title: The LD loop as an important structural element required for transmission of the allosteric signal in the HtrA (DegP) protease from Escherichia coli.
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.
authors.affiliation: Department of General and Medical Biochemistry, Faculty of Biology, University of Gdansk, Poland.
authors.forename: Donata
authors.initials: D
authors.lastname: Figaj
authors.affiliation: Department of Theoretical Chemistry, Faculty of Chemistry, University of Gdansk, Poland.
authors.forename: Artur
authors.initials: A
authors.lastname: Gieldon
authors.affiliation: Department of General and Medical Biochemistry, Faculty of Biology, University of Gdansk, Poland.
authors.forename: Marlena
authors.initials: M
authors.lastname: Bartczak
authors.affiliation: Department of General and Medical Biochemistry, Faculty of Biology, University of Gdansk, Poland.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Koper
authors.affiliation: Department of General and Medical Biochemistry, Faculty of Biology, University of Gdansk, Poland.
authors.forename: Urszula
authors.initials: U
authors.lastname: Zarzecka
authors.affiliation: Department of Molecular Biochemistry, Faculty of Chemistry, University of Gdansk, Poland.
authors.forename: Adam
authors.initials: A
authors.lastname: Lesner
authors.affiliation: Department of General and Medical Biochemistry, Faculty of Biology, University of Gdansk, Poland.
authors.forename: Barbara
authors.initials: B
authors.lastname: Lipinska
authors.affiliation: Department of General and Medical Biochemistry, Faculty of Biology, University of Gdansk, Poland. joanna.skorko-glonek@biol.ug.edu.pl.
authors.forename: Joanna
authors.initials: J
authors.lastname: Skorko-Glonek
mesh_terms: D000495:Allosteric Site / Q000235:genetics; D019943:Amino Acid Substitution; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D033903:Periplasmic Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011489:Protein Denaturation; D054730:Protein Interaction Domains and Motifs; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D033903:Periplasmic Proteins; D011994:Recombinant Proteins; C061682:DegP protease; D012697:Serine Endopeptidases
keywords: allosteric regulation; enzyme mutation; kinetic measurements; molecular biology; serine protease