Table 2.

Dissociation constants (K_d) for interactions between CBL variants, UbcH5B variants and ZAP-70 peptide

Immobilized Protein	Analyte	Kd (μM)1
GST-CBL47–435
WT	UbcH5B	311 ± 25
M222F	UbcH5B	320 ± 27
M222E	UbcH5B	70 ± 8
Y371F	UbcH5B	250 ± 6
Y368F	UbcH5B	138 ± 4
Y368F pY371	UbcH5B	28 ± 1
Y368F pY371 K389A	UbcH5B	81 ± 2
Y368F pY371 V431A	UbcH5B	43 ± 2
WT	UbcH5B K4A	619 ± 140
M222F	UbcH5B K4A	595 ± 120
M222E	UbcH5B K4A	225 ± 10
WT	UbcH5B + ZAP-70 peptide2	244 ± 7
WT	ZAP-70 peptide	17 ± 0.4
Y368F pY371	ZAP-70 peptide	11 ± 1
GST-CBL354–435
WT	UbcH5B	42 ± 2
pY3713	UbcH5B	20 ± 1
Y368F	UbcH5B	38 ± 2
Y368F pY371	UbcH5B	18 ± 1
Y371F	UbcH5B	50 ± 3

s.e.m. are indicated. Number of replicates, representative sensorgrams and binding curves are shown in Supplementary Fig. 3.

¹Due to the weak E2 binding, we could not saturate E2 concentrations for some of these measurements. The K_d values were approximated by fitting data obtained from 0–90 μM E2 using steady state affinity analyses.

²Saturated ZAP-70 peptide (100 μM) was included in the analyte.

³This sample contained trace amounts of GST-CBL_354–435 pY368.