Factors Defining the Functional Oligomeric State of Escherichia coli DegP Protease

Escherichia coli DegP protein is a periplasmic protein that functions both as a protease and as a chaperone. In the absence of substrate, DegP oligomerizes as a hexameric cage but in its presence DegP reorganizes into 12 and 24-mer cages with large chambers that house the substrate for degradation or refolding. Here, we studied the factors that determine the oligomeric state adopted by DegP in the presence of substrate. Using size exclusion chromatography and electron microscopy, we found that the size of the substrate molecule is the main factor conditioning the oligomeric state adopted by the enzyme. Other factors such as temperature, a major regulatory factor of the activity of this enzyme, did not influence the oligomeric state adopted by DegP. In addition, we observed that substrate concentration exerted an effect only when large substrates (full-length proteins) were used. However, small substrate molecules (peptides) always triggered the same oligomeric state regardless of their concentration. These results clarify important aspects of the regulation of the oligomeric state of DegP

Characterization of the Autocleavage Process of the Escherichia coli HtrA Protein: Implications for its Physiological Role▿ †

The Escherichia coli HtrA protein is a periplasmic protease/chaperone that is upregulated under stress conditions. The protease and chaperone activities of HtrA eliminate or refold damaged and unfolded proteins in the bacterial periplasm that are generated upon stress conditions. In the absence of substrates, HtrA oligomerizes into a hexameric cage, but binding of misfolded proteins transforms the hexamers into bigger 12-mer and 24-mer cages that encapsulate the substrates for degradation or refolding. HtrA also undergoes partial degradation as a consequence of self-cleavage of the mature protein, producing short-HtrA protein (s-HtrA). The aim of this study was to examine the physiological role of this self-cleavage process. We found that the only requirement for self-cleavage of HtrA into s-HtrA in vitro was the hydrolysis of protein substrates. In fact, peptides resulting from the hydrolysis of the protein substrates were sufficient to induce autocleavage. However, the continuous presence of full-length substrate delayed the process. In addition, we observed that the hexameric cage structure is required for autocleavage and that s-HtrA accumulates only late in the degradation reaction. These results suggest that self-cleavage occurs when HtrA reassembles back into the resting hexameric structure and peptides resulting from substrate hydrolysis are allosterically stimulating the HtrA proteolytic activity. Our data support a model in which the physiological role of the self-cleavage process is to eliminate the excess of HtrA once the stress conditions cease

Cryo-electron microscopy of a reaction mixture containing DegP_S210A and β-casein at high temperatures.

<p>Representative micrographs of vitrified samples of a mixture of DegP_S210A and β-casein at 37°C (A) and 42°C (B). Contrast of both images was inverted for easy comparison with negatively stained micrographs in previous figures. The right panels in (A) and (B) show a comparison of two-dimensional projections (column labeled as “Projection”) calculated from the X-ray structure of DegP₂₄ (PDB ID: 3CS0) with class averages (column labeled as “Average”) of the corresponding views calculated from particle images extracted from the micrographs.</p

Effect of temperature on the oligomeric state of DegP_S210A:substrate complexes.

<p>Representative electron micrographs of reaction mixtures containing DegP_S210A alone (top panels), DegP_S210A+β-casein (middle panels) or DegP_S210A+β-casein peptides (bottom panels) at either 37°C (left panels) or 42°C (right panels). Complexes were assembled, applied to continuous carbon grids and negatively stained at the indicated temperatures.</p

Oligomeric state of DegP_S210A in the presence of substrates of different sizes.

<p><b>A</b>. Elution profiles from a Superdex-200 column of solutions containing either DegP_S210A alone (42 µM) (black line) or DegP_S210A (42 µM) mixed with β-casein (42 µM) (blue line) or β-casein peptide fragments (168 µM) (red line). The expected elution volumes (Ve) for DegP₆ (11.5 ml), DegP₁₂ (9.9 ml) and DegP₂₄ (8.4 ml) are indicated by the dashed lines in the plot. Numbers above the peaks refer to the gel lanes in the SDS-PAGE gel in panel B. <b>B</b>. SDS-PAGE analysis of fractions from the elution profiles in panel A. Lane 1, 2 and 3 resolved samples from fractions indicated with the corresponding numbers in the elution profiles in panel A. Similarly, lane 4 & 5 also contained samples from fractions indicated with the corresponding number in the elution profiles in panel A. However, these fractions were previously concentrated by Trichloroacetic acid precipitation (TCA ppt) to visualize the β-casein peptides. Lane 6 shows a sample of β-casein peptides used to form complexes. <b>C</b>. The DegP_S210A:substrate complexes purified by size exclusion chromatography in (A) were visualized by negative staining EM. The left panels shows representative negatively stained electron micrographs obtained from the central fraction of the peaks labeled as “1&4” (top), “2” (middle) and “5” in the elution profiles in (A). The right panels shows a comparison of two-dimensional projections (column labeled as “Projection”) calculated from the X-ray structure of DegP₂₄ (PDB ID: 3CS0) (middle panel) or the cryo-EM structure of DegP₁₂ (PDB ID: 2ZLE) (bottom panel) with class averages (column labeled as “Average”) of the corresponding views calculated from particle images extracted from the micrographs in the left panel.</p

Oligomeric state of DegP_S210A in the presence of PZ model peptides.

