Allosteric regulation of rhomboid intramembrane proteolysis

Abstract

Proteolysis within the lipid bilayer is poorly understood, in particular the regulation of substrate cleavage. Rhomboids are a family of ubiquitous intramembrane serine proteases that harbour a buried active site and are known to cleave transmembrane substrates with broad specificity. In vitro gel and Förster resonance energy transfer (FRET)-based kinetic assays were developed to analyse cleavage of the transmembrane substrate psTatA (TatA from Providencia stuartii). We demonstrate significant differences in catalytic efficiency (kcat/K0.5) values for transmembrane substrate psTatA (TatA from Providencia stuartii) cleavage for three rhomboids: AarA from P. stuartii, ecGlpG from Escherichia coli and hiGlpG from Haemophilus influenzae demonstrating that rhomboids specifically recognize this substrate. Furthermore, binding of psTatA occurs with positive cooperativity. Competitive binding studies reveal an exosite-mediated mode of substrate binding, indicating allostery plays a role in substrate catalysis. We reveal that exosite formation is dependent on the oligomeric state of rhomboids, and when dimers are dissociated, allosteric substrate activation is not observed. We present a novel mechanism for specific substrate cleavage involving several dynamic processes including positive cooperativity and homotropic allostery for this interesting class of intramembrane proteases.

Keywords: GlpG; allostery; intramembrane protease; kinetics; rhomboid protease.

Figure 1. psTatA cleavage by three prokaryotic rhomboids demonstrates substrate specificity

1. Topology of three studied rhomboids. hiGlpG and ecGlpG are the simplest and commonly found in prokaryotes. AarA has 7 TMD similar to rhomboids typically found in eukaryotes.

2. Hill plots of formation of rhomboid-cleaved psTatA versus psTatA concentration (n = 2, mean ± SD). The product concentration was calculated as the percentage of cleaved substrate from the blot image.

3. Western blot analysis of rhomboid-mediated cleavage of psTatA. psTatA at the concentration range of 0.5–15 μM was incubated with 0.33 μM (hiGlpG and ecGlpG) or 0.13 μM (AarA) at 37°C for 4 h or 15 min, and the cleavage product (C) was separated from the uncleaved substrate (U) with SDS-Tricine gel and developed with Western blot using anti-His antibodies. The star represents a contaminant band from the substrate sample.

Source data are available online for this figure.

Figure 2. FRET-based AarA activity assay

A FRET-based activity assay was used to measure the catalytic parameters for AarA-mediated cleavage of psTatA-FRET.

1. Schematic illustration of the FRET-based cleavage assay. TatA structure determined by NMR (Rodriguez et al, 2013).

2. AarA activity dependence on pH. The proteolysis reaction was performed at 37°C for 2 h in the presence of 0.18 μM of rhomboid protease and 2 μM of full-length psTatA-FRET in 150 mM NaCl, 20% glycerol, 0.1% DDM and 2 mM DTT reaction buffer, at pH interval spanning from 2 to 9 with 50 mM of a broad-range pH triple buffer (boric acid:citric acid:phosphate) (Carmody, 1961). The highest initial velocity was calculated to determine the percent activity along the pH gradient. The same pH activity dependence is observed when using a single buffer (Supplementary Fig S3A).

3. Hill plot of AarA-mediated cleavage of psTatA-FRET. psTatA-FRET at the concentration range from 0.13 to 7 μM was incubated with 0.135 μM of AarA at 37°C for 2 h. Velocities were calculated as described in Materials and Methods (n = 5, mean ± SD). See Table2.

Figure 3. Michaelis–Menten graphs of rhomboid-mediated FL-casein cleavage indicate a different mode of cleavage for soluble substrate FL-casein

Michaelis–Menten plots for AarA(red), ecGlpG(blue) and hiGlpG(green) cleavage of FL-casein. Rhomboid purification, as well as the activity measurements, were carried out in buffer containing 0.1% dodecyl-maltoside (DDM) to obtain the dimeric state of the enzymes. 0.18 μM of rhomboid protease was incubated with 0.1–5.8 μM of FL-casein at 37°C for 2 h. For AarA and hiGlpG n = 3, mean ± SD. For ecGlpG n = 5, mean ± SD (see legend for Table1).

