Staphylococcal SplB serine protease utilizes a novel molecular mechanism of activation

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


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Catalytic Domain
  • Chymotrypsin / chemistry
  • Chymotrypsin / genetics
  • Chymotrypsin / metabolism
  • Crystallography, X-Ray
  • Enzyme Precursors / metabolism
  • Hydrogen Bonding
  • Models, Molecular
  • Protein Sorting Signals / physiology
  • Protein Structure, Tertiary
  • Serine Proteases / chemistry*
  • Serine Proteases / genetics
  • Serine Proteases / metabolism*
  • Staphylococcus aureus / enzymology*
  • Staphylococcus aureus / genetics
  • Structure-Activity Relationship


  • Bacterial Proteins
  • Enzyme Precursors
  • Protein Sorting Signals
  • Serine Proteases
  • Chymotrypsin

Associated data

  • PDB/4K1S
  • PDB/4K1T