title: Architecture of the catalytic HPN motif is conserved in all E2 conjugating enzymes.
abstract: E2 conjugating enzymes are the central enzymes in the ubiquitination pathway and are responsible for the transfer of ubiquitin and ubiquitin-like proteins on to target substrates. The secondary structural elements of the catalytic domain of these enzymes is highly conserved, including the sequence conservation of a three-residue HPN (His-Pro-Asn) motif located upstream of the active-site cysteine residue used for ubiquitin conjugation. Despite the vast structural knowledge of E2 enzymes, the catalytic mechanism of these enzymes remains poorly understood, in large part due to variation in the arrangements of the residues in the HPN motif in existing E2 structures. In the present study, we used the E2 enzyme HIP2 to probe the structures of the HPN motif in several other E2 enzymes. A combination of chemical-shift analysis, determination of the histidine protonation states and amide temperature coefficients were used to determine the orientation of the histidine ring and hydrogen-bonding arrangements within the HPN motif. Unlike many three-dimensional structures, we found that a conserved hydrogen bond between the histidine imidazole ring and the asparagine backbone amide proton, a common histidine protonation state, and a common histidine orientation exists for all E2 enzymes examined. These results indicate that the histidine within the HPN motif is orientated to structurally stabilize a tight turn motif in all E2 enzymes and is not orientated to interact with the asparagine side chain as proposed in some mechanisms. These results suggest that a common catalysis mechanism probably exists for all E2 conjugating enzymes to facilitate ubiquitin transfer.
authors.affiliation: Department of Biochemistry, The University of Western Ontario, London, ON, Canada, N6A 5C1.
authors.forename: Benjamin W
authors.initials: BW
authors.lastname: Cook
authors.forename: Gary S
authors.initials: GS
authors.lastname: Shaw
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D017124:Conserved Sequence; D006639:Histidine / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D025801:Ubiquitin / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry* / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D025801:Ubiquitin; D006639:Histidine; C100879:UBE2K protein, human; D044763:Ubiquitin-Conjugating Enzymes