Search for putative heme binding sites in the integrated membrane protein of human erythrocytes SLC4A1 and protein kinases SYK and LYN

Keywords: SLC4A1, band 3 protein, phosphorylation, SYK, LYN, heme binding, erythrocytes, hemolysis, molecular docking, HeMoQuest, HemeBind


SLC4A1 protein or band 3 protein (band 3) is one of the most common erythrocyte membrane proteins. Along with the function of the anion exchanger, it contributes to the control of cell shape and lifespan through the formation of various complexes with cytoskeletal components and enzymes. Under oxidative stress, the protein oxidative modifications occur, in particular, due to the binding of hemoglobin aggregates, but the direct effect of heme as the major product of hemoglobin degradation on the band 3 protein activity has not been described in the literature. It is known that hemolytic conditions are accompanied by phosphorylation of the SLC4A1 protein, non-receptor tyrosine protein kinases LYN (by residue Y359), and SYK (by residues Y8 and Y21), while SYK kinase inhibitors have a stabilizing effect on erythrocytes. The regulatory effect of heme on Src kinases, which include SYK and LIN kinases, has been clarified, but the sites of their interaction with heme have not been investigated. Therefore, the aim of this study was to predict putative heme binding sites in the integral protein of the erythrocyte membrane SLC4A1, as well as in the protein kinases SYK and LYN and their complexes with SLC4A1, modeled in silico. Sequence analysis of proteins with HeMoQuest revealed several nonapeptides with potential heme binding sites in the SLC4A1 protein, including the cytosolic His98 and Tyr553 and Tyr555 residues in the region between the TM5 and TM6 transmembrane segments. These residues, as well as the amino acids Tyr216, His303, and His192, were also predicted as heme-binding sites by HemeBind tool. The largest number of putative heme binding sites was found for SYK protein kinase, including the two cytosolic residues Tyr216 and His303. Molecular docking of the SLC4A1 protein revealed a heme binding site in the cavity between His192 and region 173–176 in the cytosolic domain structure (PDB ID 4KY9 and 1HYN), also as part of a simulated complex with SYK or LYN kinases. It should be noted that site 175–185 is known as the ankyrin binding site. Docking heme to the membrane domain (PDB ID 4YZF) revealed a putative heme binding site near Lys539 in TM5, which, according to the literature, belongs to one of the reactive centers sensitive to the anionic transport inhibitor DIDS. Molecular docking to SYK protein kinase with ATP in the active site (PDB ID 4FL2) revealed two putative heme binding sites – near Tyr64 and near His243, but when ATP was removed from pdb-file, the heme occupied a nucleotide binding site in the cavity near Lys402 and His531. LYN protein kinase (PDB ID 5XY1) had a heme binding site near Tyr321 in the structure with inhibitor molecule (piperazine derivative). After removing the inhibitor, the heme occupied its area near Glu290 and Ala371. In most model complexes, the band 3 protein was revealed to be a more likely heme binding site than LYN and SYK protein kinases, but kinases with free active sites can apparently bind a heme instead of substrate, which will interfere phosphorylation. Disruption of band 3 protein under heme accumulation can inhibit an anion transport or complicate the formation of SLC4A1 complexes with cytoskeletal proteins that together with the effects on phosphorylation could be a mechanism for reducing erythrocyte stability.


Download data is not yet available.

Author Biographies

T. Barannik, V.N. Karazin Kharkiv National University

Svobody Sq., 4, Kharkiv, Ukraine, 61022,

M. Lebedieva, V.N. Karazin Kharkiv National University

Svobody Sq., 4, Kharkiv, Ukraine, 61022,


Belcher J.D., Beckman J.D., Balla G. et al. (2010). Heme degradation and vascular injury. Antioxid. Redox Signal, 12(2), 233–248.

Brunati A.M., Bordin L., Clari G. et al. (2000). Sequential phosphorylation of protein band 3 by SYK and LYN tyrosine kinases in intact human erythrocytes: identification of primary and secondary phosphorylation sites. Blood, 96(4), 1550–1557.

Chang H.C., Huang D.Y., Wu M.S. et al. (2017). Spleen tyrosine kinase mediates the actions of EPO and GM-CSF and coordinates with TGF-β in erythropoiesis. Biochim. Biophys. Acta – Mol. Cell Res., 1864(4), 687–696.

Chu H., Low P.S. (2006). Mapping of glycolytic enzyme-binding sites on human erythrocyte band 3. Biochem J., 400(1), 143–151.

