Multifaceted Substrate Capture Scheme of a Rhomboid Protease

Rhomboid proteases are integral membrane serine proteases that catalyze peptide bond hydrolysis in biological membranes. Little is currently known about the interaction of enzyme and substrate. Coarse-grained molecular dynamics simulations in hydrated lipid bilayers are employed herein to study the interaction of the E. coli rhomboid protease GlpG (ecGlpG) with the transmembrane domain (TMD) of the substrate Spitz. Spitz does not associate with ecGlpG exclusively at the putative substrate gate near TMD 5. Instead, there are six prominent and stable interaction sites, including one between TMDs 1 and 3, with the closest enzyme–substrate proximity occurring at the ends of helical TMDs or in loops. Bilayer thinning is observed proximal to ecGlpG, but there is no evidence of additional thinning of the bilayer upon interaction with substrate. We suggest that the initial interaction between enzyme and substrate, or substrate capture event, is not limited to a single site on the enzyme, and may be driven by juxtamembrane electrostatic interactions. The findings are of additional interest because catalytically inactive rhomboids (iRhoms) are now known to interact with the substrates of their catalytically active counterparts and to antagonize the enzyme-driven pathways

Headgroup-Dependent Membrane Catalysis of Apelin−Receptor Interactions Is Likely

Apelin is the peptidic ligand for the G-protein-coupled receptor APJ. The apelin−APJ system is important in cardiovascular regulation, fluid homeostasis, and angiogenesis, among other roles. In this study, we investigate interactions between apelin and membrane-mimetic micelles of the detergents sodium dodecyl sulfate (SDS), dodecylphosphocholine (DPC), and 1-palmitoyl-2-hydroxy-<i>sn</i>-glycero-3-[phospho-<i>rac</i>-(1-glycerol)] (LPPG). Far-ultraviolet circular dichroism spectropolarimetry and diffusion-ordered spectroscopy indicate that apelin peptides bind to micelles of the anionic detergents SDS and LPPG much more favorably than to zwitterionic DPC micelles. Nuclear magnetic resonance spectroscopy allowed full characterization of the interactions of apelin-17 with SDS micelles. Titration with paramagnetic agents and structural determination of apelin-17 in SDS indicate that R6−K12 is highly structured, with R6−L9 directly interacting with headgroups of the micelle. Type I β-turns are initiated between R6 and L9, and a well-defined type IV β-turn is initiated at S10. Furthermore, binding of apelin-17 to SDS micelles causes structuring of M15-F17, with no evidence for direct binding of this region to the micelles. These results are placed into the context of the membrane catalysis hypothesis for peptide−receptor binding, and a hypothetical mechanism of APJ binding and activation by apelin is advanced

Scanning electron micrographs (SEM) of aggregates (spheroids) and fibrils/fiber formed from the W3 protein.

<p>The protein sample was immobilized either using a wet fixation method to preserve spheroids (panel A and B) or using a dry fixation method to retain fibrils (panel C and D). For panel B, the protein solution was swirled with a pipette tip to increase fiber formation before fixation. For imaging the macro-fiber, dry fibers were not treated by any solvent, but they were broken in liquid nitrogen to show fiber cross section (panel E to H). Panel D, F and H corresponds to the boxed areas of panel C, E and G, respectively, with a higher magnification.</p

Construction of AcSp1-derived recombinant proteins.

<p>(A) Schematic illustration of construction strategy of the recombinant genes. Plasmid pW_n (n = 1, 2, or 3) was digested with restriction enzymes <i>Bam</i>HI and <i>Bsg</i>I, and the resulting <i>Bam</i>HI-<i>Bsg</i>I fragment containing the W_n coding sequence was ligated with a <i>Bam</i>HI-<i>Bse</i>RI fragment isolated from plasmid pW_1a containing the W_1a coding sequence. The resulting plasmid pW_n+1 has the W_n and W_1a coding sequences seamlessly fused to produce the W_n+1 coding sequence. (B) Schematic illustration of the W₁ to W₄ proteins with their sizes shown. (C) Amino acid sequences. The 106-aa H₆-SUMO tag was added to the N-terminus of the W₁ to W₄ proteins. The 200-aa W_1a sequence was derived from the consensus repeat of the AcSp1 protein of <i>Argiope trifasciata </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050227#pone.0050227-Hayashi1" target="_blank">[25]</a>. The 199-aa W₁ sequence (not shown) was the same as W_1a except the absence of the N-terminal S residue.</p

Comparison of physical properties of W₄ fiber with those of other artificial and natural spider silk fibers.

<p>Properties of the recombinant AcSp1 (aciniform) fiber, which was formed from protein W₄, were determined in this study. Properties of the recombinant MaSp1 (dragline) fiber, which was formed from protein 4RepCT, were obtained from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050227#pone.0050227-Grip1" target="_blank">[29]</a>. Properties of the fiber formed from native sized MaSp1 (dragline) protein were obtained from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050227#pone.0050227-Xia1" target="_blank">[5]</a>. Properties of natural aciniform silk were obtained from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050227#pone.0050227-Hayashi1" target="_blank">[25]</a>.</p

Theoretical and observed average mass of W_1–4.

<p>Average Mass_theor: theoretical average mass; Average Mass_obs: observed average mass. Observed average mass was calculated from charge 7, 8 and 9 for W₁, charge 16, 17 and 18 for W₂; charge 30, 32 and 34 for W₃; charge 44, 45 and 46 for W₄.</p

Protein expression and purification.

<p>Each fusion protein was expressed in <i>E. coli</i> and analyzed by SDS-PAGE followed by Coomassie blue staining, with protein size markers shown in lane M. (A) Expression and purification of the fusion protein H₆-SUMO-W₁. Lanes 1 and 2 are total cellular proteins before and after IPTG-induced protein expression, respectively. The induced cell lysate was separated into soluble (lane 3) and insoluble (lane 4) fractions, and the soluble fraction was passed through a nickel-beads affinity column. Lane 5 shows unbound proteins that flew-through the column, and lanes 6–8 are three consecutive fractions eluted from the column. (B) Removal of the H₆-SUMO tag. The fusion protein H₆-SUMO-W₁ (lanes 1) was digested with a SUMO protease at 4°C for 6 hours (lane 2) and overnight (lane 3). The resulting sample was passed through a nickel-beads affinity column, and the tag-free W₁ protein was collected as a purified protein in the flew-through fraction (lane 4). (C) The W₁, W₂, W₃ and W₄ proteins that were expressed and purified as in panes A and B.</p

Analysis of protein secondary structures.

<p>(A) CD spectra of the W₁, W₂, W₃, and W₄ proteins in solution. (B) Raman spectra of a fiber formed from the W₄ protein. (C) Spectral decomposition in the amide I region of the W₄ silk.</p

Electrospray ionization mass spectrum of W_1–4.

<p>Numbers above each peak in blue are different charges states.</p