Biopolym. Cell. 2025; 41(1):32-41.
http://dx.doi.org/10.7124/bc.000B0F
Structure and Function of Biopolymers
Primary insights into structure and structururally determined features of C2 domain of Bcr
1, 2Kravchuk I. V., 1Gurianov D. S., 1Antonenko S. V., 1Telegeev G. D.
  1. Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03143
  2. Department of Microbiology and Parasitology with the Basics of Immunology, O.O. Bogomolets National Medical University
    13, Taras Shevchenko Blvd., Kyiv, Ukraine, 01601

Abstract

Aim. This study aimed to estimate the structure and lipid–binding capacity of the C2 domain of Bcr. Methods. The secondary and tertiary structures were predicted using various bioinformatic tools. A DNA fragment encoding the C2 domain was introduced into the expression vector pET28 using sequence and ligation–independent cloning techniques. The purified recombinant C2 domain was used to obtain circular dichroism spectra and perform a lipid–binding assay. Results. The structure predictions indicate that the C2 domain of Bcr likely adopts a typical beta–sandwich structure with type II topology. The circular dichroism data on the purified recombinant C2 domain confirmed the expected predominance of beta–structures. Binding to eight phospholipids was identified. Conclusions. The recombinant C2 domain of Bcr has a structure comparable to other well–studied C2 domains. Binding to various lipids suggests a possible specific localization of the Bcr protein on different cellular membrane structures.
Keywords: C2 domain, Bcr, circular dichroism, lipid binding
Full text: (PDF, in English)

