Abstract
Ubiquitination regulates nearly every aspect of cellular life. It is catalysed by a cascade of three enzymes and results in the attachment of the C-terminal carboxylate of ubiquitin to a lysine side chain in the protein substrate. Chain extension occurs via addition of subsequent ubiquitin molecules to either one of the seven lysine residues of ubiquitin, or via its N-terminal α-amino group to build linear ubiquitin chains. The pKa of lysine side chains is around 10.5 and hence E3 ligases require a mechanism to deprotonate the amino group at physiological pH to produce an effective nucleophile. In contrast, the pKa of N-terminal α-amino groups of proteins can vary significantly, with reported values between 6.8 and 9.1, raising the possibility that linear chain synthesis may not require a general base. In this study we use NMR spectroscopy to determine the pKa for the N-terminal α-amino group of methionine1 of ubiquitin for the first time. We show that it is 9.14, one of the highest pKa values ever reported for this amino group, providing a rational for the observed need for a general base in the E3 ligase HOIP, which synthesizes linear ubiquitin chains.
Highlights
The modification of proteins with polyubiquitin chains has profound consequences for their behaviour and can target proteins to the proteasome for degradation, activate signalling cascades or regulate the DNA damage response amongst many other functions
Our structural work on the catalytic core of the RBR ligase HOIP – which is the only E3 ligase capable of synthesizing linear ubiquitin chains8 - provided a molecular explanation for the observed high chain linkage specificity of HOIP, and highlighted a histidine residue in the active site, His[887], that was ideally positioned to carry out the role of a general base[21]
The pKa of 9.14 for the N-terminal amino group of ubiquitin, determined from the 15N chemical shift, is at the upper limit of experimental values for proteins reported in the literature, which span 6.8 to 9.1, and is higher than that determined for model peptides[20,35]
Summary
The modification of proteins with polyubiquitin chains has profound consequences for their behaviour and can target proteins to the proteasome for degradation, activate signalling cascades or regulate the DNA damage response amongst many other functions. It is generally recognised that the best reporter is the ionization site itself, where the frequency change associated with the ionization is likely to be largest, followed by the nucleus or nuclei that are covalently bound to the ionization site and other closely adjacent nuclei In view of these considerations we have adopted an indirect-detection approach to determine the pKa of the α-amino group of Met[1] by monitoring the 15N resonance frequency, detected indirectly via the non-labile alpha hydrogen of the terminal residue. This approach is based on that used by André et al to determine the pKa values of side-chain ionizable groups of lysine and arginine residues[28], and by Lorieau et al for the amino group of the influenza hemagglutinin fusion peptide[29]
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