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Biophysical and functional characterization of N-terminal domain of Human Interferon Regulatory Factor 6

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Abstract

Background

Interferon regulatory factor 6 (IRF6) has a key function in palate fusion during palatogenesis during embryonic development, and mutations in IRF6 cause orofacial clefting disorders.

Methods and results

The in silico analysis of IRF6 is done to obtain leads for the domain boundaries and subsequently the sub-cloning of the N-terminal domain of IRF6 into the pGEX-2TK expression vector and successfully optimized the overexpression and purification of recombinant glutathione S-transferase-fused NTD-IRF6 protein under native conditions. After cleavage of the GST tag, NTD-IRF6 was subjected to protein folding studies employing Circular Dichroism and Intrinsic fluorescence spectroscopy at variable pH, temperature, and denaturant. CD studies showed predominantly alpha-helical content and the highest stability of NTD-IRF6 at pH 9.0. A comparison of native and renatured protein depicts loss in the secondary structural content. Intrinsic fluorescence and quenching studies have identified that tryptophan residues are majorly present in the buried areas of the protein and a small fraction was on or near the protein surface. Upon the protein unfolding with a higher concentration of denaturant urea, the peak of fluorescence intensity decreased and red shifted, confirming that tryptophan residues are majorly present in a more polar environment. While regulating IFNβ gene expression during viral infection, the N-terminal domain binds to the promoter region of Virus Response Element-Interferon beta (VRE-IFNβ). Along with the protein folding analysis, this study also aimed to identify the DNA-binding activity and determine the binding affinities of NTD-IRF6 with the VRE-IFNβ promoter region. The protein–DNA interaction is specific as demonstrated by gel retardation assay and the kinetics of molecular interactions as quantified by Biolayer Interferometry showed a strong affinity with an affinity constant (KD) value of 7.96 × 10–10 M.

Conclusion

NTD-IRF6 consists of a mix of α-helix and β-sheets that show temperature-dependent cooperative unfolding between 40 °C and 55 °C. Urea-induced unfolding shows moderate tolerance to urea as the mid-transition concentration of urea (Cm) is 3.2 M. The tryptophan residues are majorly buried as depicted by fluorescence quenching studies. NTD-IRF6 has a specific and high affinity toward the promoter region of VRE-IFNβ.

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Abbreviations

NTD:

N-terminal domain

SEC:

Size exclusion chromatography

GST:

Glutathione S-transferase

IFNβ:

Interferon beta

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Acknowledgements

Human IRF6 cloned in pUC57 vector, a kind gift by Prof. W. Royer (UMASS, U.S.A.), and Human VRE-IFNβ cloned in pTZ57R/T vector was kindly gifted by Dr. Krishna Prakash, CUSB, Bihar, India. For funding, B.K.S. is thankful to the CSIR fellowship, Dr. T.K. is thankful to the DBT Bio-CARe project, and P.M. is thankful to the UGC non-net fellowship for supporting this work.

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Authors and Affiliations

  1. Department of Life Science, School of Earth, Biological and Environmental Sciences, Central University of South Bihar, Gaya, Bihar, 824236, India

    Binita Kumari Sinha, Priyabrata Meher & Tara Kashav

  2. Department of Biotechnology, School of Earth, Biological and Environmental Sciences, Central University of South Bihar, Gaya, Bihar, India

    Krishna Prakash

  3. School of Life Sciences, Jawaharlal Nehru University, New Delhi, India

    Devbrat Kumar & Samudrala Gourinath

  4. Department of Biochemical Engineering and Biotechnology, IIT Delhi, New Delhi, India

    Shilpi Kumari

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  1. Binita Kumari Sinha
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  7. Tara Kashav
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Contributions

BKS has performed all the experiments and in silico work along with writing the original draft of the manuscript. DK gave valuable suggestions for purification, CD analysis, and fluorescence experiments. PM and SK have contributed to NTD cloning. KP gave valuable suggestions for the EMSA assay. SG guided and provided the facility for purification and characterization studies. TK was responsible for the concept, planning the experiments, and discussing and finalizing the manuscript.

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Correspondence to Tara Kashav.

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Sinha, B.K., Kumar, D., Meher, P. et al. Biophysical and functional characterization of N-terminal domain of Human Interferon Regulatory Factor 6. Mol Biol Rep 51, 380 (2024). https://doi.org/10.1007/s11033-024-09205-1

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