Grey J.L., Kodippili G.C., Simon K., Low P.S. (2012). Identification of contact sites between ankyrin and band 3 in the human erythrocyte membrane. Biochemistry, 51(34), 6838–6846.

Grosdidier A., Zoete V., Michielin O. (2011). SwissDock, a protein-small molecule docking web service based on EADock DSS. Nucleic Acids Res., 39 (Web Server issue), W270–277.

Johansson M.U., Zoete V., Michielin O., Guex N. (2012). Defining and searching for structural motifs using DeepView/Swiss-PdbViewer. BMC Bioinformatics, 13:173.

Kodippili G.C., Spector J., Hale J. et al. (2012). Analysis of the mobilities of band 3 populations associated with ankyrin protein and junctional complexes in intact murine erythrocytes. J. Biol. Chem., 287(6), 4129–4138.

Liu R., Hu J. (2011). HemeBIND: a novel method for heme binding residue prediction by combining structural and sequence information. BMC Bioinformatics, 12(207), 1–6.

Pantaleo A., Ferru E., Pau M.C. et al. (2016). Band 3 erythrocyte membrane protein acts as redox stress sensor leading to its phosphorylation by p (72) Syk. Oxid. Med. Cell Longev., 2016, 6051093.

Paul George A.A., Lacerda M., Syllwasschy B.F. et al. (2020). HeMoQuest: A webserver for qualitative prediction of transient heme binding to protein motifs. BMC Bioinformatics, 21, 124.

Pettersen E.F., Goddard T.D., Huang C.C. et al. (2004). UCSF Chimera – a visualization system for exploratory research and analysis. J. Comput. Chem., 25(13), 1605–1612.

Pierce B.G., Wiehe K., Hwang H. et al. (2014). ZDOCK server: interactive docking prediction of protein-protein complexes and symmetric multimers. Bioinformatics, 30(12), 1771–1773.

Reithmeier R.A., Casey J.R., Kalli A.C. et al. (2016). Band 3, the human red cell chloride/bicarbonate anion exchanger (AE1, SLC4A1), in a structural context. Biochim. Biophys. Acta, 1858(7 Pt A), 1507–1532.

Roumenina L.T., Rayes J., Lacroix-Desmazes S., Dimitrov J.D. (2016). Heme: modulator of plasma systems in hemolytic diseases. Trends Mol. Med., 22(3), 200–213.

Schneidman-Duhovny D., Inbar Y., Nussinov R., Wolfson H.J. (2005). PatchDock and SymmDock: servers for rigid and symmetric docking. Nucleic Acids Res., 33 (Web Server issue), W363–367.

Slavova-Azmanova N.S., Kucera N., Louw A. et al. (2014). LYN kinase plays important roles in erythroid expansion, maturation and erythropoietin receptor signalling by regulating inhibitory signalling pathways that control survival. Biochem J., 459(3), 455–466.

Walder J.A., Chatterjee R., Steck T.L. et al. (1984). The interaction of hemoglobin with the cytoplasmic domain of band 3 of the human erythrocyte membrane. J. Biol. Chem., 259(16), 10238–10346.

Weber R.E., Voelter W., Fago A. et al. (2004). Modulation of red cell glycolysis: interactions between vertebrate hemoglobins and cytoplasmic domains of band 3 red cell membrane proteins. Am. J. Physiol. Regul. Integr. Comp. Physiol., 287(2), R454–464.

Welbourn E.M., Wilson M.T., Yusof A. et al. (2017). The mechanism of formation, structure and physiological relevance of covalent hemoglobin attachment to the erythrocyte membrane. Free Radic. Biol. Med., 103, 95–106.

Wißbrock A., Paul George A.A., Brewitz H.H. et al. (2019).The molecular basis of transient heme-protein interactions: analysis, concept and implementation. Biosci. Rep., 39(1), BSR20181940.

Yao X., Balamurugan P., Arvey A. et al. (2010). Heme controls the regulation of protein tyrosine kinases Jak2 and Src. Biochem. Biophys. Res. Commun., 403(1), 30–35.

Zhang L., Guarente L. (1995). Heme binds to a short sequence that serves a regulatory function in diverse proteins. EMBO J., 14, 313–320.

How to Cite
Barannik, T., & Lebedieva, M. (2021). Search for putative heme binding sites in the integrated membrane protein of human erythrocytes SLC4A1 and protein kinases SYK and LYN. The Journal of V.N.Karazin Kharkiv National University. Series «Biology», 36, 4-15.