1. The LD loop as an important structural element required for transmission of the allosteric signal in the HtrA (DegP) protease from Escherichia coli.
- Author
-
Figaj D, Gieldon A, Bartczak M, Koper T, Zarzecka U, Lesner A, Lipinska B, and Skorko-Glonek J
- Subjects
- Allosteric Site genetics, Amino Acid Substitution, Enzyme Stability, Escherichia coli genetics, Escherichia coli Proteins genetics, Escherichia coli Proteins metabolism, Heat-Shock Proteins genetics, Heat-Shock Proteins metabolism, Kinetics, Models, Molecular, Mutagenesis, Site-Directed, Periplasmic Proteins genetics, Periplasmic Proteins metabolism, Protein Denaturation, Protein Interaction Domains and Motifs, Protein Structure, Quaternary, Protein Structure, Secondary, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Serine Endopeptidases genetics, Serine Endopeptidases metabolism, Escherichia coli enzymology, Escherichia coli Proteins chemistry, Heat-Shock Proteins chemistry, Periplasmic Proteins chemistry, Serine Endopeptidases chemistry
- Abstract
High-temperature requirement A (HtrA; DegP) from Escherichia coli, an important element of the extracytoplasmic protein quality-control system, is a member of the evolutionarily conserved family of serine proteases. The characteristic feature of this protein is its allosteric mode of activation. The regulatory loops, L3, L2, L1 and LD, play a crucial role in the transmission of the allosteric signal. Yet, the role of LD has not been fully elucidated. Therefore, we undertook a study to explain the role of the individual LD residues in inducing and maintaining the proteolytic activity of HtrA. We investigated the influence of amino acid substitutions located within the LD loop on the kinetics of a model substrate cleavage as well as on the dynamics of the oligomeric structure of HtrA. We found that the mutations that were expected to disturb the loop's structure and/or interactions with the remaining regulatory loops severely diminished the proteolytic activity of HtrA. The opposite effect, that is, increased activity, was observed for G174S substitution, which was predicted to strengthen the interactions mediated by LD. HtrAG174S protein had an equilibrium shifted toward the active enzyme and formed preferentially high-order oligomeric forms., (© 2016 Federation of European Biochemical Societies.)
- Published
- 2016
- Full Text
- View/download PDF