title: Long-range stabilization of anthrax protective antigen upon binding to CMG2.
abstract: 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 pKa values and HDX rates of histidines located in separate domains change upon receptor binding. These results indicate that when one end of the protein is anchored, the structure of PA is tightened, noncovalent 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.
authors.affiliation: Case Center for Proteomics and Bioinformatics, ‡Department of Pharmacology, and §Department of Ophthalmology and Visual Sciences, Case Western Reserve University , Cleveland, Ohio 44106, United States.
authors.forename: Vennela
authors.initials: V
authors.lastname: Mullangi
authors.forename: Sireesha
authors.initials: S
authors.lastname: Mamillapalli
authors.forename: David J
authors.initials: DJ
authors.lastname: Anderson
authors.forename: James G
authors.initials: JG
authors.lastname: Bann
authors.forename: Masaru
authors.initials: M
authors.lastname: Miyagi
mesh_terms: D000942:Antigens, Bacterial / Q000737:chemistry / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010447:Peptide Hydrolases / Q000378:metabolism; D011485:Protein Binding; D018000:Receptors, Peptide / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: C447150:ANTXR2 protein, human; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D018000:Receptors, Peptide; C030325:anthrax toxin; D010447:Peptide Hydrolases