The serine protease HhoA from Synechocystis sp. strain PCC 6803: substrate specificity and formation of a hexameric complex are regulated by the PDZ domain

Abstract

Enzymes of the ATP-independent Deg serine endopeptidase family are very flexible with regard to their substrate specificity. Some family members cleave only one substrate, while others act as general proteases on unfolded substrates. The proteolytic activity of Deg proteases is regulated by PDZ protein interaction domains. Here we characterized the HhoA protease from Synechocystis sp. strain PCC 6803 in vitro using several recombinant protein constructs. The proteolytic activity of HhoA was found to increase with temperature and basic pH and was stimulated by the addition of Mg(2+) or Ca(2+). We found that the single PDZ domain of HhoA played a critical role in regulating protease activity and in the assembly of a hexameric complex. Deletion of the PDZ domain strongly reduced proteolysis of a sterically challenging resorufin-labeled casein substrate, but unlabeled beta-casein was still degraded. Reconstitution of the purified HhoA with total membrane proteins isolated from Synechocystis sp. wild-type strain PCC 6803 and a DeltahhoA mutant resulted in specific degradation of selected proteins at elevated temperatures. We concluded that a single PDZ domain of HhoA plays a critical role in defining the protease activity and oligomerization state, combining the functions that are attributed to two PDZ domains in the homologous DegP protease from Escherichia coli. Based on this first enzymatic study of a Deg protease from cyanobacteria, we propose a general role for HhoA in the quality control of extracytoplasmic proteins, including membrane proteins, in Synechocystis sp. strain PCC 6803.

FIG. 1.

Engineering and purification of HhoA deletion constructs. (A) Domain structure of the full-length HhoA protease and truncated constructs. Gray oval, putative signal peptides; light gray boxes, protease domains of the S1B subfamily of serine proteases, with the amino acid residues of the catalytic triad indicated; dark gray boxes, PDZ domains; His, six-His tag and linker introduced by the expression vector. (B) Ni²⁺ affinity-purified recombinant proteins (4 μg) separated by SDS-PAGE and visualized by Coomassie blue staining. Lane 1, HhoAΔN34; lane 2, HhoA_S237AΔN34; lane 3, HhoAPD; lane 4, HhoA_S237APD; lane M, markers.

FIG. 2.

Proteolytic activities of purified HhoA deletion constructs with various model substrates. In all assays 100 pmol purified HhoA protein was incubated with the substrate for 2 h at 40°C in 50 mM HEPES (pH 8.0) and 20 mM MgCl₂. (A) Degradation assays with β-casein and BSA visualized in a Coomassie blue-stained SDS-PAGE gel. A representative gel for three independent replicates is shown. Lane 0, elution buffer; lane 1, HhoAΔN34; lane 2, HhoA_S237AΔN34; lane 3, HhoAPD; lane 4, HhoA_S237APD. (B) Proteolytic activity against resorufin-labeled casein as determined by absorption at 574 nm. The chemical structure of the resorufin label is shown in the inset. The values are means ± standard deviations (n = 4).

FIG. 3.

Analysis of HhoA complex formation. The elution diagrams for size exclusion chromatography of recombinant HhoAΔN34 (solid line) and HhoA_S237APD (dotted line) are shown. For HhoAΔN34, the proteolytic activity of 10 μl of selected fractions against resorufin-labeled casein is shown (gray columns and right axis).

FIG. 4.

Characterization of HhoA protease activity. For all assays 100 pmol purified HhoAΔN34 was incubated with 8 μg resorufin-labeled casein, and the experiment was repeated with at least three independent protein purifications. (A) Degradation kinetics at 40°C in 50 mM HEPES (pH 8.0) and 20 mM CaCl₂. The values are means ± standard deviations (n = 4), normalized to the activity after 4 h. (B) Effect of pH. HhoAΔN34 was incubated in 50 mM morpholineethanesulfonic acid (MES) (pH 6.0 to 6.5) or 50 mM HEPES (pH 7.0 to 8.5) supplemented with 20 mM MgCl₂ for 2 h at 40°C. The values are means ± standard deviations (n = 7), normalized to the activity at pH 8.0. (C) Effect of temperature. HhoAΔN34 was incubated in 50 mM HEPES (pH 8.0) and 20 mM CaCl₂ for 2 h. The values are means ± standard deviations (n = 6) normalized to the activity at 40°C. (D) Effect of divalent cations. HhoAΔN34 was incubated for 2 h at 40°C in 50 mM HEPES (pH 8.0) supplemented with 20 mM MgCl₂ or CaCl₂ (black bars) and with 20 mM EDTA (gray bars). The values are means ± standard deviations (n = 6 for black bars and n = 3 for gray bars). ddH2O, double-distilled water.

FIG. 5.

Activity of HhoAΔN34 against isolated total membrane proteins. Isolated total membranes of WT or ΔhhoA mutant cells were incubated without (lanes −) or with (lanes +) recombinant HhoAΔN34 at 15 and 45°C in 50 mM Tris (pH 7.5) supplemented with 20 mM CaCl₂, separated by SDS-PAGE, and visualized by Coomassie blue staining. The open arrows indicate bands of added HhoAΔN34 and its degradation fragments, and the filled arrows indicate protein bands specifically degraded by HhoAΔN34. Lane M contained markers.