References

[1] Sherbenou DW, Druker BJ. Applying the discovery of the Philadelphia chromosome. J Clin Invest. 2007; 117(8):2067-74.
[2] Advani AS, Pendergast AM. Bcr-Abl variants: biological and clinical aspects. Leuk Res. 2002; 26(8):713-20.
[3] Pane F, Intrieri M, Quintarelli C, Izzo B, Muccioli GC, Salvatore F. BCR/ABL genes and leukemic phenotype: from molecular mechanisms to clinical correlations. Oncogene. 2002; 21(56):8652-67.
[4] Haskovec C, Ponzetto C, Polák J, Maritano D, Zemanová Z, Serra A, Michalová K, Klamová H, Cermák J, Saglio G. P230 BCR/ABL protein may be associated with an acute leukaemia phenotype. Br J Haematol. 1998; 103(4):1104-8.
[5] An X, Tiwari AK, Sun Y, Ding PR, Ashby CR Jr, Chen ZS. BCR-ABL tyrosine kinase inhibitors in the treatment of Philadelphia chromosome positive chronic myeloid leukemia: a review. Leuk Res. 2010; 34(10):1255-68.
[6] Leak S, Horne GA, Copland M. Targeting BCR-ABL1-positive leukaemias: a review article. Camb Prism Precis Med. 2023; 1:e21.
[7] Rossari F, Minutolo F, Orciuolo E. Past, present, and future of Bcr-Abl inhibitors: from chemical development to clinical efficacy. J Hematol Oncol. 2018; 11(1):84.
[8] Zaker E, Nouri N, Sorkhizadeh S, Ghasemirad H, Hajijafari AH, Zare F. The importance of personalized medicine in chronic myeloid leukemia management: a narrative review. Egypt J Med Hum Genet. 2023; 24(1):31.
[9] Jones DT. Protein secondary structure prediction based on position-specific scoring matrices. J Mol Biol. 1999; 292(2):195-202.
[10] Buchan DWA, Jones DT. The PSIPRED Protein Analysis Workbench: 20 years on. Nucleic Acids Res. 2019; 47(W1):W402-W407.
[11] Moffat L, Jones DT. Increasing the accuracy of single sequence prediction methods using a deep semi-supervised learning framework. Bioinformatics. 2021; 37(21):3744-51.
[12] Kandathil SM, Greener JG, Lau AM, Jones DT. Ultrafast end-to-end protein structure prediction enables high-throughput exploration of uncharacterized proteins. Proc Natl Acad Sci U S A. 2022; 119(4):e2113348119.
[13] Micsonai A, Wien F, Kernya L, Lee YH, Goto Y, Réfrégiers M, Kardos J. Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy. Proc Natl Acad Sci U S A. 2015; 112(24):E3095-103.
[14] Micsonai A, Wien F, Bulyáki É, Kun J, Moussong É, Lee YH, Goto Y, Réfrégiers M, Kardos J. BeStSel: a web server for accurate protein secondary structure prediction and fold recognition from the circular dichroism spectra. Nucleic Acids Res. 2018; 46(W1):W315-W322.
[15] Micsonai A, Bulyáki É, Kardos J. BeStSel: From Secondary Structure Analysis to Protein Fold Prediction by Circular Dichroism Spectroscopy. In Structural Genomics: General Applications; Chen, Y.W., Yiu, C.-P.B., Eds.; Springer US: New York, NY, 2021; pp. 175-89 ISBN 978-1-07-160892-0.
[16] Micsonai A, Moussong É, Murvai N, Tantos Á, Tőke O, Réfrégiers M, Wien F, Kardos J. Disordered-Ordered Protein Binary Classification by Circular Dichroism Spectroscopy. Front Mol Biosci. 2022; 9:863141.
[17] Micsonai A, Moussong É, Wien F, Boros E, Vadászi H, Murvai N, Lee YH, Molnár T, Réfrégiers M, Goto Y, Tantos Á, Kardos J. BeStSel: webserver for secondary structure and fold prediction for protein CD spectroscopy. Nucleic Acids Res. 2022; 50(W1):W90-W98.
[18] Li MZ, Elledge SJ. SLIC: a method for sequence- and ligation-independent cloning. Methods Mol Biol. 2012; 852:51-9.
[19] Studier FW. Protein production by auto-induction in high density shaking cultures. Protein Expr Purif. 2005; 41(1):207-34.
[20] Ernst O, Zor T. Linearization of the bradford protein assay. J Vis Exp. 2010; (38):1918.
[21] Rizo J, Südhof TC. C2-domains, structure and function of a universal Ca2+-binding domain. J Biol Chem. 1998; 273(26):15879-82.
[22] Larsen AH, Sansom MSP. Binding of Ca2+-independent C2 domains to lipid membranes: A multi-scale molecular dynamics study. Structure. 2021; 29(10):1200-13.e2.
[23] Corbalan-Garcia S, Gómez-Fernández JC. Signaling through C2 domains: more than one lipid target. Biochim Biophys Acta. 2014; 1838(6):1536-47.
[24] Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, Tunyasuvunakool K, Bates R, Žídek A, Potapenko A, Bridgland A, Meyer C, Kohl SAA, Ballard AJ, Cowie A, Romera-Paredes B, Nikolov S, Jain R, Adler J, Back T, Petersen S, Reiman D, Clancy E, Zielinski M, Steinegger M, Pacholska M, Berghammer T, Bodenstein S, Silver D, Vinyals O, Senior AW, Kavukcuoglu K, Kohli P, Hassabis D. Highly accurate protein structure prediction with AlphaFold. Nature. 2021; 596(7873):583-9.
[25] Varadi M, Bertoni D, Magana P, Paramval U, Pidruchna I, Radhakrishnan M, Tsenkov M, Nair S, Mirdita M, Yeo J, Kovalevskiy O, Tunyasuvunakool K, Laydon A, Žídek A, Tomlinson H, Hariharan D, Abrahamson J, Green T, Jumper J, Birney E, Steinegger M, Hassabis D, Velankar S. AlphaFold Protein Structure Database in 2024: providing structure coverage for over 214 million protein sequences. Nucleic Acids Res. 2024; 52(D1):D368-D375.
[26] Bohdanowicz M, Grinstein S. Role of phospholipids in endocytosis, phagocytosis, and macropinocytosis. Physiol Rev. 2013; 93(1):69-106.
[27] Marat AL, Haucke V. Phosphatidylinositol 3-phosphates-at the interface between cell signalling and membrane traffic. EMBO J. 2016; 35(6):561-79.
[28] Posor Y, Jang W, Haucke V. Phosphoinositides as membrane organizers. Nat Rev Mol Cell Biol. 2022; 23(12):797-816.
[29] Yang Y, Lee M, Fairn GD. Phospholipid subcellular localization and dynamics. J Biol Chem. 2018; 293(17):6230-40.
[30] Lennartz MR. Phospholipases and phagocytosis: the role of phospholipid-derived second messengers in phagocytosis. Int J Biochem Cell Biol. 1999; 31(3-4):415-30.
[31] Yeung T, Gilbert GE, Shi J, Silvius J, Kapus A, Grinstein S. Membrane phosphatidylserine regulates surface charge and protein localization. Science. 2008; 319(5860):210-3.
[32] Hasegawa J, Uchida Y, Mukai K, Lee S, Matsudaira T, Taguchi T. A Role of Phosphatidylserine in the Function of Recycling Endosomes. Front Cell Dev Biol. 2021; 9:783857.
[33] Miroshnychenko D, Dubrovska A, Maliuta S, Telegeev G, Aspenström P. Novel role of pleckstrin homology domain of the Bcr-Abl protein: analysis of protein-protein and protein-lipid interactions. Exp Cell Res. 2010; 316(4):530-42.
[34] Antonenko SV, Gurianov DS, Kravchuk IV, Dybkov MV, Shvachko LP, Telegeev GD. Role of BCR and FNBP1 Proteins in Phagocytosis as a Model of Membrane Rearrangements with Chronic Myelogenous Leukemia. Cytol Genet. 2023; 57(4):291-7.
[35] Nalefski EA, Falke JJ. The C2 domain calcium-binding motif: structural and functional diversity. Protein Sci. 1996; 5(12):2375-90.
[36] Nalefski EA, Wisner MA, Chen JZ, Sprang SR, Fukuda M, Mikoshiba K, Falke JJ. C2 domains from different Ca2+ signaling pathways display functional and mechanistic diversity. Biochemistry. 2001; 40(10):3089-100.
[37] Malmberg NJ, Varma S, Jakobsson E, Falke JJ. Ca2+ activation of the cPLA2 C2 domain: ordered binding of two Ca2+ ions with positive cooperativity. Biochemistry. 2004; 43(51):16320-8.
[38] Lee JO, Yang H, Georgescu MM, Di Cristofano A, Maehama T, Shi Y, Dixon JE, Pandolfi P, Pavletich NP. Crystal structure of the PTEN tumor suppressor: implications for its phosphoinositide phosphatase activity and membrane association. Cell. 1999; 99(3):323-34.