A Small Subset of Signal Peptidase Residues are Perturbed by Signal Peptide Binding

Authors

  • Monika Musial-Siwek,

    1. Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269, USA
      1Current address: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06552, USA.
      2Current address: Office of the Dean-FASN, 360 Dr. Martin Luther King, Jr., Blvd., Hill Hall Room 325, Rutgers, The State University of New Jersey, Newark, NJ 07102, USA.
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  • 1 Philip L. Yeagle,

    1. Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269, USA
      1Current address: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06552, USA.
      2Current address: Office of the Dean-FASN, 360 Dr. Martin Luther King, Jr., Blvd., Hill Hall Room 325, Rutgers, The State University of New Jersey, Newark, NJ 07102, USA.
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    • P.L.Y. and D.A.K. contributed equally to this work.

  • and 2, Debra A. Kendall ,

    Corresponding author
    1. Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269, USA
      1Current address: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06552, USA.
      2Current address: Office of the Dean-FASN, 360 Dr. Martin Luther King, Jr., Blvd., Hill Hall Room 325, Rutgers, The State University of New Jersey, Newark, NJ 07102, USA.
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    • P.L.Y. and D.A.K. contributed equally to this work.


*Debra A. Kendall, debra.kendall@uconn.edu

Abstract

Perturbations of the chemical shifts of a small subset of residues in the catalytically active domain of Escherichia coli signal peptidase I (SPase I) upon binding signal peptide suggest the contact surface on the enzyme for the substrate. SPase I, an integral membrane protein, is vital to preprotein transport in prokaryotic and eukaryotic secretory systems; it binds and proteolyses the N-terminal signal peptide of the preprotein, permitting folding and localization of the mature protein. Employing isotopically labeled C-terminal E. coli SPase I Δ2-75 and an unlabeled soluble synthetic alkaline phosphatase signal peptide, SPase I Δ2-75 was titrated with the signal peptide and 2D 1H-15N heteronuclear single-quantum correlation nuclear magnetic resonance spectra revealed chemical shifts of specific enzyme residues sensitive to substrate binding. These residues were identified by 3D HNCACB, 3D CBCA(CO)NH, and 3D HN(CO) experiments. Residues Ile80, Glu82, Gln85, Ile86, Ser88, Gly89, Ser90, Met91, Leu95, Ile101, Gly109, Val132, Lys134, Asp142, Ile144, Lys145, and Thr234, alter conformation and are likely all in, or adjacent to, the substrate binding site. The remainder of the enzyme structure is unperturbed. Ramifications for conformational changes for substrate docking and catalysis are discussed.

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