Characterization of the binding interface between ubiquitin and class I human ubiquitin-conjugating enzyme 2b by multidimensional heteronuclear NMR spectroscopy in solution.
Ubiquitin-conjugating enzymes (Ubc) are involved in ubiquitination of proteins in the protein degradation pathway of eukaryotic cells. Ubc transfers the ubiquitin (Ub) molecules to target proteins by forming a thioester bond between their active site cysteine residue and the C-terminal glycine residue of ubiquitin. Here, we report on the NMR assignment and secondary structure of class I human ubiquitin-conjugating enzyme 2b (HsUbc2b). Chemical shift perturbation studies allowed us to map the contact area and binding interface between ubiquitin and HsUbc2b by1H-15N HSQC NMR spectroscopy. The serine mutant of the active site Cys88 of HsUbc2b was employed to obtain a relatively stable covalent ubiquitin complex of HsUbc2b(C88S). Changes in chemical shifts of amide protons and nitrogen atoms induced by the formation of the covalent complex were measured by preparing two segmentally labeled complexes with either ubiquitin or HsUbc2b(C88S)15N-labeled. In ubiquitin, the interaction is primarily sensed by the C-terminal segment Val70 - Gly76, and residues Lys48 and Gln49. The surface area on ubiquitin, as defined by these residues, overlaps partially with the presumed binding site with ubiquitin-activating enzyme (E1). In HsUbc2b, most of the affected residues cluster in the vicinity of the active site, namely, around the active site Cys88 itself, the second alpha-helix, and the flexible loop which connects helices alpha2 and alpha3 and which is adjacent to the active site. An additional site on HsUbc2b for a weak interaction with ubiquitin could be detected in a titration study where the two proteins were not covalently linked. This site is located on the backside of HsUbc2b opposite to the active site and is part of the beta-sheet. The covalent and non-covalent interaction sites are clearly separated on the HsUbc2b surface, while no such clear-cut segregation of the interaction area was observed on ubiquitin.
Miura
T
T
Pharma Preclinical Research Department, F. Hoffmann-La Roche AG, Basel, CH-4070, Switzerland.
Klaus
W
W
Gsell
B
B
Miyamoto
C
C
Senn
H
H
D000595:Amino Acid Sequence; D006801:Humans; D008025:Ligases / Q000737:chemistry / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes; D014452:Ubiquitins / Q000378:metabolism*
D011994:Recombinant Proteins; D014452:Ubiquitins; C471546:UBE2B protein, human; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases