Biophysical investigation of GpIbalpha binding to thrombin anion binding exosite II.
Substrates and cofactors of the serine protease thrombin (IIa) employ two anion binding exosites (ABE-I and -II) to aid in binding. On the surface of platelets resides the GpIbalpha/beta-GpIX-GpV membrane-bound receptor complex. IIa's ABE-II is proposed to interact with an anionic portion of GpIbalpha which enhances IIa cleavage of PAR-1 and subsequent activation of platelets. In this work, one-dimensional (1D) and two-dimensional (2D) NMR, analytical ultracentrifugation (AUC), and hydrogen-deuterium exchange (HDX) coupled with MALDI-TOF MS were performed to further characterize the features of binding to IIa's ABEs. The described work builds upon investigations performed in a prior study with fibrin(ogen)'s gamma' peptide and IIa [Sabo, T. M., Farrell, D. H., and Maurer, M. C. (2006) Biochemistry 45, 7434-7445]. 1D line broadening NMR (1H and 31P) and 2D trNOESY NMR studies indicate that GpIbalpha residues D274-E285 interact extensively with the IIa surface in an extended conformation. AUC demonstrates that both GpIbalpha (269-286) and gamma' (410-427) peptides interact with IIa with a 1:1 stoichiometry. When the HDX results are compared to those for the ABE-I targeting peptide hirudin (54-65), the data imply that GpIbalpha (269-286), GpIbalpha (1-290), and gamma' (410-427) are indeed directed to ABE-II. The ABE-II binding fragments reduce HDX for sites distant from the interface, suggesting long-range conformational effects. These studies illustrate that GpIbalpha and gamma' target ABE-II with similar consequences on IIa dynamics, albeit with differing structural features.
Sabo
T Michael
TM
Department of Chemistry, University of Louisville, 2320 South Brook Street, Louisville, Kentucky 40292, USA.
Maurer
Muriel C
MC
D000818:Animals; D000838:Anions / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002417:Cattle; D041961:Deuterium Exchange Measurement; D006629:Hirudins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D019038:Platelet Glycoprotein GPIb-IX Complex; D011485:Protein Binding; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D000838:Anions; D006629:Hirudins; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D010455:Peptides; D019038:Platelet Glycoprotein GPIb-IX Complex; C094990:adhesion receptor; C091596:hirudin (54-65); D013917:Thrombin