Journal: Nature

Article Title: Ubiquitin ligation to F-box protein targets by SCF–RBR E3–E3 super-assembly

doi: 10.1038/s41586-021-03197-9

Figure Lengend Snippet: a , Strategy to generate an ABP to visualize TS1 (TS1 ABP). The goal was to generate an ABP with a warhead between the catalytic cysteine of UBE2L3 and the C terminus of ubiquitin that would react with ARIH1 only when assembled with a neddylated CRL. An intein-based semisynthesis route was used to couple Ub(1–75)–MESNa and (E)-3-[2-(bromomethyl)-1,3-dioxolan-2-yl]prop-2-en-1-amine (BmDPA) to yield a cyclic ketal-protected ubiquitin species. Acidic deprotection of the cyclic ketal yields a reactive ubiquitin species , which when conjugated to a single-cysteine-containing version of UBE2L3 produces an ABP with a Michael acceptor between the C terminus of ubiquitin and the active site of UBE2L3. b , Quality controls comparing predicted masses for Ub–MESNa, ABP precursor and TS1 ABP entities with measurements obtained by electrospray ionization–time-of-flight mass spectrometry. c , SDS–PAGE gel confirming TS1 ABP reaction depends on the catalytic cysteine of ARIH1 (C>S refers to serine replacement). Gel image is representative of independent technical replicates ( n = 2). d , SDS–PAGE gel demonstrating TS1 ABP reaction with ARIH1 depends on neddylated CUL1–RBX1 and ARIH1 residues required for ubiquitylating client substrates bound to an F-box protein . Gel image is representative of independent technical replicates ( n = 2). e , Because structural biology is an empirical endeavour, various TS2 ABP approaches were tested to identify a strategy yielding high-quality electron microscopy data visualizing TS2. The concept was to place a warhead between a substrate and the C terminus of ubiquitin, to generate an ABP that would react only with ARIH1 super-assembled with the SCF containing the cognate F-box protein of the substrate. The fully synthetic TS2 ABP alternative (left) displayed reactivity and specificity matching the native reaction when assembled with cyclin E or p27 phosphopeptide substrate mimics, as shown by SDS–PAGE gel (right). f , In parallel, we tested a semisynthetic strategy, which led to high-resolution cryo-EM structures, and thus complexes generated with this strategy are referred to as TS2 throughout the article. Ub–MESNa and a substrate phosphopeptide with an N-terminal cysteine placed to mimic the acceptor site were fused via native chemical ligation and the free cysteine was converted to dehydroalanine. g , SDS–PAGE showing TS2 p27 ABP reaction with ARIH1 requires all elements needed for native TS2, or use of a mutant version of ARIH1 (ARIH1(F430A/E431A/E503A)) bypassing the need for NEDD8 for this reaction. Gel image is representative of independent technical replicates ( n = 2). h , SDS–PAGE testing specificity of TS2 ABPs for cognate F-box proteins. Phosphorylated cyclin E and p27 are substrates of SCF FBXW7 and SCF SKP2 , respectively. All experiments with SCF SKP2 also contained the essential protein partner CKSHS1 unless otherwise indicated. Gel image is representative of independent technical replicates ( n = 2).

Article Snippet: The basis for the formation of the TS1 ABP–UBE2L3~Ub was coupling His–Ub(1–75)–MESNa to (E)-3-[2-(bromomethyl)-1,3-dioxolan-2-yl]prop-2-en-1-amine (BmDPA) (>95% purity, ChiroBlock) to yield a reactive species .

Techniques: Mass Spectrometry, SDS Page, Electron Microscopy, Cryo-EM Sample Prep, Generated, Ligation, Mutagenesis