von Häm als Regulator nur wenig bekannt. Einige Prozesse und damit verbundene Proteine sind bekannt, bei denen Häm/Hämin eine wichtige Rolle spielt. Diese Proteinen tragen einen kurzen Sequenzabschnitt, den man als Häm-regulatorisches Motiv bezeichnet. Bisher konnte die Bindung von Häm an solche Sequenzabschnitte nur unzureichend aufgeklärt werden. Die vorliegende Arbeit beschäftigt sich mit der Analyse und Vorhersage Häm-regulatorischer Motive (HRM). Dazu wurde eine kombinatorische Peptidbibliothek mit der allgemeinen Sequenz AAXXXX[C/H/Y]XXXXBBBBRM-Linker-Harz (X = 18 proteinogene Aminosäuren außer Cys und Met, inkl. Nle) mit Hämin inkubiert und auf Hämin-bindende Motive gescreent. Für ausgewählte, resynthetisierte Vertreter wurde mittels funktioneller Studien (UV/Vis-Spektroskopie, Kompetitionsstudien mit hSlo1-Kanälen) die Bindung von Hämin an die Peptide quantifiziert. Auf Cys-basierte und im Speziellen Cys-Pro-basierte Sequenzen wurde dabei vorrangig, aber nicht ausschließlich, fokussiert. Weiterführend wurden strukturelle Untersuchungen (Resonanz-Ramanspektroskopie, NMR-Spektroskopie) zu ausgewählten Komplexen aus Hämin und Cys-basierten Sequenzen durchgeführt. Mit Hilfe von Datenbankrecherchen zu den erhaltenen Daten der kombinatorischen Peptidbibliothek konnten Vorhersagen für potentiell Hämin-bindende Proteine gemacht werden. Dipeptidylpeptidase 8 und ein daraus abgeleitetes 23 Aminosäuren-langes Peptid konnten so identifiziert und mittels enzymatischer Studien und den genannten spektroskopischen Methoden in Bezug auf ihre Häminbindung charakterisiert werden

Intracellular hemin is a potent inhibitor of the voltage-gated potassium channel Kv10.1

Heme, an iron-protoporphyrin IX complex, is a cofactor bound to various hemoproteins and supports a broad range of functions, such as electron transfer, oxygen transport, signal transduction, and drug metabolism. In recent years, there has been a growing recognition of heme as a non-genomic modulator of ion channel functions. Here, we show that intracellular free heme and hemin modulate human ether à go-go (hEAG1, Kv10.1) voltage-gated potassium channels. Application of hemin to the intracellular side potently inhibits Kv10.1 channels with an IC50 of about 4 nM under ambient and 63 nM under reducing conditions in a weakly voltage-dependent manner, favoring inhibition at resting potential. Functional studies on channel mutants and biochemical analysis of synthetic and recombinant channel fragments identified a heme-binding motif CxHx8H in the C-linker region of the Kv10.1 C terminus, with cysteine 541 and histidines 543 and 552 being important for hemin binding. Binding of hemin to the C linker may induce a conformational constraint that interferes with channel gating. Our results demonstrate that heme and hemin are endogenous modulators of Kv10.1 channels and could be exploited to modulate Kv10.1-mediated cellular functions

Structural insights into heme binding to IL-36α proinflammatory cytokine

Cytokines of the interleukin (IL)-1 family regulate immune and inflammatory responses. The recently discovered IL-36 family members are involved in psoriasis, rheumatoid arthritis, and pulmonary diseases. Here, we show that IL-36α interacts with heme thereby contributing to its regulation. Based on in-depth spectroscopic analyses, we describe two heme-binding sites in IL-36α that associate with heme in a pentacoordinated fashion. Solution NMR analysis reveals structural features of IL-36α and its complex with heme. Structural investigation of a truncated IL-36α supports the notion that the N-terminus is necessary for association with its cognate receptor. Consistent with our structural studies, IL-36-mediated signal transduction was negatively regulated by heme in synovial fibroblast-like synoviocytes from rheumatoid arthritis patients. Taken together, our results provide a structural framework for heme-binding proteins and add IL-1 cytokines to the group of potentially heme-regulated proteins

The pH-Induced Selectivity Between Cysteine or Histidine Coordinated Heme in an Artificial alpha-Helical Metalloprotein

