Table II. Binding of wild-type and mutant Tsg101 UEV domains to HIV-1 p6, Ub and a p6–Ub fusion construct.

	Relative affinitya
	p6	p6–Ub	Ubb
Wild type	27 ± 5 µMc	2.1 ± 0.7 µMc	635 ± 82 µMc
	16 ± 2 µMd	1.9 ± 0.3 µMd	354 ± 24 µMd
	2.8 ± 0.8 µMe
	4.3 ± 1.6 µMf
p6 binding
Y63A	14	32	0.9
V89A	0.8	1.2	1.0
M95A	52	290	1.6
V141A	2.5	3.5	1.4
Ub binding
V43A	1.0	3.7	3.1
F44A	1.1	1.4	1.4
N45A	1.3	7.1	8.0
D46A	0.8	4.4	5.1
W75A	1.1	1.3	1.6
F88A	1.6	4.2	3.7

^aAffinities of wild-type Tsg101 UEV domain for p6, p6–Ub and Ub are reported as dissociation constants (K_d), averaged from 16, 8 and 3 independent measurements, respectively. Affinities of mutant constructs are reported as fold decreases relative to wild-type binding.

^bExtrapolated to 50% binding.

^cMeasured at 20 mM sodium phosphate pH 7.2, 150 mM NaCl. All mutant binding affinities were measured under these conditions.

^dMeasured at 20 mM sodium phosphate pH 7.2, 50 mM NaCl.

^eMeasured at 20 mM sodium phosphate pH 7.2, 0 mM NaCl.

^fMeasured at 20 mM sodium phosphate pH 6, 50 mM NaCl, the conditions used for NMR chemical shift perturbation.