A small subset of signal peptidase residues are perturbed by signal peptide binding.
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 Delta2-75 and an unlabeled soluble synthetic alkaline phosphatase signal peptide, SPase I Delta2-75 was titrated with the signal peptide and 2D (1)H-(15)N 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.
Musial-Siwek
Monika
M
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06552, USA.
Yeagle
Philip L
PL
Kendall
Debra A
DA
D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D008565:Membrane Proteins; D021382:Protein Sorting Signals; D012697:Serine Endopeptidases; C026515:type I signal peptidase