C-terminal, endoplasmic reticulum-lumenal domain of prosurfactant protein C – structural features and membrane interactions

Authors

  • Cristina Casals,

    1.  Department of Biochemistry and Molecular Biology I & CIBER Enfermedades Respiratorias, Complutense University of Madrid, Spain
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  • Hanna Johansson,

    1.  Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, The Biomedical Centre, Uppsala, Sweden
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  • Alejandra Saenz,

    1.  Department of Biochemistry and Molecular Biology I & CIBER Enfermedades Respiratorias, Complutense University of Madrid, Spain
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  • Magnus Gustafsson,

    1.  Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, The Biomedical Centre, Uppsala, Sweden
    2.  Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden
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  • Carlos Alfonso,

    1.  Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain
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  • Kerstin Nordling,

    1.  Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, The Biomedical Centre, Uppsala, Sweden
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  • Jan Johansson

    1.  Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, The Biomedical Centre, Uppsala, Sweden
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J. Johansson, Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, The Biomedical Centre, 751 23 Uppsala, Sweden
Fax: +46 18 550762
Tel: +46 18 4714065
E-mail: jan.johansson@afb.slu.se

Abstract

Surfactant protein C (SP-C) constitutes the transmembrane part of prosurfactant protein C (proSP-C) and is α-helical in its native state. The C-terminal part of proSP-C (CTC) is localized in the endoplasmic reticulum lumen and binds to misfolded (β-strand) SP-C, thereby preventing its aggregation and amyloid fibril formation. In this study, we investigated the structure of recombinant human CTC and the effects of CTC–membrane interaction on protein structure. CTC forms noncovalent trimers and supratrimeric oligomers. It contains two intrachain disulfide bridges, and its secondary structure is significantly affected by urea or heat only after disulfide reduction. The postulated Brichos domain of CTC, with homologs found in proteins associated with amyloid and proliferative disease, is up to 1000-fold more protected from limited proteolysis than the rest of CTC. The protein exposes hydrophobic surfaces, as determined by CTC binding to the environment-sensitive fluorescent probe 1,1′-bis(4-anilino-5,5′-naphthalenesulfonate). Fluorescence energy transfer experiments further reveal close proximity between bound 1,1′-bis(4-anilino-5,5′-naphthalenesulfonate) and tyrosine residues in CTC, some of which are conserved in all Brichos domains. CTC binds to unilamellar phospholipid vesicles with low micromolar dissociation constants, and differential scanning calorimetry and CD analyses indicate that membrane-bound CTC is less structurally ordered than the unbound protein. The exposed hydrophobic surfaces and the structural disordering that result from interactions with phospholipid membranes suggest a mechanism whereby CTC binds to misfolded SP-C in the endoplasmic reticulum membrane.

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