NMR Structural Analysis and Folding Studies of the Factor XI Apple 4 Domain as a Model for Factor XI Homodimerization.

Abstract

Factor XI (FXI) is a 160kD homodimeric component of the intrinsic pathway of the blood coagulation system. The FXI Apple 4 domain (A4) mediates homodimer formation even when the cysteine responsible for the intermolecular disulfide is mutated to serine (C321S). Preliminary NMR structural studies of the isolated wild type (wt) A4 domain showed a flattened, planar structure (Samuel D, Cheng H, Riley PW, Walsh PN, Roder H. Blood 104, 481a, 2004), compared to a more globular structure consisting of two layers of β-sheet and a flanking α-helix that was observed on further analysis of wt and C321S A4. Hydrogen exchange NMR and surface NMR analysis (SEAHSQC), as well as temperature dependent hydrogen exchange NMR showed that in C321S and wt A4, the loop surrounding C321 or S321 is flexible and exposed to the solvent. Thus, the globular structure is the major species in both wt and C321S A4, but the planar structure is present as well. Fluorescence anisotropy studies of C321S A4 yielded a Kd value of ~64 nM (monomeric protein concentration), compared with ~72 nM for a dissociable mutant full-length FXI (G326C) obtained by analytical ultracentrifugation (Sinha D, Marcinkiewicz M, Lear JD, Walsh PN. Biochemistry, in press, 2005), suggesting that only the A4 domain mediates dimer formation in the intact protein. In addition, fluorescence and circular dichroism (CD) studies of the folding equilibrium of C321S A4 indicate that starting from the unfolded monomeric state, partial folding precedes dimerization. This folding mechanism for FXI A4 is consistent with the observed failure of a naturally occurring mutant (FXI F283L) to dimerize intracellularly (Meijers JC, Davie EW, Chung DW. Blood 79, 1435–1440, 1992). To determine the structural consequences of the mutation, F283L was introduced into the noncovalent dimer C321S A4, which increased the Kd of the homodimer interaction to ~5 μM, but did not change the secondary structure (from comparison of the CD spectra), or the high degree of thermostability observed for C321S A4. Since the F283 sidechain has been tentatively assigned to the surface of FXI A4 near the interface, the mutation to Leu may significantly alter the interface, thereby raising the Kd but not changing the overall secondary structure significantly. In summary, there appears to be a mechanism by which a conformational change or productive folding process occurs in the monomer state that activates it for native dimer formation. Also, inserting the F283L (type III) patient mutation does affect the dimer stability, but not the overall secondary structure.