Activation of factor XIII is accompanied by a change in oligomerization state

Abstract

Factor XIII A (FXIIIA) is a member of the transglutaminase enzyme family that cross-links both intra- and extracellular protein substrates. To prevent undesired cross-linking, FXIIIA is expressed as an inactive zymogen and exists intracellularly as an A2 homodimer. In plasma, FXIII A2 is complexed with two protective factor XIII B subunits (A2B2) that dissociate upon activation of the zymogen. Based on limited experimental data, activated FXIII was considered a dimer of two catalytically active A subunits. However, accumulating but indirect evidence has suggested activation may lead to a monomeric state instead. In the present study, we employed analytical ultracentrifugation (AUC) to directly explore the oligomerization state of zymogen as well as active FXIIIA in solution. We first confirmed that the zymogen was a FXIIIA2 dimer. When we activated FXIIIA non-proteolytically (by high mM Ca2+), the protein dissociated to monomers. More importantly, FXIIIA incubation with its physiological partner, the protease thrombin, led to a monomeric state as well. AUC studies of partially cleaved FXIIIA further suggested that thrombin cleavage of a single activation peptide in a zymogen dimer is sufficient to weaken inter-subunit interactions, initiating the transition to monomer. The enzymatic activity of the thrombin-cleaved species was higher than non-proteolytically activated enzyme, suggesting that displacement of the activation peptide renders the FXIIIA more accessible to substrates. Thus, results provide evidence that FXIII undergoes a change in oligomerization state as part of the activation process, and emphasize the role of the activation peptide in preventing FXIIIA catalytic activity.

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

“…Bulk solvent/water permeates interfaces between all subunits, bringing a semblance of symmetry to the disruption/disassembly process in the physiological environment. We can, however, conclude that the process of dissociation is strong enough to separate FXIII-A monomers from each other ( Supplemental video 2 ; Supplemental Figure 12 ), providing further support that the activated FXIII-A molecule is a monomer(31). Given the binding affinity of the FXIII subunits and their conformational motions, FXIII mutations that affect interface residues or conformational flexibility are likely to undermine complex assembly, resulting in either loosely or too tightly bound complex.…”

“…This can be explained by overall negative charge carried by FXIII-A 2 dimer surface, which relies on positive electrostatic patches on the FXIII-B subunit to adhere to the mica surface in a complexed state ( Figure 1d ). The differences in height might be attributed to adsorption effects on the structure of the protein(31, 32). Wrapping of FXIII-B subunits around FXIII-A 2 dimer occurred from one side, giving the molecule a bi-partite appearance, suggesting partial asymmetry in the complex.…”

“…(iii) The FXIII-AB heterodimer separates into individual, free subunits. During this event, unfavorable conformational entropies are counteracted by favorable enthalpic changes, which explain the final disruption of FXIII-AB heterodimer into calcium saturated, activated and open FXIII-A* monomer(31)). At the conditions used (T=30°C), all three events were spontaneous (ΔG<0).…”

The Plasma Factor XIII Heterotetrameric Complex Structure: Unexpected Unequal Pairing within a Symmetric Complex

“…This can be explained by an overall negative charge carried by FXIII-A 2 dimer surface, which relies on positive electrostatic patches on the FXIII-B subunit to adhere to the mica surface in a complexed state ( Figure 1). The differences in height might be attributed to adsorption effects on the structure of the protein [31,32]. Wrapping of FXIII-B subunits around FXIII-A 2 dimer occurred from one side, giving the molecule a bi-partite appearance, suggesting partial asymmetry in the complex.…”

“…(iii) The FXIII-AB heterodimer separates into individual, free subunits. During this event, unfavorable conformational entropies are counteracted by favorable enthalpic changes, which explain the final disruption of FXIII-AB heterodimer into calcium saturated, activated and open FXIII-A * monomer [31]). At the conditions used (T = 30 • C), all three events were spontaneous (∆G < 0).…”