The plasma Factor XIII heterotetrameric complex structure: unexpected unequal pairing within a symmetric complex

Abstract

AbstractFactor XIII (FXIII) is a predominant determinant of clot stability, strength, and composition. Plasma FXIII circulates as a pro-transglutaminase with 2 catalytic A subunits and 2 carrier-protective B subunits in a heterotetramer (FXIII-A2B2). FXIII-A2and -B2subunits are synthesized separately and then assembled in plasma. Following proteolytic activation by thrombin and calcium-mediated dissociation of the B-subunits, activated FXIII (FXIIIa) covalently cross-links fibrin, promoting clot stability. The zymogen and active states of the FXIII-A subunits have been structurally characterized; however, the structure of FXIII-B subunits and the FXIII-A2B2complex have remained elusive. Using integrative hybrid approaches including atomic force microscopy, cross-linking mass spectrometry, and computational approaches, we have constructed the first all-atom model of the FXIII-A2B2complex. We also used molecular dynamic simulations in combination with isothermal titration calorimetry to characterize FXIII-A2B2assembly, activation, and dissociation. Our data reveal unequal pairing of individual subunit monomers in an otherwise symmetric complex, and suggest this unusual structure is critical for both assembly and activation of this complex. Our findings enhance understanding of mechanisms associating FXIII-A2B2mutations with disease and have important implications for the rational design of molecules to alter FXIII assembly and/or activity to reduce bleeding and thrombotic complications.