Abstract
The post-translational modification of proteins by ubiquitin is central to the regulation of eukaryotic cells. Substrate-bound ubiquitin chains linked by lysine 11 and 48 target proteins to the proteasome for degradation and determine protein abundance in cells, while other ubiquitin chain linkages regulate protein interactions. The specificity of chain-linkage type is usually determined by ubiquitin-conjugating enzymes (E2s). The degradative E2, Ube2K, preferentially catalyses formation of Lys48-linked chains, but like most E2s, the molecular basis for chain formation is not well understood. Here we report the crystal structure of a Ube2K~ubiquitin conjugate and demonstrate that even though it is monomeric, Ube2K can synthesize Lys48-linked ubiquitin chains. Using site-directed mutagenesis and modelling, our studies reveal a molecular understanding of the catalytic complex and identify key features required for synthesis of degradative Lys48-linked chains. The position of the acceptor ubiquitin described here is likely conserved in other E2s that catalyse Lys48-linked ubiquitin chain synthesis.
Highlights
The post-translational modification of proteins by ubiquitin is central to the regulation of eukaryotic cells
Using site-directed mutagenesis and modelling, our studies reveal a molecular understanding of the catalytic complex and identify key features required for synthesis of degradative lysine 48 (Lys48)-linked chains
Two conjugates associate with the ubiquitin moiety of one conjugate packed against the MGF-motif[27] of the ubiquitin associating domain (UBA) domain of the neighbouring molecule (Fig. 1a)
Summary
The post-translational modification of proteins by ubiquitin is central to the regulation of eukaryotic cells. Substrate-bound ubiquitin chains linked by lysine 11 and 48 target proteins to the proteasome for degradation and determine protein abundance in cells, while other ubiquitin chain linkages regulate protein interactions. The ubiquitin conjugating enzyme Ube2K ( known as E2-25 K and Huntington Interacting Protein 2) is a Class II E2 that preferentially synthesizes Lys48-linked chains on monoubiquitylated substrates, or forms Lys48-linked diubiquitin in the absence of an E321. This Ubc[1] homologue[22] is highly induced in the brains of individuals with Alzheimer’s disease, and is upregulated in neuronal cells after exposure to the amyloid-β peptide[23]. The UBA domain is not required for the synthesis of Lys48-linked chains[25], but may act to tether the E2 to ubiquitylated substrates thereby enhancing elongation of Lys[48] chains[26]
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