Conformational analysis of γ′ peptide (410-427) interactions with thrombin anion binding exosite II

T. Michael Sabo, David Farrell, Muriel C. Maurer

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

Thrombin utilizes two anion binding exosites to supplement binding of fibrinogen to this serine protease. Approximately 7-15% of the fibrinogen γ chain exists as the highly anionic γ′ variant ( 408-VRPEHPAETEYSDSLYSPEDDL427). 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 γ′ peptide (410-427) and to evaluate the requirement of sulfonation for effective thrombin interaction. The results indicate the γ′ residues 414-427 make significant contact with the enzyme, a β-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 YP 422 being the more important player. Hydrogen-deuterium exchange (HDX) coupled with MALDI-TOF MS was implemented to examine the location of the γ′ peptide-thrombin interface and to screen for changes in solvent exposure at distant sites. The HDX results demonstrate that the γ′ peptide interacts with or is in close proximity to thrombin residues R93, R97, R173, and R175. The binding of the γ′ 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 γ′ peptide and PPACK, generating an enzyme whose solvent-exposed regions are even further stabilized from HDX.

Original languageEnglish (US)
Pages (from-to)7434-7445
Number of pages12
JournalBiochemistry
Volume45
Issue number24
DOIs
StatePublished - Jun 20 2006

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Thrombin
Anions
Peptides
Deuterium
Hydrogen
Sulfonation
R 97
Fibrinogen
Autolysis
Phosphotyrosine
Phosphorylation
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Serine Proteases
Enzymes
Catalytic Domain
Nuclear magnetic resonance

ASJC Scopus subject areas

  • Biochemistry

Cite this

Conformational analysis of γ′ peptide (410-427) interactions with thrombin anion binding exosite II. / Sabo, T. Michael; Farrell, David; Maurer, Muriel C.

In: Biochemistry, Vol. 45, No. 24, 20.06.2006, p. 7434-7445.

Research output: Contribution to journalArticle

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