Increasing Affinity of Interferon-𝜸 Receptor 1 to Interferon-𝜸 by Computer-Aided Design

Table 2

Calculated and experimental values of the changes of free energy, ΔΔ𝐺, of the interaction between IFN-γ-Rx mutants and IFN-γ-SC relative to the wild-type receptor.

Construct		The best ΔΔ𝐺^c	ΔΔ𝐺 from MD^d	Experimental ΔΔ𝐺^e	esd^f
ID^a	Mutation^b	(kJ/mol)	(kJ/mol)	(kJ/mol)	(kJ/mol)
1	N65R	−5.4	17.3	2.1	—
2	N70G	−5.4	0.3	−0.6	—
3	S95R	−8.3	11.8	2.1	—
4	N96F	−13.0	−0.6	−0.2	—
5	N96W	−9.9	−6.1	−3.9	0.2
6	K115Y	−0.3	−9.6	0.7	—
7	T166M	−5.8	−5.4	2.0	—
8	T166Y	−9.8	0.9	2.5	—
9	H222R	−6.9	−15.8	−0.1	0.2
10	N96W + H222R	−7.1	−7.1	−5.0	0.2
11	N70G + S95R	−7.3	2.7	1.5	—
12	N70G + H222R	−4.6	−7.3	−0.3	—
13	S95R + H222R	−11.4	−10.8	1.5	—
14	N70G + S95R + H222R	−15.8	−5.6	0.5	0.1
15	Y66L	2.1	11.8	0.0	—
16	S71E	9.6	19.6	1.6	—
17	H222D	6.7	5.8	2.0	—

aMutants 1–14 are single, double, and triple mutants designed to increase affinity to IFN-𝛾 compared to WT. Mutants 15–17 were designed to lower the affinity between IFN-𝛾 and IFN-𝛾-Rx but not to destabilize the unbound IFN-𝛾-Rx.
bResidues are numbered as in the UniProt entry P15260.
cFor mutants 1–14, the most negative (most stabilizing) values obtained at the four crystal interfaces by FoldX [33]. For mutants 15–17, the ΔΔ𝐺 listed are for the least positive (least destabilizing) interface.
dAveraged ΔΔ𝐺 values calculated by FoldX on structures taken from snapshots of 10 to 20 ns MD runs by GROMACS [36].
eΔΔG values determined from experimental SPR values of dissociation equilibrium constants 𝐾_𝑑 as ΔΔ𝐺=−RTln{(𝐾_𝑑)_WT/(𝐾_𝑑)_mut}.
fEstimated standard deviations for the experimental values of ΔΔ𝐺 with the number of independent SPR measurements 𝑁>2 (Table 3).