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

The LA loop as an important regulatory element of the HtrA (DegP) protease from Escherichia coli : structural and functional studies

Bacterial HtrAs are serine proteases engaged in extracytoplasmic protein quality control and are required for the virulence of several pathogenic species. The proteolytic activity of HtrA (DegP) from Escherichia coli, a model prokaryotic HtrA, is stimulated by stressful conditions; the regulation of this process is mediated by the LA, LD, L1, L2, and L3 loops. The precise mechanism of action of the LA loop is not known due to a lack of data concerning its three-dimensional structure as well as its mode of interaction with other regulatory elements. To address these issues we generated a theoretical model of the three-dimensional structure of the LA loop as per the resting state of HtrA and subsequently verified its correctness experimentally. We identified intra- and intersubunit contacts that formed with the LA loops; these played an important role in maintaining HtrA in its inactive conformation. The most significant proved to be the hydrophobic interactions connecting the LA loops of the hexamer and polar contacts between the LA′ (the LA loop on an opposite subunit) and L1 loops on opposite subunits. Disturbance of these interactions caused the stimulation of HtrA proteolytic activity. We also demonstrated that LA loops contribute to the preservation of the integrity of the HtrA oligomer and to the stability of the monomer. The model presented in this work explains the regulatory role of the LA loop well; it should also be applicable to numerous Enterobacteriaceae pathogenic species as the amino acid sequences of the members of this bacterial family are highly conserved

Analysis of the redox state and structural changes in the presence of DTT over a temperature range of 20–45°C.

<p>Left axis: relative change in near-UV circular dichroism (CD) ellipticity integrated at the range of 255–295 nm of HtrA-S210A in the presence of DTT (10 mM) as a fraction of ΔCys variant (HtrA-C57A/C69A/S210A) ellipticity subtracted from HtrA-S210A ellipticity in the absence of DTT. Right axis: amount of HtrA-S210A reduced by DTT as a fraction of total HtrA-S210A in the sample, as calculated from integrated area under reversed-phase high performance liquid chromatography (RP-HPLC) peaks. The error bars represent the standard deviation values from at least two independent measurements.</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

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

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

Comparison of the fluorescence properties of the single-Trp HtrA variants.

<p><i>K</i>_SV—Stern-Volmer (dynamic quenching) constant, <i>V</i>—static (sphere of action) quenching constant, <i>k</i>_q—bimolecular quenching constant. The Trp residues according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117413#pone.0117413.ref049" target="_blank">49</a>] and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117413#pone.0117413.ref050" target="_blank">50</a>] are classified as: (1) = exposed [1.5 < <i>k</i>_q < 5], (2) = moderately exposed to the solvent [0.6 < <i>k</i>_q < 1.5], (3) = moderately buried [0.2 < <i>k</i>_q < 0.6].</p><p>Comparison of the fluorescence properties of the single-Trp HtrA variants.</p

Quenching of the tryptophan fluorescence of the single-Trp HtrA variants by acrylamide.

<p>(A) typical Stern-Volmer quenching plots of ΔCysW (C57A/C69A/F63W/S210A) and oxidized control (F63W/S210A) HtrA variants at 25°C. <i>F</i>₀ and <i>F</i> are the fluorescence intensities in the absence and presence of acrylamide, respectively. (B) the bimolecular quenching constant (<i>k</i>_q) values for HtrA variants as a function of temperature. The exposition classes are given according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117413#pone.0117413.ref049" target="_blank">49</a>] and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117413#pone.0117413.ref050" target="_blank">50</a>]. (C) Arrhenius plots for the <i>k</i>_q values from panel B. All data were obtained as described in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117413#pone.0117413.ref024" target="_blank">24</a>] and correspond to mean ± standard deviation values of at least three different experiments.</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