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Biophysical characterization of the DNA binding motif of human phospholipid scramblase 1

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Abstract

Human phospholipid scramblase 1 (hPLSCR1) is a 37 kDa multi-compartmental protein, which was initially identified as a Ca2+-dependent phospholipid translocator upon localizing to the plasma membrane. However, under certain physiological conditions, hPLSCR1 is localized to the nucleus where it interacts with the IP3R1 promoter (IP3R1P) and regulates its expression. In this study, the DNA binding properties of hPLSCR1 and ∆100-hPLSCR1 (N-terminal 100 amino acids deleted from hPLSCR1) were investigated by using a synthetic IP3R1P oligonucleotide and nonspecific scrambled-sequence oligonucleotides. Our results revealed that hPLSCR1 and ∆100-hPLSCR1 were bound to IP3R1P oligos in a 1:1 stoichiometry. In addition, ∆160-hPLSCR1 could not bind to IP3R1P oligonucleotide suggesting that the proposed DNA binding motif is the actual DNA binding motif. Specific binding of hPLSCR1 and ∆100-hPLSCR1 to IP3R1P oligos was demonstrated by fluorescence anisotropy assay. ITC analysis revealed that hPLSCR1 binds to IP3R1P oligos with Kd = 42.91 ± 0.23 nM. Binding of IP3R1P oligos induces β-sheet formation in hPLSCR1 and increases the thermal stability of hPLSCR1 and ∆100-hPLSCR1. Binding of IP3R1P oligos to hPLSCR1 altered the B-form of the DNA, which was not observed with ∆100-hPLSCR1. Results from this study suggest that (i) ∆100-hPLSCR1 possesses a minimal DNA binding region and (ii) structural alterations of IP3R1P oligo by hPLSCR1 require proline-rich N-terminal region.

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Acknowledgements

SR is supported by Senior Research Fellowship from CSIR. US acknowledge the Ministry of Human Resource and Development, Government of India for fellowship. Authors acknowledge the Department of Science and Technology and the Indian Institute of Technology Madras for their financial support for CD spectroscopy, ITC, and DSC instruments.

Funding

This work was funded by the Council of Scientific and Industrial Research (CSIR), Government of India (Grant ID: 37/1651/15-EMR-II).

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Authors and Affiliations

  1. Applied and Industrial Microbiology Laboratory, Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai, 600036, India

    Sarika Rayala, Ulaganathan Sivagnanam & Sathyanarayana N. Gummadi

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  1. Sarika Rayala
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  2. Ulaganathan Sivagnanam
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  3. Sathyanarayana N. Gummadi
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SNG coordinated the study. SNG and SR designed the experiments and SR performed all the experiments. US performed gel filtration chromatography and electrophoretic mobility shift assay. SR, US and SNG prepared the manuscript. All authors declare no competing interests.

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Correspondence to Sathyanarayana N. Gummadi.

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Rayala, S., Sivagnanam, U. & Gummadi, S.N. Biophysical characterization of the DNA binding motif of human phospholipid scramblase 1. Eur Biophys J 51, 579–593 (2022). https://doi.org/10.1007/s00249-022-01621-0

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