International audienceDe Novo metalloprotein design assesses the relationship between metal active site architecture and catalytic reactivity. Herein, we use an alpha-helical scaffold to control the iron coordination geometry when a heme cofactor is allowed to bind to either histidine or cysteine ligands, within a single artificial protein. Consequently, we uncovered a reversible pH-induced switch of the heme axial ligation within this simplified scaffold. Characterization of the specific heme coordination modes was done by using UV-Vis and Electron Paramagnetic Resonance spectroscopies. The penta- or hexa-coordinate thiolate heme (9 ≤ pH ≤ 11) and the penta-coordinate imidazole heme (6 ≤ pH ≤ 8.5) reproduces well the heme ligation in chloroperoxidases or cyt P450 monooxygenases and peroxidases, respectively. The stability of heme coordination upon ferric/ferrous redox cycling is a crucial property of the construct. At basic pHs, the thiolate mini-heme protein can catalyze O2 reduction when adsorbed onto a pyrolytic graphite electrode

The pH- Induced Selectivity Between Cysteine or Histidine Coordinated Heme in an Artificial α- Helical Metalloprotein

De Novo metalloprotein design assesses the relationship between metal active site architecture and catalytic reactivity. Herein, we use an α- helical scaffold to control the iron coordination geometry when a heme cofactor is allowed to bind to either histidine or cysteine ligands, within a single artificial protein. Consequently, we uncovered a reversible pH- induced switch of the heme axial ligation within this simplified scaffold. Characterization of the specific heme coordination modes was done by using UV/Vis and Electron Paramagnetic Resonance spectroscopies. The penta- or hexa- coordinate thiolate heme (9- ¤pH- ¤11) and the penta- coordinate imidazole heme (6- ¤pH- ¤8.5) reproduces well the heme ligation in chloroperoxidases or cyt- P450 monooxygenases and peroxidases, respectively. The stability of heme coordination upon ferric/ferrous redox cycling is a crucial property of the construct. At basic pHs, the thiolate mini- heme protein can catalyze O2 reduction when adsorbed onto a pyrolytic graphite electrode.A pH- dependent preference for the heme ligation is achieved when using self- assembling α- helical coiled coils having Cys and His as binding sites. EPR and UV/Vis absorption spectroscopies uncovered a switch in heme axial ligand from His (6- ¤pH- ¤8.5) to Cys (9- ¤pH- ¤11), mimicking the heme sites in cyt- P450 monooxygenase, chloroperoxidase and peroxidases, all three in a single artificial protein.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/166347/1/ange202012673.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/166347/2/ange202012673_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/166347/3/ange202012673-sup-0001-misc_information.pd

High-affinity binding and catalytic activity of His/Tyr-based sequences: Extending heme-regulatory motifs beyond CP

Background & motivationPeptides and proteins can interact with heme through His, Tyr, or Cys in heme-regulatory motifs (HRMs). The Cys-Pro dipeptide is a well investigated HRM, but for His and Tyr such a distinct motif is currently unknown. In addition, many heme-peptide complexes, such as heme-amyloid β, can display a peroxidase-like activity, albeit there is little understanding of how the local primary and secondary coordination environment influences catalytic activity. We thus systematically evaluated a series of His- and Tyr-based peptides to identify sequence features for high-affinity heme binding and their impact on the catalytic activity of heme.MethodsWe employed solid-phase peptide synthesis to produce 58 nonapeptides, which were investigated by UV/vis, resonance Raman, and 2D NMR spectroscopy. A chromogenic assay was used to determine the catalytic activity of the heme-peptide complexes.ResultsHeme-binding affinity and binding mode were found to be dependent on the coordinating amino acid and spacer length between multiple potential coordination sites in a motif. In particular, HXH and HXXXH motifs showed strong heme binding. Analysis of the peroxidase-like activity revealed that some of these peptides and also HXXXY motifs enhance the catalytic activity of heme significantly.ConclusionsWe identify HXH, HXXXH, and HXXXY as potential new HRMs with functional properties. Several peptides displayed a strikingly high peroxidase-like activity.General significanceThe identification of HRMs allows to discover yet unknown heme-regulated proteins, and consequently, enhances our current understanding of pathologies involving labile heme

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