title: Determination of the pKaof the N-terminal amino group of ubiquitin by NMR.
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 pKaof 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 pKaof 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 pKafor 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 pKavalues 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.
authors.affiliation: MRC Biomedical NMR Centre, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.
authors.forename: Alain
authors.initials: A
authors.lastname: Oregioni
authors.affiliation: Molecular Structure of Cell Signalling Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Stieglitz
authors.affiliation: MRC Biomedical NMR Centre, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.
authors.forename: Geoffrey
authors.initials: G
authors.lastname: Kelly
authors.affiliation: Molecular Structure of Cell Signalling Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.
authors.forename: Katrin
authors.initials: K
authors.lastname: Rittinger
authors.affiliation: MRC Biomedical NMR Centre, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.
authors.forename: Tom
authors.initials: T
authors.lastname: Frenkiel
mesh_terms: D020134:Catalytic Domain; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D025801:Ubiquitin