Table 2. Affinity of CC2‐LZ and NOAZ for K48‐, K63‐linked and linear poly‐ubiquitin chainsa
CC2‐LZ KD (chain concentration, μM)NOAZ KD (chain concentration, μM)Gain of affinity with ZF
K63‐Poly‐Ub3–7350.3× 117
K48‐Poly‐Ub3–7459.5× 4.7
K63/K48 specificity1.332
K63‐Tetra‐Ub250.22× 114
Linear tetra‐ubiquitinb2.21.7× 1.3
Linear nona‐ubiquitin2.01.9× 1.05
  • ZF, zinc finger; Ub, ubiquitin.

  • Italics represent gain of specificity or affinity, whereas bold represents actual affinity values.

  • The EC50 (defined as the concentration of competitor necessary to displace 50% of the preformed complex) of the CC2‐LZ and CC2‐LZ‐ZF (NOAZ) peptides for K48‐, K63‐linked or linear poly‐ubiquitin chains were measured by fluorescence competition as described under section Materials and methods. The EC50 were transformed into KD after fitting experimental curves obtained with two different concentrations of CC2‐LZ(F312W) (0.5 and 3 μM) as described in Supplementary data. The curves corresponding to K48‐ or K63‐linked polyubiquitin3–7, K63 tetra‐ubiquitin and linear nona‐ubiquitin are shown in Supplementary Figure S3.

  • b The affinity of CC2‐LZ and NOAZ for GST‐(linear tetra‐Ub) was also measured by fluorescence polarization as described under section Materials and methods. Similar KD values of 3 and 3.9 μM were obtained for CC2‐LZ and NOAZ, respectively, again showing no gain of affinity with the ZF.