Analysis of the link between the redox state and enzymatic activity of the HtrA (DegP) protein from Escherichia coli

Bacterial HtrAs are proteases engaged in extracytoplasmic activities during stressful conditions and pathogenesis. A model prokaryotic HtrA (HtrA/DegP from Escherichia coli) requires activation to cleave its substrates efficiently. In the inactive state of the enzyme, one of the regulatory loops, termed LA, forms inhibitory contacts in the area of the active center. Reduction of the disulfide bond located in the middle of LA stimulates HtrA activity in vivo suggesting that this S-S bond may play a regulatory role, although the mechanism of this stimulation is not known. Here, we show that HtrA lacking an S-S bridge cleaved a model peptide substrate more efficiently and exhibited a higher affinity for a protein substrate. An LA loop lacking the disulfide was more exposed to the solvent; hence, at least some of the interactions involving this loop must have been disturbed. The protein without S-S bonds demonstrated lower thermal stability and was more easily converted to a dodecameric active oligomeric form. Thus, the lack of the disulfide within LA affected the stability and the overall structure of the HtrA molecule. In this study, we have also demonstrated that in vitro human thioredoxin 1 is able to reduce HtrA; thus, reduction of HtrA can be performed enzymatically

Kinetic values of proteolysis catalysed by HtrA.

<p><i>V</i>_max—apparent maximum cleavage rate, <i>K</i>’—apparent Michaelis constant, <i>n</i>—Hill coefficient.</p><p>Kinetic values of proteolysis catalysed by HtrA.</p

Kinetics of proteolysis catalyzed by HtrA.

<p>Initial rates of ΔCys (HtrA-C57A/C69A-6×His tag) and oxidized control (wt HtrA-6×His tag) variant (0.1 μM monomer) cleavage of different concentrations of PepPEG (NWVSAA↓KFE- Y^NO₂-O2Oc-O2Oc-IYQV) were measured at 20°C, as described in “Materials and Methods”. The empirical data were plotted against the Hill equation. The error bars represent the standard deviation values from three independent measurements.</p

Enzymatic reduction of HtrA by human Trx1.

<p>(A) Reversed-phase high performance liquid chromatography (RP-HPLC) analysis of HtrA-S210A redox state depending on the presence of Trx1, DTT, or 22-peptide (10-fold molar excess) in comparison to ΔCys (HtrA-C57A/C69A/S210A) variant. The elution volumes of Trx1, HtrA red (the reduced form or cysteine-less variant), and HtrA ox (the oxidized form) are shown as vertical dotted lines. A representative elution profile is shown; a.u., arbitrary units. (B) fraction of HtrA-S210A reduced by Trx1 over 15 minutes at 37°C as a fraction of total HtrA-S210A in the sample, as calculated from the integrated area under RP-HPLC peaks. The error bars represent the standard deviation values from at least two independent measurements.</p

Second derivative and deconvoluted infrared absorbance spectra of oxidized and reduced HtrA-S210A at 20°C.

<p>Superimposition of HtrA-S210A reduced in the presence of DTT (red; green color) and oxidized HtrA-S210A version (ox; red color). Top: the second derivative spectra calculated over a 5-data point range (5 cm^-1). Bottom: the deconvoluted spectra calculated with a half-band width at 19 cm^-1 and a resolution enhancement factor of 3. At least 16 spectra were collected; a.u., arbitrary units.</p

Bacterial strains and plasmids.

<p>Bacterial strains and plasmids.</p

Sensorgrams of HtrA affinity to β-casein.

<p>(A) affinity of ΔCys (HtrA-C57A/C69A/S210A–6×His tag) variant. (B) affinity of oxidized control (HtrA-S210A–6×His tag) variant. Increasing amounts (100–300 nM) of HtrA variants were run over the surface of a sensor chip with immobilized β-casein, as described in “Materials and Methods”. A representative sensorgram is shown; RU, response units.</p

Comparison of evolutionarily conserved sequence within the LA loop surrounding disulfide bonds in the Enterobacteriaceae family.

<p>The sequence alignment to residues 40–72 from <i>E</i>. <i>coli</i> HtrA generated by Clustal 2.1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117413#pone.0117413.ref056" target="_blank">56</a>]. The classification of residue hydrophobicity is according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117413#pone.0117413.ref057" target="_blank">57</a>].</p

The oligomeric states of the HtrA protein variants in the presence or absence of a peptide substrate.

<p>ΔCys (C57A/C69A/S210A) and oxidized control (S210A) HtrA variants were incubated with a 2.5-, 5-, or 10-fold molar excess of the 22-peptide NWVSAAKFESTDGSTDYGIYQV (2.5 ×, 5 ×, or 10 ×, respectively) or without ligand (0), subjected to cross-linking with bis[sulfosuccinimidyl] suberate (BS³), and then analyzed using size exclusion chromatography as described in “Materials and Methods”. The void volume (V₀) and elution volumes of molecular weight standards (Bio-Rad) used for column calibration are shown as vertical dotted lines. The positions of HtrA dodecamers (12), hexamers (6), and trimers (3) are indicated with arrows. A representative elution profile is shown; a.u., arbitrary units.</p

Interactions between LA’, L1, and L2 loops.

<p>The LA loop model in inactive conformation (PDB entry: 1ky9) according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117413#pone.0117413.ref023" target="_blank">23</a>]. The positions of subunits A, E, and F within the hexamer are shown in the left top corner. His105, Asp135, and Ser210 constitute the catalytic triad. The selected contacts between the loops are shown as dotted lines. The picture was generated with the aid of Swiss-PdbViewer 4.1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117413#pone.0117413.ref044" target="_blank">44</a>].</p