Dimer Dissociation and Unfolding Mechanism of Coagulation Factor XI Apple 4 Domain: Spectroscopic and Mutational Analysis

Department of Biochemistry and The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140, USA.
Journal of Molecular Biology (Impact Factor: 3.96). 04/2007; 367(2):558-73. DOI: 10.1016/j.jmb.2006.12.066
Source: PubMed

ABSTRACT The blood coagulation protein factor XI (FXI) consists of a pair of disulfide-linked chains each containing four apple domains and a catalytic domain. The apple 4 domain (A4; F272-E362) mediates non-covalent homodimer formation even when the cysteine involved in an intersubunit disulfide is mutated to serine (C321S). To understand the role of non-covalent interactions stabilizing the FXI dimer, equilibrium unfolding of wild-type A4 and its C321S variant was monitored by circular dichroism, intrinsic tyrosine fluorescence and dynamic light scattering measurements as a function of guanidine hydrochloride concentration. Global analysis of the unimolecular unfolding transition of wild-type A4 revealed a partially unfolded equilibrium intermediate at low to moderate denaturant concentrations. The optically detected equilibrium of C321S A4 also fits best to a three-state model in which the native dimer unfolds via a monomeric intermediate state. Dimer dissociation is characterized by a dissociation constant, K(d), of approximately 90 nM (in terms of monomer), which is in agreement with the dissociation constant measured independently using fluorescence anisotropy. The results imply that FXI folding occurs via a monomeric equilibrium intermediate. This observation sheds light on the effect of certain naturally occurring mutations, such as F283L, which lead to intracellular accumulation of non-native forms of FXI. To investigate the structural and energetic consequences of the F283L mutation, which perturbs a cluster of aromatic side-chains within the core of the A4 monomer, it was introduced into the dissociable dimer, C321S A4. NMR chemical shift analysis confirmed that the mutant can assume a native-like dimeric structure. However, equilibrium unfolding measurements show that the mutation causes a fourfold increase in the K(d) value for dissociation of the native dimer and a 1 kcal/mol stabilization of the monomer, resulting in a highly populated intermediate. Since the F283 side-chain does not directly participate in the dimer interface, we propose that the F283L mutation leads to increased dimer dissociation by stabilizing a monomeric state with altered side-chain packing that is unfavorable for homodimer formation.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The H2A-H2B histone heterodimer folds via monomeric and dimeric kinetic intermediates. Within ∼5 ms, the H2A and H2B polypeptides associate in a nearly diffusion limited reaction to form a dimeric ensemble, denoted I₂ and I₂*, the latter being a subpopulation characterized by a higher content of nonnative structure (NNS). The I₂ ensemble folds to the native heterodimer, N₂, through an observable, first-order kinetic phase. To determine the regions of structure in the I₂ ensemble, we characterized 26 Ala mutants of buried hydrophobic residues, spanning the three helices of the canonical histone folds of H2A and H2B and the H2B C-terminal helix. All but one targeted residue contributed significantly to the stability of I₂, the transition state and N₂; however, only residues in the hydrophobic core of the dimer interface perturbed the I₂* population. Destabilization of I₂* correlated with slower folding rates, implying that NNS is not a kinetic trap but rather accelerates folding. The pattern of Φ values indicated that residues forming intramolecular interactions in the peripheral helices contributed similar stability to I₂ and N₂, but residues involved in intermolecular interactions in the hydrophobic core are only partially folded in I₂. These findings suggest a dimerize-then-rearrange model. Residues throughout the histone fold contribute to the stability of I₂, but after the rapid dimerization reaction, the hydrophobic core of the dimer interface has few fully native interactions. In the transition state leading to N₂, more native-like interactions are developed and nonnative interactions are rearranged.
    Journal of Molecular Biology 11/2011; 415(3):600-14. DOI:10.1016/j.jmb.2011.11.032 · 3.96 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The type three secretion system (T3SS) is a large and complex protein nano-machine that many gram-negative pathogens employ to infect host cells. A key structure of this machine is a proteinaceous pore that inserts into the target membrane and forms a channel for bacterial toxins to flow from bacteria into the host cell. The pore is mainly formed from two large membrane proteins called translocators. Importantly, effective secretion and thus pore formation of the translocators depends on their binding to and being transported by small specialized chaperones after synthesis in the bacterial cytosol. Recent crystal structures have shown these chaperones are formed from modular tetratricopeptide repeats. However, each crystal structure produced different homodimeric structures, suggesting flexibility in their topology that may be of importance to function. Given the crucial role of the translocator chaperones, we investigated the conformational stability of the chaperone LcrH (Yersinia pestis). Mutational analysis coupled with analytical centrifugation and equilibrium denaturations showed that LcrH is a weak and thermodynamically unstable dimer (K(D) ≈ 15 µM, ΔG = 7.4 kcalmol(-1)). The modular TPR structure of the dimer allows it to readily unfold in a non-cooperative manner to a one-third unfolded dimeric intermediate (ΔG = 1.7 kcalmol(-1)), before cooperatively unfolding to a monomeric denatured state (ΔG = 5.7 kcalmol(-1)). Thus under physiological conditions the chaperone is able to populate C-terminally unravelled partially folded states, whilst being held together by its dimeric interface. Such ability suggests a fly-casting mechanism as a route to binding their far larger translocator cargo.
    Journal of Biological Chemistry 12/2012; DOI:10.1074/jbc.M112.395889 · 4.60 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Factor XI (FXI) is the zymogen of an enzyme (FXIa) that contributes to hemostasis by activating factor IX. Although bleeding associated with FXI deficiency is relatively mild, there has been resurgence of interest in FXI because of studies indicating it makes contributions to thrombosis and other processes associated with dysregulated coagulation. FXI is an unusual dimeric protease, with structural features that distinguish it from vitamin K-dependent coagulation proteases. The recent availability of crystal structures for zymogen FXI and the FXIa catalytic domain have enhanced our understanding of structure-function relationships for this molecule. FXI contains 4 "apple domains" that form a disk structure with extensive interfaces at the base of the catalytic domain. The characterization of the apple disk structure, and its relationship to the catalytic domain, have provided new insight into the mechanism of FXI activation, the interaction of FXIa with the substrate factor IX, and the binding of FXI to platelets. Analyses of missense mutations associated with FXI deficiency have provided additional clues to localization of ligand-binding sites on the protein surface. Together, these data will facilitate efforts to understand the physiology and pathology of this unusual protease, and development of therapeutics to treat thrombotic disorders.
    Blood 04/2010; 115(13):2569-77. DOI:10.1182/blood-2009-09-199182 · 9.78 Impact Factor


Available from
Jun 6, 2014