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

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 2Δ 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. Keywords membrane protein; NMR; signal peptidase; signal peptide; substrate binding Virtually all proteins destined for extracytoplasmic locations are synthesized as preproteins with a cleavable N-terminal extension sequence called the signal peptide. The signal peptide earmarks the preprotein for entry into the appropriate transport pathway and promotes the membrane translocation process via interactions with components of the transport machinery. In the penultimate step of transport, recognition by a signal peptidase is critical for proteolytic removal of the signal peptide and sets the stage for folding of the mature protein and its final localization.The Escherichia coli signal peptidase I (SPase I) is an integral membrane protein of 37 kDa with two transmembrane segments (residues 4-28 and 58-76), a periplasmic N-terminus and C-terminal catalytic domain (residues 77-323) (1,2). The enzyme is thought to catalyze peptide bond hydrolysis via a catalytic dyad involving the hydroxyl of Ser90 for nucleophilic attack and the ε-amine of Lys145 as the general base to cleave the preprotein. Crystal structures of the soluble catalytic domain (SPase I Δ2-75) in the apo-form (3) and with bound inhibitors

Energetics of SecA Dimerization

“…Three lysines were added at each terminus to enhance solubility of the peptide. Addition of these lysines does not perturb the ability of this peptide to interact with the signal peptidase 79. KAP25 stimulates SecA ATPase activity, indicating that it binds to SecA in a functionally-relevant manner.…”

Structural studies of a signal peptide in complex with signal peptidase I cytoplasmic domain: The stabilizing effect of membrane‐mimetics on the acquired fold

“…Furthermore, it is consistent with our previous work showing the interaction of this same peptide with SecA, 15 another component of the Sec-dependent transport pathway, and a comparable peptide with SPase I Δ2-75. 10 Moreover, we found that the enzyme slowly cleaves the KAP25 signal peptide with about 7, 19 and 27% cleavage observed at 24, 36 and 48 hours of incubation, respectively (data not shown). This slow extent of cleavage is consistent with previous studies that report a decrease in preprotein digestion by SPase I and the soluble form in the absence of Triton X-100 and for a small peptide relative to the preprotein.…”

“…To circumvent this issue, we previously reported that the addition of three lysine residues to each termini is a good strategy to design highly soluble signal peptides, 10,15 which can be studied by NMR or other biophysical techniques. In this study, we used a comparable approach to examine the E. Coli alkaline phosphatase signal peptide (KAP25).…”

“…8,9 In addition, NMR analysis indicates the SPase I Δ2-75 amide resonances shift upon peptide binding, a finding which has facilitated docking of the amino acids at these positions. 10 Although there is considerable evidence regarding the importance of the signal peptide -1 and -3 residues for SPase I interaction, little is known about how the signal peptide interacts with the enzyme for cleavage. The structure of the Methanococcus jannaschii SecYEG channel suggests that the signal peptide region of the preprotein enters the SecYEG protein conducting channel by promoting the displacement of a domain of SecY that serves as the channel plug.…”

Microseparation of membrane proteins

“…Such a complex system plays an essential role in many biological activities such as cell -cell communication [3,4], receptor-mediated signal transduction [5,6], enzyme catalysis [7,8], selective transport of metabolites [9,10], and pharmacological actions [11,12]. Thus, membrane protein studies are important for understanding their functions in different biological processes.…”