An internal histidine residue from the bacterial surface protein, PAM, mediates its binding to the kringle-2 domain of human plasminogen.
The determinants of binding of a peptide lacking C-termini-exposed lysine residues to a kringle domain were investigated using an up-regulated lysine binding kringle (K2Pg[C4G/E56D/K72Y]) of plasminogen and a peptide (a1-PAM) with a sequence derived from a surface-exposed M-like streptococcal protein. Significant kringle-induced chemical shifts in a His side-chain of a1-PAM were revealed by two-dimensional NMR. Further studies using isothermal titration calorimetry (ITC) provided support for the involvement of His12 in the peptide/ protein complex. In an effort to screen a1-PAM-derived truncation peptides, a combinatorial mixture, a1deltaa2-PAM[H12X] (where X=Pro, Arg, His, Trp, Lys, Ala, Phe, Asp and Gly), was analyzed using the surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI) platform. The major peptide that remained bound to the surface of the K2Pg[C4G/ E56D/K72Y]-containing chip was that containing His12, corresponding to the wild-type sequence. Minor peaks, representing binding, were obtained for Lys12-, Arg12- and Trp12-containing peptides. Individual peptides containing these amino acids were then examined using ITC and the binding constants obtained correlated with the relative strengths of binding estimated from the SELDI-based screen.
Schenone
M M
MM
Department of Chemistry and Biochemistry, University of Notre Dame, IN, USA.
Warder
S E
SE
Martin
J A
JA
Prorok
M
M
Castellino
F J
FJ
D001426:Bacterial Proteins; D002151:Calorimetry; D002352:Carrier Proteins / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D018082:Kringles; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry; D010958:Plasminogen / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013816:Thermodynamics; D013997:Time Factors
D001426:Bacterial Proteins; D002352:Carrier Proteins; D008024:Ligands; D010455:Peptides; C084529:plasminogen-binding protein, bacteria; D006639:Histidine; D010958:Plasminogen