Conformational analysis of gamma' peptide (410-427) interactions with thrombin anion binding exosite II.
Thrombin utilizes two anion binding exosites to supplement binding of fibrinogen to this serine protease. Approximately 7-15% of the fibrinogen gamma chain exists as the highly anionic gamma' variant (408VRPEHPAETEY(S)DSLY(S)PEDDL427). This segment has been demonstrated to target thrombin ABE-II and can accommodate sites of phosphorylation in place of sulfonation without sacrificing binding affinity. The present work employed 1D and 2D solution NMR to characterize the structural features of the bound gamma' peptide (410-427) and to evaluate the requirement of sulfonation for effective thrombin interaction. The results indicate the gamma' residues 414-427 make significant contact with the enzyme, a beta-turn exists between residues 422-425 in the presence of thrombin, and there is a large cluster of through-space interactions involving residues 418-422. Effective contact with ABE-II requires the presence of at least one phosphotyrosine residue with Y(P)422 being the more important player. Hydrogen-deuterium exchange (HDX) coupled with MALDI-TOF MS was implemented to examine the location of the gamma' peptide-thrombin interface and to screen for changes in solvent exposure at distant sites. The HDX results demonstrate that the gamma' peptide interacts with or is in close proximity to thrombin residues R93, R97, R173, and R175. The binding of the gamma' peptide also protects other regions of thrombin from deuterium exchange. Affected regions include segments of ABE-I, the autolysis loop, the edge of the active site region, and the A-chain. Finally, thrombin forms a ternary complex with the gamma' peptide and PPACK, generating an enzyme whose solvent-exposed regions are even further stabilized from HDX.
Sabo
T Michael
TM
Department of Chemistry, University of Louisville, 2320 South Brook Street, Louisville, Kentucky 40292, USA.
Farrell
David H
DH
Maurer
Muriel C
MC
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D018360:Crystallography, X-Ray; D004220:Disulfides / Q000737:chemistry; D005340:Fibrinogen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D010434:Pepsin A / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013917:Thrombin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D004220:Disulfides; D010455:Peptides; C027539:fibrinopeptides gamma; D005340:Fibrinogen; D013917:Thrombin; D010434:Pepsin A