Mapping the Oligomeric Assembly of Plasma Factor XIII A2B2 by Hydrogen-Deuterium Exchange Coupled with MALDI-TOF Mass Spectrometry

Abstract

Factor XIII (FXIII) is responsible for catalyzing the formation of covalent γ-glutamyl-e-lysyl crosslinks in fibrin chains during blood coagulation. In plasma, unactivated FXIII exists as a heterotetramer consisting of catalytic A2 and regulatory B2 subunits. Each B subunit has 10 tandem repeats called sushi domains. In the presence of thrombin and calcium, the FXIII B2 units are released and the FXIII A2 units are activated. X-ray crystal structures have been solved for FXIII A2, but none are yet available for FXIII A2B2. The current work employed amide proton hydrogen-deuterium exchange coupled with MALDI-TOF mass spectrometry to map sites of contact between the FXIII B2 and A2 subunits. Native gel electrophoresis demonstrated that FXIII A2B2 could be created from mixtures of recombinant A2 and B2 subunits. The high disulfide content of the B2 sushi domains hindered pepsin cleavage of the FXIII B2 dimer and the A2B2 heterotetramer. In the presence of the B2 subunits, sequence coverage for a pepsin digest of FXIII A2 decreased 3-fold further supporting heterotetramer formation. HDX studies then revealed the regions of FXIII A2 that were protected from HDX in the presence of B2. Decreases in percent deuteration were observed for 220-230 (glutamine substrate recognition site), 240-247 (A2 dimer interface), 298-305 (catalytic core domain), 328-338 (catalytic core domain), 607-619 (beta barrel 1), and 632-646 (beta barrel 2). These results support the proposal that the B2 subunits straddle across the A2 subunits protecting the transglutaminase from becoming prematurely activated. Proteolytic or nonproteolytic activation of the A2 units then leads to conformational changes that emphasize the dimer interface, the catalytic core regions, and influences emanating from the calcium binding site. (NIH HL068440)