<p><b>A</b>. Names and sequences of the three PZ model peptides used to form complexes with DegP_S210A in the size exclusion chromatography experiment shown in (B). Each peptide contains a protease motif at the N-terminus (labeled as “P”) and a PDZ motif at the C-terminus (labeled as “Z”). The +/− sign next to the “P” and “Z” labels indicated whether the motif is recognized (+) or not recognized (−) by the corresponding domain. <b>B</b>. Elution profiles from a Superdex-200 column of solutions containing DegP_S210A alone (94 µM) or a mixture of DegP_S210A (94 µM) with one of the model peptides at a concentration of 376 µM. The expected elution volumes (Ve) for DegP₆ (11.5 ml) and DegP₁₂ (9.9 ml) are indicated by the dashed lines in the plot.</p

Filtered β-casein peptide fragments induce DegP₁₂ cages.

<p><b>A</b>. A 15% SDS-PAGE resolving a preparation of full-length β-casein (lines labeled as “β-casein”) and β-casein peptide fragments (lines labeled as “β-casein peptides”) before (−) and after (+) they were filtered through a 30 kDa-cutoff filter to remove full-length β-casein. Lines containing full-length β-casein were stained with Coomassie brilliant blue and lines containing the β-casein peptide fragments were silver stained to visualize that no full-length β-casein or proteolytically active DegP remained after filtration. <b>B</b>. Elution profiles from a Superdex-200 column of solutions containing DegP_S210A (42 µM) mixed with a preparation of β-casein peptide fragments (168 µM) that has been filtered through a 30 kDa-cutoff filter to remove traces of full-length β-casein. The expected elution volumes (Ve) for DegP₆ (11.5 mL), DegP₁₂ (9.9 mL) and DegP₂₄ (8.4 mL) are indicated by the dashed lines in the plot. <b>C</b>. Negatively stained electron micrograph obtained from the fraction at 10 mL from the size exclusion chromatography profile shown in panel B.</p

Oligomeric state of DegP_S210A in the presence of α-casein, malate dehydrogenase and their corresponding peptide fragments.

<p><b>A</b>. Coomassie brilliant blue stained 15% SDS-PAGE resolving the full-length α-casein and malate dehydrogenase (MDH) used to induce the formation of large oligomeric states in DegP_S210A. Peptides generated from both proteins by incubation with DegP are also shown in the gel to provide an estimation of their molecular weight. <b>B</b>. Elution profiles from a Superdex-200 column of solutions containing either DegP_S210A alone or DegP_S210A mixed with α-casein or α-casein peptide fragments. The concentration of each component of the reaction is indicated. The expected elution volumes (Ve) for DegP₆ (11.5 ml), DegP₁₂ (9.9 ml) and DegP₂₄ (8.4 ml) are indicated by the dashed lines in the plot. Numbers above the peaks refer to the electron micrographs in panel D. <b>C</b>. Elution profiles from a Superdex-200 column of solutions containing either DegP_S210A alone or DegP_210A mixed with MDH or MDH peptide fragments. The expected elution volumes (Ve) for DegP₆ (11.5 ml), DegP₁₂ (9.9 ml) and DegP₂₄ (8.4 ml) are indicated by the dashed lines in the plot. Numbers above the peaks refer to the electron micrographs in panel D. <b>D</b>. Visualization of complexes of DegP_S210A with α-casein and MDH substrates purified by size exclusion chromatography. The images are representative fields of negatively stained electron micrographs obtained from fractions labeled from 1 to 4 in the elution profiles shown in panels B and C. The arrowheads in panel 3 indicate some DegP₁₂ cages present in this fraction.</p

Oligomeric state of DegP_S210A at various substrate concentrations.

<p>Elution profiles of DegP_S210A in the presence of various concentrations of β-casein peptides (A) or full-length β-casein (B). The expected elution volumes (Ve) for DegP₆ (11.5 ml), DegP₁₂ (9.9 ml) and DegP₂₄ (8.4 ml) are indicated by the dashed lines in the plot. (C) Dynamic light scattering analysis of DegP_S210A in the presence of variable concentrations of full-length β-casein or β-casein peptides. The graph shows the distribution of hydrodynamic radius of particles for various mixtures of DegP_S210A and full-length β-casein or DegP_S210A and β-casein peptides.</p