RING-Between-RING (RBR) E3 ligases mediate ubiquitin transfer through an obligate E3- ubiquitin thioester intermediately prior to substrate ubiquitination. While RBRs share a conserved catalytic module, substrate recruitment mechanisms remain enigmatic and the relevant domains have yet to be identified for any member of the class. Here we characterize the interaction between the auto-inhibited RBR, HHARI (AriH1), and its target protein, 4EHP, using quantitative XL-MS.

To determine domain rearrangements and interdomain contacts due to phospho-mimetic activation and to direct substrate binding, we performed cross-linking mass spectrometry of HHARI in the presence or absence of 4EHP. This approach relies on the physical proximity of residues, providing information on the dynamics and topology of crosslinked regions. Four separate reaction conditions were performed: (1) Wild-type HHARI; (2) Wild-type HHARI + 4EHP; (3) Activated HHARI; (4) Activated HHARI + 4EHP. Each reaction was performed in triplicate. A Q Exactive HF-X (Thermo Fisher Scientific) was used to perform mass spectrometry in positive ion mode. For cross-link identification experiments, data dependent acquisition (DDA) mode was used with a maximum of 20 tandem MS (MS/MS) spectra acquired per MS spectrum (scan range of m/z 400–1,600). Cross-linked peptides were identified using Kojak (http://www.kojak-ms.org/) and viewed using ProXL (https://proxl.yeastrc.org/proxl/p/hhari and https://proxl-ms.org/). Peptide identifications were used to make spectral libraries for quantification in Skyline. For quantification experiments, single-injection data independent acquisition (DIA) runs were performed. MS spectra were acquired at 30,000 resolution with a scan range from 595 to 1205 m/z, automatic gain control of 3e6 and a maximum fill time of 45 ms. Bibliospec DDA spectrum libraries for all Kojak searches were built using Skyline. For this purpose, Kojak results were converted to proxl.xml format according to ProXL’s instructions at https://proxl-web-app.readthedocs.io/en/latest/using/upload_data.html. The resulting proxl.xml was input into Skyline’s Bibliospec library builder. Cross-linked peptides identified by Kojak in DDA data were used to generate targets in Skyline for subsequent quantification of DIA data acquired on the same samples.

Reactions were 50 μL total volume in 20 mM HEPES pH 7.5, 100 mM NaCl, 1 mM DTT plus 0.5 mM DSS (disuccinimidyl suberate, ThermoFisher Scientific, Waltham, MA). HHARI only reactions contained 0.2 μg/μl (3.6 μM) HHARI. HHARI plus 4EHP reactions additionally contained 0.08 μg/μL (3.9 μM) 4EHP. Ska core complex was purified as described by and spiked into all reactions at 0.02 μg/μL (1:10 Ska complex:HHARI weight to weight ratio) as a normalization control. Crosslinking was carried out for 15 mins at room temperature before quenching by addition of 5 μL 1 M NH4HCO3. Four separate reaction conditions were performed: (1) Wild-type HHARI; (2) Wild-type HHARI + 4EHP; (3) Activated HHARI; (4) Activated HHARI + 4EHP. Each reaction was performed in triplicate.