The LD loop as an important structural element required for transmission of the allosteric signal in the HtrA (DegP) protease from Escherichia coli

• Published in: The FEBS journal 2016-09, Vol.283 (18), p.3471-3487
• Main Authors: Figaj, Donata, Gieldon, Artur, Bartczak, Marlena, Koper, Tomasz, Zarzecka, Urszula, Lesner, Adam, Lipinska, Barbara, Skorko‐Glonek, Joanna
• Format: Article
• Language: English
• Subjects: allosteric regulation; Allosteric Site - genetics; Amino Acid Substitution; Amino acids; E coli; enzyme mutation; Enzyme Stability; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Heat-Shock Proteins - chemistry; Heat-Shock Proteins - genetics; Heat-Shock Proteins - metabolism; kinetic measurements; Kinetics; Models, Molecular; molecular biology; Mutagenesis, Site-Directed; Periplasmic Proteins - chemistry; Periplasmic Proteins - genetics; Periplasmic Proteins - metabolism; Proteases; Protein Denaturation; Protein Interaction Domains and Motifs; Protein Structure, Quaternary; Protein Structure, Secondary; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Serine Endopeptidases - chemistry; Serine Endopeptidases - genetics; Serine Endopeptidases - metabolism; serine protease
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/febs.13822

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. HtrA is a protease characterized by an allosteric mode of activation. The regulatory loops, L3, L2, L1, and LD, are necessary for the transmission of allosteric signal. We found that disturbance of LD's structure and/or interactions with the remaining regulatory loops severely diminished the activity of HtrA. The G174S substitution, predicted to strengthen the activation cluster, led to increased activity and preferential formation of high‐order oligomers by HtrA.