Conformational Analysis of γ‘ Peptide (410−427) Interactions with Thrombin Anion Binding Exosite II

Abstract

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.

Ligand Binding to Anion-binding Exosites Regulates Conformational Properties of Thrombin

“…In addition, modest protection could at times be documented for the thrombin fragments 85-99 and 135-149D. Previous HDX studies also reported effects on these three thrombin fragments (15,28). The solvent protection observed for thrombin fragments 173-180 and 173-181 in the presence of PPACK was further maintained upon the addition of Hirudin(54 -65).…”

“…4) verified that ligand binding to ABE II is not dependent on the presence of an intact ABE I. Prior NMR studies on the binary fibrinogen ␥Ј(410 -427) to ␥-thrombin complex could only demonstrate that thrombin contact sites were maintained in the presence of a species with a disrupted ABE I (15). The current HDX studies on ␥-thrombin revealed that the GpIb␣ and fibrinogen ␥Ј ligands were still protecting ABE II from hydrogen-deuterium exchange (see Fig.…”

“…All of the bovine thrombin peptides in the peptic digest were previously identified by Croy et al (28), and the identities were verified in our laboratory by using post-source decay sequencing (15). All of the spectra were calibrated using two reference peptides, including the singly protonated monoisotopic mass 888.4943 Da (46 -52 residues) and singly protonated pentaisotopic mass 2106.1505 Da (85-99 residues).…”

“…All of the spectra were calibrated using two reference peptides, including the singly protonated monoisotopic mass 888.4943 Da (46 -52 residues) and singly protonated pentaisotopic mass 2106.1505 Da (85-99 residues). The amount of deuterium uptake by each peptide was quantified as described by Sabo et al (15,16).…”

“…Our previous HDX data demonstrated that binding of either GpIb␣(269 -286) or fibrinogen ␥Ј(410 -427) to ABE II led to distinctive interexosite communication over to ABE I (15,16). By contrast, other HDX studies showed that binding of Hirudin (54 -65) to ABE I did not cause the same kind of extensive long range effect to ABE II (16).…”