Role of calcium in the conformational dynamics of factor XIII activation examined by hydrogen–deuterium exchange coupled with MALDI-TOF MS

Abstract

Factor XIII catalyzes formation of γ-glutamyl-ε-lysyl crosslinks within fibrin clots. FXIII A2 can be activated proteolytically with thrombin and low mM Ca2+ or nonproteolytically with high monovalent/divalent cations along with low mM Ca2+. Physiologically, FXIII A2 is poised to respond to transient influxes of Ca2+ in a Na+ containing environment. A successful strategy to monitor FXIII conformational events is hydrogen-deuterium exchange (HDX) coupled with mass spectrometry. FXIII A2 was examined in the presence of different cations (Ca2+, Mg2+, Ba2+, Cu2+, Na+, TMAC+, and EDA2+) ranging from 1–2 mM, physiological Ca2+ concentration, to 50–500mM for nonproteolytic activation. Increases in FXIII solvent exposure could already be observed at 1 mM Ca2+ for the dimer interface, the catalytic site, and glutamine substrate regions. By contrast, solvent protection was observed at the secondary cleavage site. These events occurred even though 1mM Ca2+ is insufficient for FXIII activation. The metals 1mM Mg2+, 1mM Ba2+, and 1mM Cu2+ each led to conformational changes, many in the same FXIII regions as Ca2+. FXIII could also be activated nonproteolytically with 500mM tetramethylammonium chloride (TMAC+) and 500mM ethylenediamine (EDA2+), both with 2mM Ca2+. These different HDX studies help reveal the first FXIII segments that respond to physiological Ca2+ levels.

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

“…Finally, in the presence of control 100 m m Mg 2+ no detectable activity was observed (Fig. , v), consistent with previous conclusions that among divalent cations, Mg 2+ was the least efficient in supporting FXIIIA transglutaminase activity .…”

“…However, with a FXIIIA‐catalyzed reaction where two polypeptide chains are cross‐linked, significant rearrangements in the enzyme would be required to bind substrate and to release the product. Indeed, solution‐based hydrogen‐deuterium exchange and chemical modification experiments indicated conformational alterations with opening of the FXIIIA dimer interface upon activation . Consistent with those conclusions, nonproteolytically activated FXIIIA that had been covalently inhibited was later crystallized with an exposed active site and proposed to be monomeric .…”

Hydrogen Exchange Mass Spectrometry: Are We Out of the Quicksand?

“…One solution that may seem to simplify the problem is to use MALDI instead of LC electrospray MS, therefore eliminating the entire LC step. While MALDI can be used successfully in HX MS (e.g., [51–55]), the disadvantage is that there is more back-exchange than well controlled LC electrospray MS experiments and that all peptides one wishes to study are in a single spectrum. Lack of chromatography limits the protein size that can be analyzed owing to peak capacity problems and ionization efficiencies.…”

Structure of Active Coagulation Factor XIII Triggered by Calcium Binding: Basis for the Design of Next‐Generation Anticoagulants

“…These findings are in agreement with mass spectrometry data obtained for hydrogen/deuterium exchange in solution. [13,14] Structural rearrangements upon activation occur only within the catalytic domain, while the b-sandwich and the two b-barrel domains conserve their overall fold. The electron density of the loop connecting the catalytic and bbarrel 1 domain (residues 502-515) is not visible, possibly because of the pronounced flexibility of this portion.…”