Modeling the IFNa 2IFNAR2 Complex

Double-mutant cycle analysis measures the coupling energy between two residues. Coupled mutations are most often spatially close, making it feasible to apply this information as distance constraints between the two residues for docking. In some sense, this is similar to the use of NOEs in calculating an NMR structure. Extensive double-mutant cycle mapping between residues on IFNa 2 and on IFNAR2-EC yielded a number of interacting residue pairs (Fig. 4). Docking of IFNa 2 and IFNAR2 using these distance constraints resulted in a structural model of the complex, which accounted well for the single-mutation data (Chill et al. 2003; Roisman et al. 2001). The striated motif observed for the IFNAR2-EC binding surface interacts with a highly complementary array of hydrophobic and hydrophilic patches on the binding surface of IFNa 2. Receptor residues RE50, RK48, RH76, and RE77 of the hydrophilic strip interact with a matching array of alternating charges upon the ligand formed by residues aR33, aD35, aS152, and aR149. The resulting overall pattern of four intermolecular electrostatic interactions of alternating polarity on the surface of both receptor and ligand is particularly striking (Fig. 4), and provides a good explanation for the fast rate of association observed for this complex. At the heart of the binding interface are the two complementary hydrophobic strips, with receptor residues RM46, RP49, RV80, RV82, RW100, and RI103 interacting with ligand residues aM16 and aA19 of the A helix, aL26, and aL30 of the AB loop, and aA145 and aM148 of the E-helix.

As mentioned above, the split of binding energies of mutations on IFNAR2 was significantly different for binding to IFNa 2 versus to IFNp. Superimposing the unbound structure of IFNP onto the structure of IFNa 2 in the model of the complex placed W22 of IFNP at the same location occupied by A19 in IFNa2 (Fig. 4) and suggests a direct interaction between PW22 and R2W100. As mentioned above, the mutation R2W100A had a much larger effect on IFNP binding than on IFNa 2 binding. A clear validation of the similarity of the IFNa 2 and IFNP binding sites on IFNAR2 was obtained by mutating Ala 19 on IFNa 2 to Trp and showing a clear interaction between the A19 W mutation on IFNa 2 and W100 on IFNAR2. (Slutzki et al. 2006). This suggests that differential activation between IFNa 2 and IFNP is apparently not a result of differences in the structure of this complex.

A number of attempts have been made to model the IFN-IFNAR1 interaction also (Cajean-Feroldi et al. 2004; Mogensen et al. 1999). However, because of lack of cohesive structural data on the IFNAR1 receptor, and the partial mapping of the IFN binding site on IFNAR1, these models are still quite speculative and their validation will have to await further experimental studies.

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