Figure 4. Dimeric but not monomeric rhomboids can cleave the transmembrane substrate psTatA

A–C Gel filtration experiments of (A) hiGlpG, (B) ecGlpG and (C) AarA either in 0.1% dodecyl-maltoside (DDM, red) or exchanged into 0.2% decyl-maltoside (DM, blue), carried out with a Superdex 200 (16/60) column. A third detergent exchange with AarA purified in 0.2% DM was conducted in gel filtration buffer with 0.2% DM (DM/DM, green) (C). Standards for gel filtration column are as follows: 1. Thyroglobulin, 51.1 ml (MW 670 kDa, Stokes radius 86 Å); 2. IgG, 67.3 ml (MW 158 kDa, Stokes radius 51 Å); 3. Ovalbumin, 83.4 ml (MW 44 kDa, Stokes radius 28 Å); 4. Myoglobin, 94.4 ml (MW 17 kDa, Stokes radius 19 Å); 5. Vitamin B12, 111.9 ml (MW 1.3 kDa, Stokes radius 1.6 Å). Vo, void volume 46.51 ml, Vt, total column volume 128 ml. D–F The cleavage of psTatA by (D) hiGlpG, (E) ecGlpG or (F) AarA was assessed in activity buffer containing either 0.1% DDM or 0.2% DM detergent (assay det). Prior to this, enzyme was purified with a Ni-NTA column in either 0.1% DDM or 0.2% DM detergents (Ni det). AarA S150A mutant was used as a negative control. G To further assess the physiological relevance of dimerization, inside-out vesicles containing full-length ecGlpG harbouring a Tobacco Etch Virus (TEV) protease site between the membrane and cytoplasmic domains were incubated with or without TEV protease and then subjected to ultracentrifugation. H The supernatant was separated with 16% SDS-Tricine gel (Cyto, ecGlpG cytoplasmic domain). TEV protease was removed using Ni-NTA resin, and the flow through was applied on a Superdex 75 (10/30) gel filtration column. Known retention volumes for monomer (M) and dimer (D) of the cytoplasmic domain, (Lazareno-Saez et al, 2013), are indicated on the gel filtration trace. Inset represents the SDS-PAGE of the main peak. Gel filtration analysis of the supernatant after ultracentrifugation demonstrates that the cytoplasmic domain elutes as a dimer only. Along with our previous work, these data clearly demonstrate rhomboids are dimeric in the lipid bilayer. Source data are available online for this figure.

Figure 5. Competitive studies of rhomboid-mediated FL-casein cleavage in the presence of psTatA

Competitive studies were conducted with three prokaryotic rhomboid enzymes to assess the mode of psTatA binding to the enzyme.

1. Competitive inhibition of ecGlpG by psTatA, with FL-casein as substrate. The protease reaction between ecGlpG (0.179 μM) with FL-casein (0.179–3.85 μM) was performed in the presence of different concentrations of psTatA (0, 3, 5 and 10 μM). The reaction was started with the protease. Fluorescence emission at 513 nm was measured at 37°C every 5 min for 2 h in a fluorescence microplate reader with an excitation wavelength of 503 nm. Fluorescence detection of each substrate concentration in the presence of corresponding psTatA concentration without enzyme was used as a negative control (n = 2, mean ± SD). Initial velocities were determined for each substrate concentration. Michaelis–Menten plots were subjected to global fit to distinguish the kinetic model and determine the kinetic parameters.

2. Non-competitive inhibition of AarA by psTatA, with FL-casein as substrate. The assay was conducted, and the kinetic model was determined similar to that for ecGlpG. The concentration of psTatA used is shown beside the corresponding curve.

Figure 6. A schematic illustration of rhomboid exosite-mediated substrate recognition and allosteric regulation of cleavage

We propose a preliminary model of transmembrane substrate cleavage by rhomboid protease. Dimerization allows the formation of an exosite, either via conformational changes upon dimerization, or the exosite is located at the interface (not depicted in this illustration). The initial binding of the substrate's transmembrane (purple) segment by its exosite recognition motif (red) to the rhomboid intramembrane-located exosite will allow the cleavage at a low rate (k_cat-α) and induce subtle conformational changes in the active site (A) resulting in the optimal active site arrangement and increased rate of catalysis (k_cat-β). The overall rate measured is therefore an apparent k_cat (k_cat-app).