Abstract

Abstract 19

Coagulation factor XI (FXI) is a uniquely dimeric coagulation protein, which in its activated form (FXIa) activates FIX to FIXa. We have previously shown that the dimeric structure of FXI is essential for normal autoactivation and activation by thrombin and FXIIa, but not for the expression of enzymatic activity against FIX (Wu W, et al J. Biol. Chem. 283:18655-18664, 2008). A comparison of three separate structures of FXI/XIa from our laboratory (i.e., the crystal structure of the catalytic domain of FXIa in complex with the kunitz protease inhibitor domain of protease nexin-2; the crystal structure of full-length, dimeric FXI; and the NMR structure of the FXI A4 domain) predicts a major conformational change accompanying the conversion of FXI to FXIa. We now show that when FXI binds to the negatively charged polymer, dextran sulfate and is autoactivated to generate FXIa, changes of intrinsic fluorescence are observed, i.e, a decrease in fluorescence intensity and a red shift of emission wavelength, which also suggests that a conformational change accompanies FXI activation. To investigate the mechanism of FXI zymogen activation and the allosteric transition accompanying the conversion of FXI to FXIa, which exposes binding sites for FXIa ligands, we have carried out fluorescence resonance energy transfer (FRET) studies to characterize the conformational changes accompanying zymogen activation. Using a sensitive free thiol quantitation assay, we confirmed the presence of a single free cysteine residue (Cys11) per subunit of recombinant FXI, which was quantitatively labeled with the thiol reactive fluorescence dye IAEDANS (5-({2-[(iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid). Fluorescence excitation of AEDANS-labeled FXI at 280 nm shows a prominent dansyl emission peak (∼450 nm) in addition to the Trp emission peak (∼325 nm) indicative of efficient FRET from Trp donors to the AEDANS acceptor. Controls using a C11S mutant of FXI showed ∼10-fold lower levels of AEDANS labeling, confirming that Cys11 is the predominant labeling site. Autoactivation of FXI in the presence of dextran sulfate results in a major decrease in donor emission, but has little effect on acceptor emission. This indicates that, for wild-type FXI, FRET is dominated by transfer within the A1 domain originating from Trp55, which is located at a distance of 18 Å from Cys11, far closer than any other tryptophan. The changes in Trp emission, which are similar in the presence and abence of AEDANS, allow us to follow the kinetics of zymogen activation. The S557A active-site mutant of FXI, which cannot undergo autoactivation, showed no fluorescence changes upon addition of dextran sulfate, confirming that the observed decrease in Trp fluorescence is due to formation of active FXIa enzyme. In an effort to observe specific inter-domain FRET, we prepared an AEDANS labeled W55H mutant of FXI, which eliminates the Trp donor in the A1 domain that dominates energy transfer in wild-type FXI. Our data show that autoactivation of the W55H mutant is accompanied by a significant increase in AEDANS emission that can be attributed to the movement of the labeled Cys11 (in A1) relative to Trp228 in the A3 domain of the opposite dimer subunit. In the crystal structure of FXI, the distance for this donor-acceptor pair is 29 Å (compared to a distance of 40 Å for the second closest Trp, Trp407 in the catalytic domain), making it a sensitive and specific FRET probe for monitoring changes in domain arrangement associated with enzyme activation and ligand interactions. A comparison of the FXI crystal structure with our model of FXIa showed that the distance between the active site serines (Ser557) of each catalytic triad is shortened from ∼118 Å in the zymogen to 40–75 Å in the enzyme. Since the distance between the two scissile bonds of each subunit of FXI is also ∼75 Å, we propose that during autoactivation, either the active site of each catalytic domain of FXIa is positioned to cleave the Arg369-Ile370 bond of the opposite subunit (intersubunit transactivation) or a FXIa dimer positions its two active sites adjacent to the two scissile bonds of a separate FXI dimer (intermolecular activation). These studies support a model in which the autoactivating transition from zymogen to enzyme is accompanied by the movement of each catalytic domain of the dimer to facilitate efficient autoactivation of FXI.

Disclosures:

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.