Discovery of substrate for type I signal peptidase SpsB from Staphylococcus aureus.
Based on the kinetic model of substrate phage proteolysis, we have formulated a strategy for best manipulating the conditions in screening phage display libraries for protease substrates (Sharkov, N. A., Davis, R. M., Reidhaar-Olson, J. F., Navre, M., and Cai, D. (2001) J. Biol. Chem. 276, 10788-10793). This strategy is exploited in the present study with signal peptidase SpsB from Staphylococcus aureus. We demonstrate that highly active substrate phage clones can be isolated from a phage display library by systematically tuning the selection stringency in screening. Several of the selected clones exhibit superior reactivity over a control, the best clone, SIIIRIII-8, showing >100-fold improvement. Because no conserved sequence features were readily revealed that could allow delineation of the active and unreactive clones, the sequences identified in five of the active clones were tested as synthetic dodecamers, Ac-AGX(8)GA-NH(2). Using electrospray ionization mass spectrometry, we show that four of these peptides can be cleaved by SpsB and that Ala is the P1 residue exclusively and Ala or Leu the P3 residue, in keeping with the (-3, -1) rule for substrate recognition by signal peptidase. Our successful screening with SpsB demonstrated the general applicability of the screening strategy and allowed us to isolate the first peptide substrates for the enzyme.
Sharkov
Nikolai A
NA
Affymax Research Institute, Palo Alto, California 94304, USA.
Cai
Danying
D
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis / Q000138:chemical synthesis; D019151:Peptide Library; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000201:enzymology*; D013379:Substrate Specificity
D017931:DNA Primers; D008565:Membrane Proteins; D010446:Peptide Fragments; D019151:Peptide Library; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C026515:type I signal peptidase