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

Structure determination of peptide-heme complexes

Heme has been traditionally viewed as a stable protein cofactor in hemoproteins. Only recently it is also being viewed as a signaling and regulatory molecule that binds temporarily to proteins and regulates their activation or inhibition. Information on the structural characteristics of heme regulatory motifs (HRMs) is scarce. Based on known HRMs from literature, nona-peptides containing the three heme-iron coordinating amino acids cysteine, histidine and tyrosine were employed for structural characterization by NMR and other biophysical techniques. Diamagnetic gallium protoporphyrin IX was used as a substitute for paramagnetic heme in NMR spectroscopy. Among the known cysteine-based HRMs, the Cys-Pro containing peptides are well structured as compared to the His and Tyr-based peptides. Furthermore, the distance between two heme-iron coordinating amino acids in a protein plays a critical role in the formation of penta- or hexacoordination. The observations of the peptide studies were further investigated at the protein level. As one example, the heme regulation of the voltage-gated potassium channel Kv1.4 has been studied at the structural level. These channels are involved in the control of neuronal excitability and regulation of neurotransmitter release. The N-terminal stretch of this channel opens and closes (inactivation) in response to the current flow and thus shapes the action potential. Heme binds to this N-terminal stretch through a CXXH motif and a distant histidine, reversibly inhibits the inactivation and thus might play an important role of counteracting the effects associated with neuropathological conditions. This interaction has been investigated using NMR spectroscopy and a model for inhibition of channel inactivation has been proposed. Finally, our peptide-based structure investigation has paved way for the identification of several yet unknown heme-regulated proteins

Heme interaction of the intrinsically disordered N-terminal peptide segment of human cystathionine-β-synthase

Cystathionine-β-synthase (CBS) belongs to a large family of pyridoxal 5’-phosphate (PLP)-dependent enzymes, responsible for the sulfur metabolism. The heme-dependent protein CBS is part of regulatory pathways also involving the gasotransmitter hydrogen sulfide. Malfunction of CBS can lead to pathologic conditions like cancer, cardiovascular and neurodegenerative disorders. Truncation of residues 1–40, absent in X-ray structures of CBS, reduces but does not abolish the activity of the enzyme. Here we report the NMR resonance assignment and heme interaction studies for the N-terminal peptide stretch of CBS. We present NMR-spectral evidence that residues 1–40 constitute an intrinsically disordered region in CBS and interact with heme via a cysteine-proline based motif