Noncovalent interactions of the Apple 4 domain that mediate coagulation factor XI homodimerization.
The Apple 4 (A4) domain of human plasma factor XI (FXI) was used to investigate the process of FXI noncovalent dimer formation. Recombinant 6-histidine-tagged A4 domain proteins were prepared utilizing a bacterial expression system. Purification was accomplished under denaturing conditions, followed by a refolding protocol to facilitate correct disulfide bond formation. Analysis of the A4 domain (C321S mutant) by size exclusion chromatography indicated the presence of a slowly equilibrating reversible monomer-dimer equilibrium. The elution profiles reveal highly symmetrical peaks for both dimeric and monomeric species with elution times that were highly reproducible for varying amounts of both the dimeric and monomeric species. The monomer-dimer equilibrium was found to be dependent upon changes in both pH and salt concentration. Under conditions approximating physiologic salt concentration and pH (20 mm HEPES, 100 mm NaCl, and 1 mm EDTA, pH 7.4), it was determined that the monomer-dimer equilibrium was characterized by a dissociation constant (K(D)) value of 229 +/- 26 nm with a calculated Delta G value of 9.1 kcal/mol. This report identifies electrostatic contributions and the presence of a hydrophobic component that mediate interactions at the A4 domain interface. The rate of dissociation for the recombinant A4 domain C321S mutant was examined by monitoring the increase in 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt fluorescence under dissociating conditions, giving a value for a dissociation rate constant (k(off)) of 4.3 x 10(-3) s(-1).
Dorfman
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Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Walsh
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PN
D000817:Anilino Naphthalenesulfonates; D019281:Dimerization; D005172:Factor XI / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D012965:Sodium Chloride / Q000494:pharmacology; D055672:Static Electricity
D000817:Anilino Naphthalenesulfonates; D011994:Recombinant Proteins; D012965:Sodium Chloride; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D005172:Factor XI