Long-range stabilization of anthrax protective antigen upon binding to CMG2

Area of Science:

• Biochemistry
• Structural Biology
• Microbiology

Background:

• Protective antigen (PA) facilitates the cellular entry of anthrax toxins, edema factor (EF) and lethal factor (LF).
• PA requires binding to a host receptor, like capillary morphogenesis protein-2 (CMG2), to initiate toxin delivery.
• Previous studies indicated receptor binding might reduce PA structural flexibility.

Purpose of the Study:

• To investigate the structural and stability changes in protective antigen (PA) upon binding to the von Willebrand factor A (vWA) domain of CMG2.
• To determine the extent and mechanism of PA stabilization induced by CMG2 binding.

Main Methods:

• Histidine hydrogen-deuterium exchange (His-HDX) mass spectrometry to monitor protein structural changes.
• Fluorescence spectroscopy as a function of denaturant to assess protein stability.
• Protease susceptibility assays to evaluate structural integrity.

Main Results:

• CMG2 vWA domain binding significantly increases the overall stability of PA.
• The stabilizing effect of CMG2 binding extends up to 70 Å from the receptor-binding interface.
• Receptor binding alters the pKa values and hydrogen-deuterium exchange rates of histidines in distinct PA domains, indicating a global structural response.

Conclusions:

• CMG2 binding induces a global tightening of the PA structure, strengthening noncovalent interactions and increasing protein stability.
• This stabilization mechanism suggests that CMG2 could be a valuable component for developing more stable protective antigen-based anthrax vaccines.