Sept. 3, 2014
Protective antigen (PA) mediates entry of edema factor (EF) and lethal factor (LF) into the cytoplasmic space of the cells through the formation of a membrane spanning pore. To do this, PA must initially bind to a host cellular receptor. Recent mass spectrometry analysis of PA using histidine hydrogen-deuterium exchange (His-HDX) has shown that binding of the von Willebrand factor A (vWA) domain of the receptor capillary morphogenesis protein-2 (CMG2) lowers the exchange rates of the imidazole C2-hydrogen of several histidines, suggesting that receptor binding decreases the structural flexibility of PA. Here, using His-HDX and fluorescence as a function of denaturant, and protease susceptibility, we show that binding of the vWA domain of CMG2 largely increases the stability of PA and the effect reaches up to 70 Å from the receptor binding interface. We also show that the pKas and HDX rates of histidines located in separate domains change upon receptor binding. These results indicate that by anchoring one end of the protein, the structure of PA is tightened, non-covalent interactions are strengthened and the global stability of the protein increases. These findings suggest that CMG2 may be used to stabilize PA in future anthrax vaccines.
Figure 1. Structure of the PA.CMG2 complex (PDB entry 1T6B). Eight histidine residues are shown (green sticks). Domain 1, Ser15.Ala258 (magenta); domain 2, Tyr259.Thr487 (blue); domain 3, Thr488.Arg595 (pink); domain 4, Phe596.Ile734 (yellow); vWA domain of CMG2 (orange). All the histidine residues in PA except His304 and His310 are shown. His304 and His310 are not observed in the structure. The 188.8.131.52 loop of domain 2 is also indicated.
Results from: Mullangi V, Mamillapalli S, Anderson DJ, Bann JG, Miyagi M.Biochemistry. 2014 Sep 3. [Epub ahead of print]