Ubiquitination is the covalent attachment of the 76-residue protein ubiquitin to another protein. Ubiquitin forms polyubiquitin, which are linkages formed by isopeptide bonds between a lysine ϵ-amine of one ubiquitin to the C-terminus of another ubiquitin. The particular biological role of polyubiquitination is dictated by the ubiquitin lysine that is involved in the isopeptide linkage. The K48-linkage dictates the proteasomal degradation of the protein to which it is attached. Regulation of protein degradation is important for a variety of biological processes, notably controlling degradation of cyclins that dictate the growth and replication of cells. Ubiquitination has been implicated in many illnesses, including several forms of cancer, Alzheimer's disease, and Parkinson's disease.
Ubiquitin and ubiquitin chains form through a hierarchical pathway. Ubiquitin is first activated by a ubiquitin activating enzyme (E1), which transfers the activated ubiquitin to a ubiquitin conjugating enzyme (E2). Ubiquitin is attached to E2 by a thioester bond between the ubiquitin C-terminus and an E2 active site cysteine. A third group of proteins, ubiquitin ligases (E3s), mediate the transfer of ubiquitin from E2 to a target protein.
One E2 that specifically catalyzes the formation of K48-linked polyubiquitin is Ube2g2. In order for the E2 and E3 to form an isopeptide linkage, they must bring together two ubiquitins and link them in a specific fashion. If the E2/E3 complex is to extend a chain, the complex must recognize the linkages already present in this chain in order to ensure the fidelity of chain elongation. We studied the binding specificity of Ube2g2 to monoubiquitin and both K48-linked and K63-linked ubiquitin dimers by Nuclear Magnetic Resonance Spectroscopic (NMR) techniques of Chemical Shift Perturbation (CSP) and Paramagnetic Relaxation Enhancement (PRE). We found that Ube2g2 bound the distal subunits of both ubiquitin dimers with a weaker affinity to the proximal ubiquitin subunits. PRE experiments also suggested that ubiquitin and ubiquitin dimers bind Ube2g2 with different conformations.
One characteristic that distinguishes Ube2g2 from other E2s is the inclusion of a thirteen-residue sequence insertion near its active site cysteine, known to be a loop from structural studies. Through collection of relaxation parameters and subsequent analysis, it was established that this insertion loop and another loop spanning residues 130–135 are mobile. The active site cysteine sits between these two loops, suggesting that the loops play a role in the catalytic mechanism of ubiquitin transfer. Additional relaxation studies suggest that these residues remain mobile when ubiquitin binds noncovalently, indicating that loop mobility is not affected by ubiquitin binding at a remote site.
|School:||The Johns Hopkins University|
|School Location:||United States -- Maryland|
|Source:||DAI-B 71/05, Dissertation Abstracts International|
|Subjects:||Molecular biology, Biochemistry, Biophysics|
|Keywords:||Chemical shift perturbation, Ubiquitin conjugating enzyme, Ubiquitination|
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