TABLE 2.

Binding constants of the WT and its mutants for mono-Ub and various forms of di-Ub (linear and Lys-48-linked and Lys-63-linked)

ZF	Mono-Ub		Lys-48-di-Ub
	KD	ΔΔG mu-wt	KD	ΔΔG mu-wt
	μm	kJ·mol−1	μm	kJ·mol−1
WT	750 ± 30	0	113 ± 40	0
D394R	1105 ± 50	+0.95	90 ± 20	−0.5
F395V	484 ± 40	−1.05	81 ± 10	−0.8
F395I	1100 ± 80a	+0.9	40 ± 8	−2.5
F395L	650 ± 50	+0.35	64 ± 8	−1.4
K399E	650 ± 50	+0.35	37 ± 16	−2.7
Y402F	535 ± 35	−0.8	92 ± 14	−0.5
Q403A	734 ± 50	−0.05	23 ± 7	−3.9
D406V	740 ± 130	−0.03	90 ± 15	−0.5
D406R	830 ± 70	+0.25	190 ± 17	+ 1.3
D408R	740 ± 60	−0.03	105 ± 20	−0.1
T409A	494 ± 40	−1.0	30 ± 4	−3.2
V414S/M415S	1210 ± 70	+1.1	139 ± 28	+ 0.5
E416R	1380 ± 150	+1.5	130 ± 20	+ 0.3
E419R	590 ± 60	−0.6	35 ± 5	−2.8

ZF	Lys-63-di-Ub		Linear di-Ub
	KD	ΔΔGmu-wt	KD	ΔΔGmu-wt
	μm	kJ.mol−1	μm	kJ.mol−1
WT	38 ± 3	0	39 ± 6	0
D394R	106 ± 20	+2.5	101 ± 25	+2.3
F395V	12 ± 3	−2.8	12 ± 3	−2.9
F395I	17 ± 4	−2	17 ± 4	−2.0
F395L	11 ± 4	−3	11 ± 3	−3.1
K399E	19 ± 4	−1.7	13 ± 3	−2.6
Y402F	30 ± 7	−0.6	340 ± 66a	+5.2
Q403A	13 ± 3	−2.6	20 ± 5	−1.6
D406V	130 ± 6	+3	109 ± 6	+2.5
D406R	91 ± 20	+2	82 ± 20	+1.8
D408R	27 ± 8	−0.8	49 ± 11	+0.5
T409A	30 ± 4	−0.6	45 ± 9	+0.3
V414S/M415S	70 ± 7	+1.5	100 ± 27	+2.3
E416R	44 ± 11	+0.35	56 ± 8	+0.9
E419R	70 ± 18	+1.5	75 ± 16	+1.6

^a Binding curves were fitted to the Hill equation for these mutants. The Hill coefficients for F395I and Y402F were 1.7 and 4.2, respectively.