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. 2011 May 1;435(3):733-42.
doi: 10.1042/BJ20102131.

Recombinant Deg/HtrA proteases from Synechocystis sp. PCC 6803 differ in substrate specificity, biochemical characteristics and mechanism

Pitter F Huesgen  1 , Helder Miranda, XuanTam Lam, Manuela Perthold, Holger Schuhmann, Iwona Adamska, Christiane Funk
Affiliations

Recombinant Deg/HtrA proteases from Synechocystis sp. PCC 6803 differ in substrate specificity, biochemical characteristics and mechanism

Pitter F Huesgen et al. Biochem J. .

Abstract

Cyanobacteria require efficient protein-quality-control mechanisms to survive under dynamic, often stressful, environmental conditions. It was reported that three serine proteases, HtrA (high temperature requirement A), HhoA (HtrA homologue A) and HhoB (HtrA homologue B), are important for survival of Synechocystis sp. PCC 6803 under high light and temperature stresses and might have redundant physiological functions. In the present paper, we show that all three proteases can degrade unfolded model substrates, but differ with respect to cleavage sites, temperature and pH optima. For recombinant HhoA, and to a lesser extent for HtrA, we observed an interesting shift in the pH optimum from slightly acidic to alkaline in the presence of Mg2+ and Ca2+ ions. All three proteases formed different homo-oligomeric complexes with and without substrate, implying mechanistic differences in comparison with each other and with the well-studied Escherichia coli orthologues DegP (degradation of periplasmic proteins P) and DegS. Deletion of the PDZ domain decreased, but did not abolish, the proteolytic activity of all three proteases, and prevented substrate-induced formation of complexes higher than trimers by HtrA and HhoA. In summary, biochemical characterization of HtrA, HhoA and HhoB lays the foundation for a better understanding of their overlapping, but not completely redundant, stress-resistance functions in Synechocystis sp. PCC 6803.

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Figures

Figure 1
Figure 1. Purification of recombinant active and inactive variants of Synechocystis 6803 Deg/HtrA proteases
(A) Schematic representations of rHtrA, rHhoA and rHhoB constructs generated for the present study, E. coli DegP and DegS and human HtrA2/Omi. Hatched boxes mark putative transmembrane domains, white boxes show predicted signal peptides, light grey boxes indicate protease domains with the positions of catalytic histidine and and aspartic acid marked in white and the position of a catalytic serine residue mutated to alanine in inactive variants indicated in black, and dark grey boxes mark PDZ domains. Proteins are drawn to scale, with a length of 100 amino acid residues indicated. (B) Coomassie-Blue-stained SDS/PAGE gels showing the elution fractions after Ni2+-affinity chromatography, containing recombinant active rHtrA, rHhoA and rHhoB, and inactive rHtrASA, rHhoASA and rHhoBSA variants of proteases. Molecular masses in kDa are indicated on the left-hand side. (C) Coomassie-Blue-stained SDS/PAGE gels showing the elution fractions after Ni2+-affinity chromatography, containing recombinant active rHtrAΔPDZ, rHhoAΔPDZ and rHhoBΔPDZ, and inactive rHtrASAΔPDZ, rHhoASAΔPDZ and rHhoBSAΔPDZ variants of proteases lacking PDZ domains.
Figure 2
Figure 2. Proteolytic activity of Synechocystis 6803 Deg/HtrA proteases against substrates with different folding states
Naturally unfolded β-casein, globular folded BSA, folded lysozyme and partially denatured reduced lysozyme were used as model substrates. (A) Protease constructs with intact PDZ domains. (B) Protease constructs without PDZ domains, e.g. C-terminally truncated after the protease domain.
Figure 3
Figure 3. Cleavage site specificity of Synechocystis 6803 Deg/HtrA proteases
(A) Degradation of β-casein by recombinant rHtrA, rHhoA and rHhoB, analysed by Coomassie-Blue-stained SDS/PAGE. M indicates the molecular-mass protein marker; molecular mass in kDa is indicated on the left-hand side. Arrows with numbers highlight bands that were excised from the gel for N- and C-terminal protein sequencing. Proteolytic assays using rHtrA were performed in 50 mM Mes, pH 5.5, and 20 mM CaCl2; assays with rHhoA and rHhoB were carried out in 50 mM Tris/HCl, pH 7.0, and 20 mM CaCl2. Representatives of at least three replicates are shown. (B) Amino acid sequence of mature β-casein. Black bars show residues identified by N-terminal sequencing of fragments excised from gels in (A), broken bars indicate residues identified by C-terminal sequencing and arrows indicate deducted cleavage sites.
Figure 4
Figure 4. Complex formation by inactive variants of Synechocystis 6803 Deg/HtrA proteases in the absence and the presence of substrate
Size-exclusion chromatography of (A) rHtrASA (B) rHhoASA and (C) rHhoBSA. Black lines show elution profiles obtained in the absence of substrate, and grey lines show profiles after pre-incubation with β-casein in a 3:1 molar ratio of protease/substrate (A and B) or a 1:3 ratio (C). Lower panels show aliquots of the size-exclusion chromatography fractions separated by SDS/PAGE and visualized by Coomassie-Blue staining. The exclusion volume (V0) of the columns and the elution volumes of selected marker proteins are indicated with black arrowheads. Ve, elution volume.
Figure 5
Figure 5. Schematic overview of the oligomerization results
Our present results demonstrated that, in contrast to trimeric rHhoB, trimeric rHtrA and hexameric rHhoA form higher-order oligomers in the presence of β-casein. Deletion of the PDZ domain locks all three proteases in the trimer state.
Figure 6
Figure 6. Biochemical properties of rHtrA, rHhoA and rHhoB assayed with a fluorigenic casein substrate
In all panels, data points obtained for rHtrA are represented by diamonds, rHhoA by triangles and rHhoB by circles. Open symbols connected by broken lines indicate the absence of divalent ions, and filled symbols connected by continuous lines indicate the presence of divalent ions. Please note that detector-gain settings may differ between panels. Results are means±S.D. (n=3). (A) Effect of Ca2+ ion concentration on proteolytic activity. Activity was measured by the initial rate of fluorescence generation in 10 mM Mops buffer, pH 6.5, and normalized to the signal observed without CaCl2. (B) Effect of pH on protease activity in the absence of divalent ions. Activity was measured by the initial rate of fluorescence generation. RFU, relative fluorescence units. (C) Effect of pH on protease activity in the presence of 10 mM CaCl2. (D) Effect of temperature on protease activity as measured by the total fluorescence after 40 min, normalized to the fluorescence observed at 10°C.
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