Small, N-terminal tags activate Parkin E3 ubiquitin ligase activity by disrupting its autoinhibited conformation

Abstract

Parkin is an E3 ubiquitin ligase, mutations in which cause Autosomal Recessive Parkinson's Disease. Many studies aimed at understanding Parkin function, regulation and dysfunction are performed using N-terminal epitope tags. We report here that the use of small tags such as FLAG, cMyc and HA, influence the physical stability and activity of Parkin in and out of cells, perturbing the autoinhibited native state of Parkin, resulting in an active-for-autoubiquitination species.

Figure 1. Chemical composition of small epitope tags.

(A) The chemical composition of the cMyc tag, (B) the FLAG tag, and (C) the HA tag. The three-letter code amino acid sequence is shown beneath each linear representation.

Figure 2. Epitope-tagged Parkin species are not in a native conformation.

(A) Limited proteolytic digest of Parkin species in the presence of increasing concentrations of subtilisin (w/w). The boxed area indicates the degradation of each species. The graph below shows the quantification of the gels with each species normalised to the total amount of full-length species present in the absence of subtilisin. Each curve is coloured according to the key. (B) Difference scanning fluorimetry of each Parkin species, with each thermal denaturation curve coloured according to the key and melting points (Tm) of each protein indicated.

Figure 3. Epitope tags disrupt Parkin autoinhibition in vitro.

(A) Western blot analysis of the autoubiquitination of wild type and cMyc-, FLAG, and HA-tagged Parkin. A truncation lacking the Ubl domain (ΔUblD) is the positive control. Ubiquitin conjugates are detected using anti-Parkin (left) and anti-His-Ub (right). (B) Western blot analysis of the autoubiquitination of Boston Biochem's His-Parkin, probed with anti Parkin (left) and anti-His-Ub (right). Ubiquitin conjugates are indicated.

Figure 4. Epitope tags influence Parkin ubiquitination in cells.

Western blot analysis of the ubiquitination of wild type and cMyc-, FLAG-, and HA-tagged Parkin in HEK293 cells. His-ubiquitin-conjugates were pulled out using nickel affinity and analysed using anti-Parkin (top panel). Soluble lysates were probed for levels of Parkin (middle panel) and actin levels are used as a loading control (bottom panel).