Schmalohr, Benjamin Franz: Histidine and Tyrosine-based Heme-binding Motifs for the Prediction of Heme-Regulated Proteins. - Bonn, 2021. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
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author = {{Benjamin Franz Schmalohr}},
title = {Histidine and Tyrosine-based Heme-binding Motifs for the Prediction of Heme-Regulated Proteins},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2021,
month = jun,

note = {The versatile molecule heme (iron protoporphyrin IX) fulfils numerous vital functions as a part of hemoproteins, such as hemoglobin and cytochromes, in which it is essential for oxygen transport, electron transport, and detoxification. Under certain conditions, it can be released from hemoproteins and then regulate cellular processes, but also exert toxic effects. In recent years, significant progress has been made towards the understanding of this regulatory heme. Surface-exposed sequence stretches were found to play a crucial role and with the cysteine-proline dipeptide, the first heme-regulatory motif (HRM) was identified. Histidine and tyrosine were also frequently identified in heme-regulated proteins and heme-binding peptides, but a distinct histidine/tyrosine (H/Y)-based motif has not been discovered yet.
In this thesis, H/Y-based motifs were analyzed systematically. For this purpose, four subclasses of heme-binding peptides (A-D) were established from all possible combinations of histidine and tyrosine, and divided according to spacer length (0-3 amino acids). Over 50 model peptides were synthesized and analyzed in depth by ultraviolet-visible (UV/Vis), resonance Raman, and NMR spectroscopy. It was found that motifs with spacer lengths of 1 (subclass B) and 3 (subclass D) exhibited the strongest heme-binding affinities and most binders were found in these classes. Structural studies revealed that these classes occupy mixed conformational states of penta- and hexacoordination and two NMR structures were solved. Overall, the motifs HXH, HXXXY, and HXXXH were found to be the most promising H/Y-based heme-binding motifs. These findings were combined with those of earlier studies and implemented into a web application called HeMoQuest. This tool allows users to predict HRMs from protein sequence and features a machine learning algorithm, which was trained with experimental peptide data. As an example of H/Y-based motifs, two proteins were studied herein. The first protein is Janus kinase 2 (JAK2), which is critical in nascent erythrocytes to propagate growth signals and increase hemoglobin production. Heme was confirmed to activate JAK2 and its corresponding downstream signaling in the K562 cell line. Furthermore, a YXH and a cysteine-proline (CP) motif were suggested as heme-binding sites in the catalytically active Janus homology 1 (JH1) domain. The second protein, Toll-like receptor 4 (TLR4), was found to be connected to three major heme-related pathologies, i.e. inflammation, thrombosis, and hemolysis. A systematic in silico analysis of heme binding to TLR4 was therefore performed with the aid of HeMoQuest and docking experiments. Therein, a suitable HXXXY motif on TLR4 itself and an interesting interaction with the lipopolysaccharide binding pocket was predicted.
The results presented in this thesis show distinct H/Y-based HRMs on the peptide level, which are used to successfully predict protein candidates. The combined knowledge is made available to the scientific community through a web-based algorithm. Better understanding of regulatory heme binding and heme biology may allow for targeted treatment and prevention of heme-related diseases.},

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