Stability of thioester intermediates in ubiquitin‐like modifications

Abstract

Ubiquitin-like modifications are important mechanisms in cellular regulation, and are carried out through several steps with reaction intermediates being thioester conjugates of ubiquitin-like proteins with E1, E2, and sometimes E3. Despite their importance, a thorough characterization of the intrinsic stability of these thioester intermediates has been lacking. In this study, we investigated the intrinsic stability by using a model compound and the Ubc9~SUMO-1 thioester conjugate. The Ubc9~SUMO-1 thioester intermediate has a half life of approximately 3.6 h (hydrolysis rate k 5 5.33 6 2.8 310 25 s 21 ), and the stability decreased slightly under denaturing conditions (k 5 12.5 6 1.8 3 10 25 s 21 ), indicating a moderate effect of the three-dimensional structural context on the stability of these intermediates. Binding to active and inactive E3, (RanBP2) also has only a moderate effect on the hydrolysis rate (13.8 6 0.8 3 10 25 s 21 for active E3 versus 7.38 6 0.7 3 10 25 s 21 for inactive E3). The intrinsically high stability of these intermediates suggests that unwanted thioester intermediates may be eliminated enzymatically, such as by thioesterases, to regulate their intracellular abundance and trafficking in the control of ubiquitin-like modifications.

Protein standard absolute quantification (PSAQ) method for the measurement of cellular ubiquitin pools

“…Similarly, it should be possible to adapt the methods reported here to measure the abundance of thioester-linked ubiquitin in biological samples. We cannot currently estimate the abundance of thioester linked ubiquitin because the half-life of thioester-linked ubiquitin species at near-physiological pH is short relative to the time required for sample processing and affinity capture 25 . Consistent with this expectation, we observed no dithiothreitol-releasable ubiquitin binding to the BUZ domain in preliminary experiments (data not shown).…”

E2s: structurally economical and functionally replete

“…Fundamental questions such as “How does an E3 catalyze transfer?” are not yet answered. Studies with the SUMO E2 Ubc9 show that the `natural' hydrolysis (in absence of any other nucleophile or E3) is slow, suggesting that thioesters in native E2s are relatively stable, and must be coaxed into discharging their Ub to substrate [87]. Hypotheses invoking allosteric effects on the E2 are pervasive but neither well-documented nor supported.…”

Structure of an E3:E2∼Ub Complex Reveals an Allosteric Mechanism Shared among RING/U-box Ligases

“…This behavior presumably prevents nonproductive hydrolysis of the Ub-thioester bond before assembly into an E3-ligase Ub-transfer complex. Thus, RING/U-box E3 ligases not only coordinately bind E2~Ub conjugate and substrate, but must also activate the E2~Ub thioester to enhance the direct transfer of Ub to a target lysine (Song et al, 2009; Wenzel et al, 2011). …”