Determination of the Structural Changes Accompanying Binding of Protein S to Factor IX_a

Background: Protein S (PS) is a 75-kDa, vitamin K-dependent anticoagulant that contains a Gla (enclosing γ-carboxyglutamic acids) module, a TSR (Thrombin Sensitive Region) module, four EGF (Epidermal Growth Factor)-like modules, and an SHBG (Sex Hormone Binding Globulin)-like region. Protein S deficiency is associated with risks for venous and arterial thrombosis, ischemic stroke, cerebral thrombophlebitis, myocardial infarction, and vascular calcification. Three disparate functions have been proposed for PS: (a) as a cofactor for active protein C; (b) as a cofactor of tissue factor pathway inhibitor, and (c) as a direct inhibitor of factor IX_a. We have shown that PS binds FIX_a with a K_d~40 nM. Currently, we are ascertaining the region and amino acid residues of FIX_a that bind PS.

Results and Discussion: We demonstrated before that lysine residue K126 and K132 of FIXa were very important in binding PS. However, there were no information regarding the change in the conformation of the complex when these residues were mutated. Here, we determined the structural changes in the complex by circular dichroism (CD), isothermal titration calorimetry (ITC), mass spectrometry, and molecular modeling to define the basis of the FIX_a-PS interaction.

The circular dichroism (CD) titration data of interaction between PS and FIX_a showed the conformational change of PS due to interaction with FIX_a. In addition, to understand the stability of the complex, we determined the α-helicity changes of PS in the presence and absence of FIX_a. Initially, PS exhibited only a small amount of α-helical content (~3.5%) with a peak maxima at 214 nm in CD spectra. On addition of a minute amount of FIX_a (50 nM), the PS CD spectra were blue-shifted by 4 nm and, simultaneously, increased in α-helical content by ~12%. The massive change in PS α-helical content on binding to FIX_a directly revealed the stability of the complex. A similar interaction pattern was reflected in ITC experiments. The binding of PS with FIX_a was essentially entropy-driven; binding was exothermic and favored by negative enthalpy and large positive entropy changes. The large positive entropy contribution to the binding may result from the disruption of water networks surrounding both proteins and a condensation of sodium and calcium ions from the polypeptide chains. Also, the negative Gibbs free energy change in binding revealed the spontaneous association between the two proteins.

To achieve our goal of defining the amino acid interaction pattern of PS with FIX_a, we combined liquid chromatography mass spectrometry (LCMS) and molecular modeling. To identify the residues involved in interaction, we subjected PS and FIX_a, individually and in a complex, to a subtractive acetylation-protection assay. We used LCMS to compare the acetylated lysine residues in the individual proteins with the modified lysines in the complex." This methodology required normalization to lysine residues that were not acetylated. We found that lysine 126 (K126) and lysine 132 (K132) in FIX_a were differentially protected in the complex about 2-fold compared with the free proteins. Our molecular modeling data also supported this mass spectrometric result. Modeling data pinpointed the regions of PS and FIX_a that interact and revealed a major involvement of K132. To confirm the importance of K132, we analyzed by CD spectroscopy a derivative of FIXa in which K132 was changed to A132. We observed a dramatic reduction in interaction of the mutant FIX_a with PS

Conclusion: Based on our preliminary data, we conclude that PS binds strongly with FIX_a, and the complex is stabilized by a shift in α-helical content as shown in Figure 1.

Figure.1 shows the CD spectral change of PS (curve 1, right panel) due to addition of different concentration of FIX_a (curve 2-8, right panel).

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