Fluorescence spectroscopy of soluble E. coli SPase I Δ2-75 reveals conformational changes in response to ligand binding

Abstract

The bacterial Sec pathway is responsible for the translocation of secretory preproteins. During the later stages of transport, the membrane-embedded signal peptidase I (SPase I) cleaves the signal peptide from a preprotein. We used tryptophan fluorescence spectroscopy of a soluble, catalytically active E. coli SPase I Δ2-75 enzyme to study its dynamic conformational changes while in solution and when interacting with lipids and signal peptides. We generated four single Trp SPase I Δ2-75 mutants, W261, W284, W300, and W310. Based on fluorescence quenching experiments, W300 and W310 were found to be more solvent accessible than W261 and W284 in the absence of ligands. W300 and W310 inserted into lipids, consistent with their location at the enzyme's proposed membrane-interface region, while the solvent accessibilities of W261, W284, and W300 were modified in the presence of signal peptide, suggesting propagation of structural changes beyond the active site in response to peptide binding. The signal peptide binding affinity for the enzyme was measured via FRET experiments and the Kd determined to be 4.4 μM. The location of the peptide with respect to the enzyme was also established; this positioning is crucial for the peptide to gain access to the enzyme active site as it emerges from the translocon into the membrane bilayer. These studies reveal enzymatic structural changes required for preprotein proteolysis as it interacts with its two key partners, the signal peptide and membrane phospholipids.

Keywords: acrylamide quenching; preprotein cleavage; protein transport; signal peptidase; signal peptide.

FIG 1

Wild-type and single tryptophan SPase I Δ2-75 enzymes. (A) Crystal structure of E. coli SPase I Δ2-75 (PDB 1T7D) is shown with the location of tryptophan residues highlighted in yellow and the serine-lysine catalytic dyad (S90 and K145) labeled in magenta. The enzyme active site is circled. (B) The name of the four single tryptophan mutants generated for this study and the residues substituted with phenylalanine in each are shown. (C) Fluorescence emission spectra of the wild-type and four single tryptophan SPase I Δ2-75 mutants are shown. The proteins were excited at 295 nm, and the emission spectra were measured from 310–400 nm as described in Materials and Methods.

FIG 2

In vitro processing of the substrate proOmpA nuclease A by the wild-type and single tryptophan SPase I Δ2-75 enzymes. Cleavage assays were carried out without (-) or with 0.75 μM (1X), 3.75 μM (5X) and 7.5 μM (10X) of the enzyme, and 15 μM of the substrate in 50 mM Tris-HCl, pH 8.0 and 1% Triton X-100 at 37 °C for 2 hours. Samples were run on a 12.5% SDS-PAGE gel followed by Coomassie Brilliant Blue staining. MW represents the molecular weight standard. The fusion preprotein proOmpA nuclease A and the nuclease A product are shown as preprotein (P) and mature (M), respectively.

FIG 3

Fluorescence quenching with acrylamide and 10-DN for the wild-type and single Trp SPase I Δ2-75 enzymes in aqueous and lipid environments. (A–E) 500 nM SPase I Δ2-75 wild-type (WT), W261, W284, W300, and W310 in TKE buffer, or in TKE buffer containing 0.24 mM E. coli lipids in liposomes, were quenched with increasing amounts of acrylamide at 20 °C. The reaction mixtures were excited at 295 nm and read at emission wavelengths of 310–530 nm. Stern-Volmer plots are shown wherein, F₀ and F represent the fluorescence emission intensities at 340 nm of SPase I Δ2-75 in the absence and presence of acrylamide, respectively. (F) Results from fluorescence quenching experiments of WT, W300 and W310 in liposomes in the presence (F) and absence (F₀) of 10-DN are shown.

FIG 4

Signal peptide binding on wild-type SPase I Δ2-75. (A) Sequence of the SP2 peptide used in the study is shown. A Cys residue was introduced at position two of the E. coli alkaline phosphatase signal peptide. (B) Results from FRET experiments between 500 nM IAEDANS-S302C SPase I Δ2-75 and 15 μM IANBD-SP2 are shown. The reaction mixture was excited at 336 nm and the emission spectrum was read from 346–660 nm as described in Materials and Methods. (C) The binding affinity of IAEDANS-SP2 to 500 nM wild-type SPase I Δ2-75 was determined via FRET experiments. The reaction mixture was excited at 295 nm and emission intensities were measured from 310–530 nm. The K_d for the reaction was calculated using the one-site binding model equation as described in Materials and Methods.

FIG 5

Fluorescence quenching with acrylamide for the wild-type and single Trp SPase I Δ2-75 enzymes in the presence and absence of PhoA WTSP. (A–E) 500 nM SPase I Δ2-75 wild-type (WT), W261, W284, W300, and W310 in TKE buffer, or in TKE buffer containing 10 μM PhoA WTSP, were quenched with increasing amounts of acrylamide at 20 °C. The reaction mixtures were excited at 295 nm and read at emission wavelengths of 310–530 nm. Stern-Volmer plots are shown wherein, F₀ and F represent the fluorescence emission intensities at 340 nm of SPase I Δ2-75 in the absence and presence of acrylamide, respectively.

FIG 6

Residues W261, W284 and W300 are affected by ligand binding. (A) Amino acids from the conserved Box E domain located near W261 and W284 (in red) that are crucial for ligand binding, structural stabilization and enzyme activity are highlighted in blue.^, The active site residues S90 and K145 are colored magenta, and the bound inhibitors (β-sultam and part of arylomycin A₂) are shown in yellow (PDB 3IIQ). (B) W300 forms van der Waal’s interactions with bound inhibitor. An overlay view of the crystal structures of E. coli SPase I Δ2-75 in the apoenzyme form (PDB 1KN9) in red and when bound to arylomycin A₂ and a β-sultam inhibitor (PDB 3IIQ) in blue is presented, illustrating the movement of the aromatic side chain of W300 towards a fatty acid methylene group of the arylomycin A₂ inhibitor. The distances between the active site and W300 (23 Å), and between W300 and the bound inhibitor (4.7 Å) are highlighted.