Factors Defining the Functional Oligomeric State of Escherichia coli DegP Protease

Jack Iwanczyk,Joaquin Ortega,Vivian Leong,Christophe Herman

doi:10.1371/journal.pone.0018944

Abstract

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.

Highlights

Escherichia coli DegP is an important enzyme that contributes to the maintenance of protein homeostasis in the bacterial periplasm [1,2,3]
Substrate size determines DegP oligomeric state The large DegP oligomerizes into 12-mer (DegP12) and DegP24 cage structures of the enzyme in the presence of substrates [10,11] confirmed early models of DegP as a self-compartmentalized protease [7,18], and suggested that the role of the bigger cages is to accommodate larger substrates that otherwise would not fit inside the DegP hexamer
We found that the size of the substrate molecule is the main determinant of the functional oligomeric state adopted by the enzyme

Summary

Introduction

Escherichia coli DegP ( called HtrA or protease Do) is an important enzyme that contributes to the maintenance of protein homeostasis in the bacterial periplasm [1,2,3]. The monomers oligomerize into a hexameric cage with a central cavity where the catalytic sites are sequestered [8]. Both the top and bottom of the cage are constructed with the protease domain of three DegP monomers. Each LA loop extends beyond the pillar it is forming and ends up protruding into the active site of the subunit in the opposite trimer. There, it interacts with its active site loops L1 and L2, forcing the catalytic site into a twisted inactive conformation. The hexameric form of DegP constitutes an inactive state of the protein [8]

Methods

Results

Conclusion