Facile Preparation of UFMylation Activity-Based Probes by Chemoselective Installation of Electrophiles at the C-Terminus of Recombinant UFM1

Abstract

Aberrations in protein modification with ubiquitin-fold modifier (UFM1) are associated with a range of diseases, but the biological function and regulation of this post-translational modification, known as UFMylation, remain enigmatic. To provide activity-based probes for UFMylation, we have developed a new method for the installation of electrophilic warheads at the Cterminus of recombinant UFM1. A C-terminal UFM1 acyl hydrazide was readily produced by selective intein cleavage and chemoselectively acylated by a variety of carboxylic acid anhydrides at pH 3, without detriment to the folded protein or reactions at unprotected amino acid side chains. The resulting UFM1 activity-based probes show a range of tunable reactivity and high selectivity for proteins involved in UFMylation processes; structurally related E1s, E2s, and proteases associated with Ub or other Ubls were unreactive. The UFM1 probes were active both in cell lysates and in living cells. A previously inaccessible α-chloroacetyl probe was remarkably selective for covalent modification of the active-site cysteine of de-UFMylase UFSP2 in cellulo.

Semisynthetic LC3 Probes for Autophagy Pathways Reveal a Noncanonical LC3 Interacting Region Motif Crucial for the Enzymatic Activity of Human ATG3

“…In analogy to probes that showed excellent activity in the UFM1-pathway, we selected α-chloroacetyl probe 1 and methyl fumarate probe 2 derived from GABARAP(ΔG116)-NHNH 2 for further studies into autophagy pathways (Figure 1a). 21 To assess the reactivity of the probes, we incubated 1 and 2 (15 μM) with recombinantly expressed ATG3 (15 μM) and its catalytically inactive variant C264A (Figure 1b, Figure S2). Probe 2 reacted with ATG3 efficiently leading to the formation of multiple ATG3−GABARAP bands.…”

Human ATG3 contains a non-canonical LIR motif crucial for its enzymatic activity in autophagy

“…GABARAP(ΔG116)-NHNH 2 was successfully converted to α-chlorocetyl probe 1 and methyl fumarate probe 2 , in analogy to probes that showed excellent activity in the UFM1-pathway (Fig. 1a) 22 .…”

“…Herein, we report the facile preparation of GABARAP and LC3A activity-based probes (ABPs) using a recently established hydrazide acylation protocol 21 . Access to these ABPs led us to identify an unknown non-canonical LIR motif embedded in an unusual β-sheet conformation in human ATG3.…”

Installation of electrophiles onto the C-terminus of recombinant ubiquitin and ubiquitin-like proteins

“…20 In the course of preparing ABPs for UFMylation we faced difficulties with established approaches due to the presence of a sterically-demanding Val as the penultimate C-terminal residue of UFM1. 21 We could, however, readily prepare the corresponding C-terminal acyl hydrazide from a UFM1-Mxe GyrA intein fusion and identied facile conditions for the site-specic attachment of electrophiles to the distal N-atom of the hydrazide, a process that could be conducted on the folded protein without the need for purication other than buffer exchange (Fig. 1b).…”