The role of the L2 loop in the regulation and maintaining the proteolytic activity of HtrA (DegP) protein from Escherichia coli.
The aim of this study was to characterize the role of particular elements of the regulatory loop L2 in the activation process and maintaining the proteolytic activity of HtrA (DegP) from Escherichia coli. We measured the effects of various mutations introduced to the L2 loop's region (residues 228-238) on the stability of HtrA molecule and its proteolytic activity. We demonstrated that most mutations affected the activity of HtrA. In the case of the following substitutions: L229N, N235I, I238N, the proteolytic activity was undetectable. Thus, the majority of interactions mediated by the studied amino-acid residues seem to play important role in maintaining the active conformation. Formation of contacts between the apical parts (residues 231-234) of the L2 loops within the HtrA trimer, in particular the residues D232, was shown to play a crucial role in the activation process of HtrA. Stabilization of these intermolecular interactions by substitution of D232 with valine caused a stimulation of proteolytic activity whereas deletion of this region abolished the activity. Since the pathogenic E. coli strains require active HtrA for virulence, the apical part of L2 is of particular interest in terms of structure-based drug design for treatment E. coli infections.
Department of Biochemistry, University of Gdansk, Poland.
Anna
A
Sobiecka-Szkatula
Artur
A
Gieldon
Andrea
A
Scire
Fabio
F
Tanfani
Donata
D
Figaj
Tomasz
T
Koper
Jerzy
J
Ciarkowski
Barbara
B
Lipinska
Joanna
J
Skorko-Glonek
D019943:Amino Acid Substitution; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D033903:Periplasmic Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D011489:Protein Denaturation; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017550:Spectroscopy, Fourier Transform Infrared; D014774:Virulence / Q000235:genetics / Q000502:physiology
D017931:DNA Primers; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D050505:Mutant Proteins; D033903:Periplasmic Proteins; D011994:Recombinant Proteins; C061682:DegP protease; D012697:Serine Endopeptidases