In vivo, the enzyme HsUbc7, Homo sapien Ubiquitin-Conjugating Enzyme 7, is tethered to the endoplasmic reticulum (ER) membrane where it functions to polyubiquitinate misfolded or misassembled proteins. Specific K48-linkages of the polyubiquitin chain will destine the misfolded proteins to the 26S proteasome for degradation. HsUbc7 is able to produce K48-linked Ub-dimers in vitro, an activity which is greatly enhanced in the presence of a RING-domain E3 enzyme. A unique structural feature of HsUbc7 is its 13-residue insert loop, juxtaposed to the enzyme's active site cysteine residue, which has been shown to be vital to the Ub-dimer activity. The work herein sought to characterize the protein HsUbc7 by Nuclear Magnetic Resonance Spectroscopy.
Chemical shift perturbation studies revealed a non-covalent Ub binding site on the surface of HsUbc7, remote from the enzyme's active site. A proposed structural model of the HsUbc7·non-colavent Ub complex suggests that an additional Ub binding site may exist between the non-covalent site and the active site cysteine of HsUbc7. Similar studies also mapped the RING-binding site of HsUbc7. Surprisingly, the addition of RING or Ub to HsUbc7 causes drastic spectral effects, localized to the same contiguous region of HsUbc7, indicating that HsUbc7 undergoes a conformational change upon the binding of Ub or RING. These effects are localized to the region around the active site cysteine (C89), the proposed oxy-anion hole (N81), and the important 13-residue insert loop. NMR relaxation data of HsUbc7 revealed that the 13-residue insert loop and the loop that covers the proposed catalytic residue, N81, (residues 131-136) are highly mobile on the picosecond timescale. These areas are also affected by the binding of Ub or RING, suggesting that binding alters this mobility, slowing the motion to the microsecond-millisecond timescale.
Finally, the extraction of amide bond vector orientations and generalized order parameters for the protein ubiquitin is demonstrated using RDC data collected in many alignment media, in the absence of any prior structural information. The resulting structural and dynamic bond vector descriptions were highly accurate and precise.
|School:||The Johns Hopkins University|
|School Location:||United States -- Maryland|
|Source:||DAI-B 68/04, Dissertation Abstracts International|
|Keywords:||Dipolar coupling, Ubiquitin|
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