A small subset of signal peptidase residues are perturbed by signal peptide 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 e-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 (4,5) revealed two shallow hydrophobic cavities adjacent to the Ser–Lys dyad that would be appropriate for docking residues −1 and −3 of the signal peptide. Although members of the SPase I family of enzymes are associated with diverse transport pathways in prokaryotic and eukaryotic systems, the bacterial SPase I uniquely uses a Lys for catalysis; in most higher organisms a conserved His is used instead in a Ser–His dyad. The SPase I family is further set apart from most serine proteases that use a Ser–His–Asp catalytic triad mechanism. Moreover, the enzyme is essential for viability of the bacterium. These features make bacterial SPase I a potentially valuable target for antimicrobial agents and its exposure to the periplasm makes the enzyme accessible to drugs. To pursue this possibility we must understand the mode of recognition of the signal peptide sequence of the preprotein by the enzyme including the topological alignment and the key contacts that lead to peptide proteolysis. To date, these interactions have been inferred from co-crystals of SPase I Δ2–75 with 5S, 6S β-lactam (penem) (4) or with the fatty acid derivative, Arylomycin A2 (5), both inhibitors of SPase I activity, and from molecular modeling using the β-lactamase signal peptide to predict its binding locale (5). However, no direct structural analysis of the enzyme with bound signal peptide or preprotein has been accomplished. This has proven a formidable problem, in part, because of the hydrophobic nature of the components involved. One of two β-sheet domains in SPase I Δ2–75 includes a large exposed hydrophobic surface thought to interact with the membrane surface in the cell. In addition, a key feature of functional signal peptides is the central 10–15 residue hydrophobic core region, making them highly susceptible to aggregation at the high concentration typically required for structural studies. Here we report the first direct measurements on a molecular scale of SPase I-signal peptide interactions using a non-perturbing experimental approach, nuclear magnetic resonance (NMR) spectroscopy. We have expressed and purified doubly 13C, 15N- and singly 15N-isotopically labeled C-terminal E. coli SPase I Δ2–75 and an unlabeled soluble synthetic signal peptide corresponding to the wild-type alkaline phosphatase signal sequence plus four residues of the mature protein. 2D 1H-15N heteronuclear single-quantum correlation (HSQC) spectra were collected as SPase I Δ2–75 was titrated with signal peptide. The chemical shifts of a small subset of specific residues were sensitive to the binding of the signal peptide. The subset of perturbed residues includes: Ile80, Glu82, Gln85, Ile86, Ser88, Gly89, Ser90, Met91, Leu95, Ile101, Gly109, Val132, Lys134, Asp142, Ile144, Lys145, and Thr234. These residues mapped to a defined patch on the protein. This strategy provides the first direct experimental data that reveal which residues of the enzyme are impacted upon substrate binding.