TY - JOUR
T1 - Escherichia coli DegP protease cleaves between paired hydrophobic residues in a natural substrate
T2 - The PapA pilin
AU - Jones, C. Hal
AU - Dexter, Paul
AU - Evans, Amy K.
AU - Liu, Christopher
AU - Hultgren, Scott J.
AU - Hruby, Dennis E.
PY - 2002/10
Y1 - 2002/10
N2 - The DegP protein, a multifunctional chaperone and protease, is essential for clearance of denatured or aggregated proteins from the inner-membrane and periplasmic space in Escherichia coli. To date, four natural targets for DegP have been described: colicin A lysis protein, pilin subunits and MalS from E. coli, and high-molecular-weight adherence proteins from Haemophilus influenzae. In vitro, DegP has shown weak protease activity with casein and several other nonnative substrates. We report here the identification of the major pilin subunit of the Pap pilus, PapA, as a natural DegP substrate and demonstrate binding and proteolysis of this substrate in vitro. Using overlapping peptide arrays, we identified three regions in PapA that are preferentially cleaved by DegP. A 7-mer peptide was found to be a suitable substrate for cleavage by DegP in vitro. In vitro proteolysis of model peptide substrates revealed that cleavage is dependent upon the presence of paired hydrophobic amino acids; moreover, cleavage was found to occur between the hydrophobic residues. Finally, we demonstrate that the conserved carboxyl-terminal sequence in pilin subunits, although not a cleavage substrate for DegP, activates the protease and we propose that the activating peptide is recognized by DegP's PDZ domains.
AB - The DegP protein, a multifunctional chaperone and protease, is essential for clearance of denatured or aggregated proteins from the inner-membrane and periplasmic space in Escherichia coli. To date, four natural targets for DegP have been described: colicin A lysis protein, pilin subunits and MalS from E. coli, and high-molecular-weight adherence proteins from Haemophilus influenzae. In vitro, DegP has shown weak protease activity with casein and several other nonnative substrates. We report here the identification of the major pilin subunit of the Pap pilus, PapA, as a natural DegP substrate and demonstrate binding and proteolysis of this substrate in vitro. Using overlapping peptide arrays, we identified three regions in PapA that are preferentially cleaved by DegP. A 7-mer peptide was found to be a suitable substrate for cleavage by DegP in vitro. In vitro proteolysis of model peptide substrates revealed that cleavage is dependent upon the presence of paired hydrophobic amino acids; moreover, cleavage was found to occur between the hydrophobic residues. Finally, we demonstrate that the conserved carboxyl-terminal sequence in pilin subunits, although not a cleavage substrate for DegP, activates the protease and we propose that the activating peptide is recognized by DegP's PDZ domains.
UR - http://www.scopus.com/inward/record.url?scp=0036786221&partnerID=8YFLogxK
U2 - 10.1128/JB.184.20.5762-5771.2002
DO - 10.1128/JB.184.20.5762-5771.2002
M3 - Article
C2 - 12270835
AN - SCOPUS:0036786221
SN - 0021-9193
VL - 184
SP - 5762
EP - 5771
JO - Journal of bacteriology
JF - Journal of bacteriology
IS - 20
ER -