Insights into the molecular basis and mechanism of heme-triggered TLR4 signalling: The role of heme-binding motifs in TLR4 and MD2.

Haemolytic disorders, such as sickle cell disease, are accompanied by the release of high amounts of labile heme into the intravascular compartment resulting in the induction of proinflammatory and prothrombotic complications in affected patients. In addition to the relevance of heme-regulated proteins from the complement and blood coagulation systems, activation of the TLR4 signalling pathway by heme was ascribed a crucial role in the progression of these pathological processes. Heme binding to the TLR4-MD2 complex has been proposed recently, however, essential mechanistic information of the processes at the molecular level, such as heme-binding kinetics, the heme-binding capacity and the respective heme-binding sites (HBMs) is still missing. We report the interaction of TLR4, MD2 and the TLR4-MD2 complex with heme and the consequences thereof by employing biochemical, spectroscopic, bioinformatic and physiologically relevant approaches. Heme binding occurs transiently through interaction with up to four HBMs in TLR4, two HBMs in MD2 and at least four HBMs in their complex. Functional studies highlight that mutations of individual HBMs in TLR4 preserve full receptor activation by heme, suggesting that heme interacts with TLR4 through different binding sites independently of MD2. Furthermore, we confirm and extend the major role of TLR4 for heme-mediated cytokine responses in human immune cells.

Shapes and Patterns of Heme-Binding Motifs in Mammalian Heme-Binding Proteins.

Heme is a double-edged sword. On the one hand, it has a pivotal role as a prosthetic group of hemoproteins in many biological processes ranging from oxygen transport and storage to miRNA processing. On the other hand, heme can transiently associate with proteins, thereby regulating biochemical pathways. During hemolysis, excess heme, which is released into the plasma, can bind to proteins and regulate their activity and function. The role of heme in these processes is under-investigated, with one problem being the lack of knowledge concerning recognition mechanisms for the initial association of heme with the target protein and the formation of the resulting complex. A specific heme-binding sequence motif is a prerequisite for such complex formation. Although numerous short signature sequences indicating a particular protein function are known, a comprehensive analysis of the heme-binding motifs (HBMs) which have been identified in proteins, concerning specific patterns and structural peculiarities, is missing. In this report, we focus on the evaluation of known mammalian heme-regulated proteins concerning specific recognition and structural patterns in their HBMs. The Cys-Pro dipeptide motifs are particularly emphasized because of their more frequent occurrence. This analysis presents a comparative insight into the sequence and structural anomalies observed during transient heme binding, and consequently, in the regulation of the relevant protein.

Host and viral proteins involved in SARS-CoV-2 infection differentially bind heme.

In most severe cases, SARS-CoV-2-induced autoimmune reactions have been associated with hemolytic complications. Hemolysis-derived heme from ruptured red blood cells has been shown to trigger a variety of fatal proinflammatory and procoagulant effects, which might deteriorate the progression of COVID-19. In addition, the virus itself can induce proinflammatory signals via the accessory protein 7a. Direct heme binding to the SARS-CoV-2 protein 7a ectodomain and other COVID-19-related proteins has been suggested earlier. Here, we report the experimental analysis of heme binding to the viral proteins spike glycoprotein, protein 7a as well as the host protein ACE2. Thus, protein 7a chemical synthesis was established, including an in-depth analysis of the three different disulfide-bonded isomers. Surface plasmon resonance spectroscopy and in silico studies confirm a transient, biphasic binding behavior, and heme-binding affinities in the nano- to low micromolar range. These results confirm the presence of the earlier identified heme-binding motifs and emphasize the relevance for consideration of labile heme in preexisting or SARS-CoV-2-induced hemolytic conditions in COVID-19 patients.