Structural Characterization of a Thrombin-Aptamer Complex by High Resolution Native Top-Down Mass Spectrometry

Area of Science:

• Biochemistry
• Structural Biology
• Biophysics

Background:

• Native mass spectrometry (MS) is crucial for analyzing intact proteins and complexes.
• Human thrombin is a key enzyme in coagulation, and its interaction with aptamers is of therapeutic interest.

Purpose of the Study:

• To structurally characterize human thrombin and its complex with the thrombin binding aptamer (TBA) using high-resolution native top-down MS.
• To investigate the influence of solution conditions on thrombin-TBA complex formation and stoichiometry.
• To identify the primary binding site of TBA on thrombin.

Main Methods:

• High-resolution native top-down mass spectrometry (MS) with electrospray ionization (ESI).
• Accurate mass measurements for glycosylation analysis.
• Electron capture dissociation (ECD) MS on a 15 Tesla Fourier transform ion cyclotron resonance (FTICR) mass spectrometer.
• Varying solution ionic strength and pH to study binding interactions.

Main Results:

• Native human α-thrombin predominantly features a 2205 Da sialylated, symmetric biantennary oligosaccharide glycosylation without fucosylation.
• Thrombin and TBA form a 1:1 complex under near physiological conditions (pH 6.8, 200 mM NH4OAc).
• Complex stoichiometry is sensitive to ionic strength, with higher TBA binding at lower ionic strength and dissociation at high ionic strength (1 M NH4OAc).
• ECD MS identified the primary binding site of TBA to thrombin at exosite I, consistent with crystallographic data.
• Binding is mediated by electrostatic interactions, supported by thrombin's surface anion binding sites.

Conclusions:

• High-resolution native top-down MS provides detailed structural insights into native proteins and protein-DNA complexes.
• The study elucidates thrombin glycosylation and the mechanism of thrombin-TBA complex formation.
• Native MS serves as a valuable complementary technique to traditional structural biology methods.