Regulatory heme binds to specific motifs in proteins and controls a variety of biochemical processes. Several of these proteins were recently shown to form complexes with ferric and/or ferrous heme via a cysteine residue as axial ligand. The objective of this study was to examine the heme-binding properties of a series of cysteine-containing peptides with focus on CP motif sequences. The peptides displayed different binding behavior upon Fe(III) heme application with characteristic wavelength shifts of the Soret band to 370 nm or 420-430 nm and in some cases to both wavelengths. Whereas for most of the peptides containing a cysteine only a shift to 420-430 nm was observed, CP-containing peptides exhibited a preference for a shift to 370 nm. Detailed structural investigation using Raman and NMR spectroscopy on selected representatives revealed different binding modes with respect to iron ion coordination, which reflected the results of the UV-vis studies. A predicted short sequence stretch derived from dipeptidyl peptidase 8 was additionally examined with respect to CP motif binding to heme on the peptide as well as on the protein level. The heme association was confirmed with the first solution structure of a CP-peptide-heme complex and, moreover, an inhibitory effect of Fe(III) heme on the enzyme's activity. The relevance of both the use of model compounds to elucidate the molecular mechanism underlying regulatory heme binding and its potential for the investigation of regulatory heme control is discussed.

Deviant levels of available heme and related molecules can result from pathological situations such as impaired heme biosynthesis or increased hemolysis as a consequence of vascular trauma or bacterial infections. Heme-related biological processes are affected by these situations, and it is essential to fully understand the underlying mechanisms. While heme has long been known as an important prosthetic group of various proteins, its function as a regulatory and signaling molecule is poorly understood. Diseases such as porphyria are caused by impaired heme metabolism, and heme itself might be used as a drug in order to downregulate its own biosynthesis. In addition, heme-driven side effects and symptoms emerging from heme-related pathological conditions are not fully comprehended and thus impede adequate medical treatment. Several heme-regulated proteins have been identified in the past decades, however, the molecular basis of transient heme-protein interactions remains to be explored. Herein, we summarize the results of an in-depth analysis of heme binding to proteins, which revealed specific binding modes and affinities depending on the amino acid sequence. Evaluating the binding behavior of a plethora of heme-peptide complexes resulted in the implementation of a prediction tool (SeqD-HBM) for heme-binding motifs, which eventually led and will perspectively lead to the identification and verification of so far unknown heme-regulated proteins. This systematic approach resulted in a broader picture of the alternative functions of heme as a regulator of proteins. However, knowledge on heme regulation of proteins is still a bottomless barrel that leaves much scope for future research and development.

Background: The notion of heme as a regulator of many physiological processes via transient binding to proteins is one that is recently being acknowledged. The broad spectrum of the effects of heme makes it important to identify further heme-regulated proteins to understand physiological and pathological processes. Moreover, several proteins were shown to be functionally regulated by interaction with heme, yet, for some of them the hemebinding site(s) remain unknown. The presented application HeMoQuest enables identification and qualitative evaluation of such heme-binding motifs from protein sequences. Results: We present HeMoQuest, an online interface (http://bit.ly/hemoquest) to algorithms that provide the user with two distinct qualitative benefits. First, our implementation rapidly detects transient heme binding to nonapeptide motifs from protein sequences provided as input. Additionally, the potential of each predicted motif to bind heme is qualitatively gauged by assigning binding affinities predicted by an ensemble learning implementation, trained on experimentally determined binding affinity data. Extensive testing of our implementation on both existing and new manually curated datasets reveal that our method produces an unprecedented level of accuracy (92%) in identifying those residues assigned "heme binding" in all of the datasets used. Next, the machine learning implementation for the prediction and qualitative assignment of binding affinities to the predicted motifs achieved 71% accuracy on our data. Conclusions: Heme plays a crucial role as a regulatory molecule exerting functional consequences via transient binding to surfaces of target proteins. HeMoQuest is designed to address this imperative need for a computational approach that enables rapid detection of heme-binding motifs from protein datasets. While most existing implementations attempt to predict sites of permanent heme binding, this application is to the best of our knowledge, the first of its kind to address the significance of predicting transient heme binding to proteins.

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.

Many research institutions, clinical diagnostic laboratories, and blood banks are desperately searching for a possibility to identify and quantify heme in different physiological and pathological settings as well as various research applications. The reasons for this are the toxicity of the heme and the fact that it acts as a hemolytic and pro-inflammatory molecule. Heme only exerts these severe and undesired effects when it is not incorporated in hemoproteins. Upon release from the hemoproteins, it enters a biologically available state (labile heme), in which it is loosely associated with proteins, lipids, nucleic acids, or other molecules. While the current methods and procedures for quantitative determination of heme have been used for many years in different settings, their value is limited by the challenging chemical properties of heme. A major cause of inadequate quantification is the separation of labile and permanently bound heme and its high aggregation potential. Thus, none of the current methods are utilized as a generally applicable, standardized approach. The aim of this Feature is to describe and summarize the most common and frequently used chemical, analytical, and biochemical methods for the quantitative determination of heme. Based on this overview, the most promising approaches for future solutions to heme quantification are highlighted.