A C2HC zinc finger is essential for the RING-E2 interaction of the ubiquitin ligase RNF125

The activity of RING ubiquitin ligases (E3s) depends on an interaction between the RING domain and ubiquitin conjugating enzymes (E2), but posttranslational events or additional structural elements, yet largely undefined, are frequently required to enhance or regulate activity. Here, we show for the ubiquitin ligase RNF125 that, in addition to the RING domain, a C2HC Zn finger (ZnF) is crucial for activity, and a short linker sequence (Li2120-128) enhances activity. The contribution of these regions was first shown with truncated proteins, and the essential role of the ZnF was confirmed with mutations at the Zn chelating Cys residues. Using NMR, we established that the C2HC ZnF/Li2120-128 region is crucial for binding of the RING domain to the E2 UbcH5a. The partial X-ray structure of RNF125 revealed the presence of extensive intramolecular interactions between the RING and C2HC ZnF. A mutation at one of the contact residues in the C2HC ZnF, a highly conserved M112, resulted in the loss of ubiquitin ligase activity. Thus, we identified the structural basis for an essential role of the C2HC ZnF and conclude that this domain stabilizes the RING domain, and is therefore required for binding of RNF125 to an E2.


RING
. Amino acid sequence alignment of human RNF125 and RNF114. The alignment was generated with EMBOSS Needle from EMBL-EBI (www.ebi.ac.uk/Tools/psa/ emboss_needle), and coloured using Color Align Properties from the Sequence Manipulation Suite (http://www.bioinformatics.org/sms2/color_align_prop.html, Stothard P (2000) The Sequence Manipulation Suite: JavaScript programs for analyzing and formatting protein and DNA sequences. Biotechniques 28:1102-1104.) This alignment was used to determine RNF125/RNF114 boundaries in the chimeras (indicated by black arrows).
boundaries of RNF125/RNF114 chimeras in Figure 2 C-terminal truncation in RNF125 stop129 Ub WB Figure S2. Mutations in the second and third ZnFs do not affect activity of RNF125.
A. Diagram of the RNF125 mutants with alanine substitutions at cysteines that are predicted to chelate zinc in the second (RNF125 C140A/C143A ) and third (RNF125 C170A/C173A ) Zn finger (indicated by stars). B. In vitro ubiquitination reactions with RNF125 and the mutants described in A were performed and analyzed as described in Materials and Methods in the main text.
Supplementary Figure S3. Backbone assignment of 13 C 15 N-UbcH5a. HSQC spectrum showing the assigned peaks for 13 C 15 N-UbcH5a. 123 of the 132 peaks for nonproline residues (of 147 amino acids in total) could be assigned. Of these, 102 were assigned automatically. The remainder of the peaks were identified by comparison with BMRB deposited backbone assignments for UbcH5a (Saxena et al., 2005) and the 89% identical protein UbcH5b (Dominguez et al., 2004;Farrow et al., 2000). Since the UbcH5a recording by Saxena et al was performed at pH6.0 instead of pH7.0 in our experiments, we also acquired HSQCs of 15 N-UbcH5a at pH6.0, and 6.5 to further aid the assignment. Peaks that were not assigned are M1, A19, I37, M38, S80, D87, S91, S100 and D122. Some of these were not visible at pH 7.0, but could be observed at the lower pHs (S80, D87, S91 and D122). The residues in paired peaks D29 /H32, as well as C21/ D59, were assigned but treated as a combined couple due to uncertainty. The CSPs for these residues were of similar magnitude within each pair and have been included in the analysis represented in Figure 4 since the error associated with mislabelling is negligible. C. An overlay of both HSQCs shows an overlap of peaks, except for those that are absent from the central region of RNF125 start31/stop129 , which represent the N-terminal 30 amino acids that are predicted to be unstructured. D. Schematic diagrams of the constructs RNF125 stop129 and RNF125 start31/stop129 . E. A plot of the differences in chemical shifts between RNF125 stop129 and RNF125 start31/stop129 . Unassigned residues are those indicated by red boxes in Fig. S7B. A. HSQC spectra of 0.2 mM 15 N-RNF125 start31/stop129 were recorded before and after the addition of UbcH5a at molar equivalents ranging from 0.125 to 2.5 as indicated. Combined 1 H and 15 N CSPs for each titration point are plotted. Details are as in Figure 5 of the main text where the data for 1:2 ( 15 N-RNF125 start31/stop129 /UbcH5a) of this experiment are represented. Red bars represent residues that broadened beyond detection in the presence of UbcH5a.
Supplementary Figure S7