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J Biol Chem. 2014 May 30;289(22):15544-53. doi: 10.1074/jbc.M113.507616. Epub 2014 Apr 8.

Staphylococcal SplB serine protease utilizes a novel molecular mechanism of activation.

Author information

1
From the Department of Analytical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30 387 Krakow, Poland, the Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30 387 Krakow, Poland.
2
the Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30 387 Krakow, Poland.
3
From the Department of Analytical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30 387 Krakow, Poland.
4
From the Department of Analytical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30 387 Krakow, Poland, the NMR Group, Max-Planck Institute for Biochemistry, 82 152 Martinsried, Germany.
5
the NMR Group, Max-Planck Institute for Biochemistry, 82 152 Martinsried, Germany, the Deutsches Forschungszentrum für Gesundheit und Umwelt, Helmholtz Zentrum München, D-85764 Neuherberg, Germany.
6
From the Department of Analytical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30 387 Krakow, Poland, the Malopolska Centre of Biotechnology, 30 387 Krakow, Poland.
7
the Division of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, 50 370 Wroclaw, Poland, the Program in Cell Death Research, Sanford-Burnham Medical Research Institute, La Jolla, California 92037.
8
the Program in Cell Death Research, Sanford-Burnham Medical Research Institute, La Jolla, California 92037.
9
the Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30 387 Krakow, Poland, the Center of Oral Health and Systemic Disease, School of Dentistry, University of Louisville, Louisville, Kentucky 40202, and.
10
the Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30 387 Krakow, Poland, the Malopolska Centre of Biotechnology, 30 387 Krakow, Poland grzegorz.dubin@uj.edu.pl.

Abstract

Staphylococcal SplB protease belongs to the chymotrypsin family. Chymotrypsin zymogen is activated by proteolytic processing at the N terminus, resulting in significant structural rearrangement at the active site. Here, we demonstrate that the molecular mechanism of SplB protease activation differs significantly and we characterize the novel mechanism in detail. Using peptide and protein substrates we show that the native signal peptide, or any N-terminal extension, has an inhibitory effect on SplB. Only precise N-terminal processing releases the full proteolytic activity of the wild type analogously to chymotrypsin. However, comparison of the crystal structures of mature SplB and a zymogen mimic show no rearrangement at the active site whatsoever. Instead, only the formation of a unique hydrogen bond network, distant form the active site, by the new N-terminal glutamic acid of mature SplB is observed. The importance of this network and influence of particular hydrogen bond interactions at the N terminus on the catalytic process is demonstrated by evaluating the kinetics of a series of mutants. The results allow us to propose a consistent model where changes in the overall protein dynamics rather than structural rearrangement of the active site are involved in the activation process.

KEYWORDS:

Crystal Structure; Enzyme Inactivation; Protease; Serine Protease; Signal Peptide; Staphylococcus aureus; Zymogen Activation

PMID:
24713703
PMCID:
PMC4140910
DOI:
10.1074/jbc.M113.507616
[Indexed for MEDLINE]
Free PMC Article

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