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Intra-subunit Disulfide Determines the Conversion and Structural Stability of CRP Isoforms

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Abstract

C-reactive protein (CRP) is a major human acute-phase reactant that is composed of five identical subunits. CRP dissociates into subunits at inflammatory loci forming monomeric CRP (mCRP) with substantially enhanced activities, which can be further activated by reducing the intra-subunit disulfide bond. However, conformational changes underlying the activation process of CRP are less well understood. Conformational changes accompanying the conversion of CRP to mCRP with or without reduction were examined with circular dichroism spectroscopy, fluorescence spectroscopy, electron microscopy, size-exclusion chromatography, and neoepitope expression. The conversion of CRP to mCRP follows a two-stage process. In the first stage, CRP dissociates into molten globular subunits characterized by intact secondary structure elements with greatly impaired tertiary packing. In the second stage, these intermediates completely lose their native subunit conformation and assemble into high-order aggregates. The inclusion of reductant accelerates the formation of molten globular subunits in the first step and promotes the formation of more compact aggregates in the second stage. We further show a significant contribution of electrostatic interactions to the stabilization of native CRP. The conformational features of dissociated subunits and the aggregation of mCRP may have a key impact on their activities.

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Acknowledgments

We thank Dr. Yi Wu and Dr. Laijun Lai for the critical reading of the manuscript. We thank the Core Facility of School of Life Sciences, Lanzhou University for technical and instrumental support.

Funding

This work was supported by grants from the National Natural Science Foundation of China (31401101, 31570749, 31960141).

Authors’ Contributions

MW and CZ designed the research. CZ, YT, HZ, ZG, JF, GL, ZS, and YF performed the research. MW, CZ, YT, and HZ analyzed the data and wrote the paper. All authors reviewed the results and approved the final version of the manuscript.

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  1. Chun-Miao Zhang, Yu-Bo Tan and Hai-Hong Zhou contributed equally to this work.

Authors and Affiliations

  1. MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou, 730000, People’s Republic of China

    Chun-Miao Zhang, Yu-Bo Tan, Zhong-Bo Ge, Jun-Rui Feng, Guang-Bo Lv, Zhi-Yuan Sun, Yu Fu & Ming-Yu Wang

  2. Gansu Provincial Cancer Hospital, Lanzhou, 730050, People’s Republic of China

    Hai-Hong Zhou

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  1. Chun-Miao Zhang
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Zhang, CM., Tan, YB., Zhou, HH. et al. Intra-subunit Disulfide Determines the Conversion and Structural Stability of CRP Isoforms. Inflammation 43, 466–477 (2020). https://doi.org/10.1007/s10753-019-01130-x

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