Crystal structure of human ornithine decarboxylase at 2.1 å resolution: structural insights to antizyme binding

The Epstein-Barr virus thwarts immune surveillance through a Gly-Ala repeat (GAr) within the viral EpsteinBarr virus-encoded nuclear antigen 1 protein. The GAr inhibits proteasome processing, an early step in antigen peptide presentation, but the mechanism of proteasome inhibition has been unclear. By embedding a GAr within ornithine decarboxylase, a natural proteasome substrate that does not require ubiquitin conjugation, we now demonstrate inhibition in a purified system, excluding involvement of ubiquitin conjugation or of proteins extraneous to substrate and proteasome. We show further that the GAr acts as a stop-transfer signal in proteasome substrate processing, resulting in vivo in partial proteolysis that halts just short of the GAr. Similarly, introducing a GAr into green fluorescent protein destabilized by the ornithine decarboxylase degradation domain also stops the progress of proteolysis, leading to the accumulation of partial degradation products. We postulate that the ATP motor of the proteasome slips when it encounters the GAr, impeding further insertion and, in this way, halting degradation.

Section: Gar Inhibits Proteolysis By Purified Components-supporting

confidence: 73%

Repeat Sequence of Epstein-Barr Virus-encoded Nuclear Antigen 1 Protein Interrupts Proteasome Substrate Processing

Zhang

Coffino

2004

“…We inserted this module after the same three positions described above, 32, 297, or 424, to form ODC 32 ::DHFR, ODC 297 ::DHFR, and ODC 424 ::DHFR; these contain inserts positioned, respectively, near the N terminus, the middle, or the C terminus of the protein. All three regions chosen for insertion are disordered in the crystal structure (26), are poorly conserved, and should tolerate bulky inserts that do not obstruct formation of the ODC homodimer, the active form of the enzyme.…”

Section: Resultsmentioning

confidence: 99%

Proteasomes Begin Ornithine Decarboxylase Digestion at the C Terminus

Zhang¹,

MacDonald²,

Hoyt

et al. 2004

Proteasomes denature folded protein substrates and thread them through a narrow pore that leads to the sequestered sites of proteolysis. Whether a protein substrate initiates insertion from its N or C terminus or in a random orientation has not been determined for any natural substrate. We used the labile enzyme ornithine decarboxylase (ODC), which is recognized by the proteasome via a 37-residue C-terminal tag, to answer this question. Three independent approaches were used to assess orientation as follows. 1) The 461-residue ODC protein chain was interrupted at position 305. The Cterminal fragment was degraded by purified proteasomes, but because processivity requires continuity of the polypeptide chain, the N-terminal fragment was spared. 2) A proteasome-inhibitory viral sequence prevented degradation when introduced near the C terminus but not when inserted elsewhere in ODC. 3) A bulky tightly folded protein obstructed in vivo degradation most effectively when positioned near the C terminus. These data demonstrate that the proteasome initiates degradation of this native substrate at the C terminus. The co-localization of entry site and degradation tag to the ODC C terminus suggests that recognition tags determine the site for initiating entry. Flexibility of a polypeptide terminus may promote the initiation of degradation.

“…These include eukaryotic ODCs, plant ADC, and a number of bacterial enzymes that encompass a wide range of substrate specificities (arginine, ornithine, lysine, diaminopimelate, and carboxynorspermidine decarboxylases) (20). The x-ray structures of several eukaryotic ODCs (mouse, T. brucei, and human) (23)(24)(25)(26)(27) and of bacterial diaminopime-late decarboxylase have been solved (28). The N-terminal domain forms a ␤/␣-barrel, and the C-terminal domain is folded into a ␤-barrel structure.…”

mentioning

confidence: 99%

Paramecium bursaria Chlorella Virus-1 Encodes an Unusual Arginine Decarboxylase That Is a Close Homolog of Eukaryotic Ornithine Decarboxylases

Shah¹,

Coleman

Mir³

et al. 2004

Paramecium bursaria chlorella virus (PBCV-1) is a large double-stranded DNA virus that infects chlorellalike green algae. The virus encodes a homolog of eukaryotic ornithine decarboxylase (ODC) that was previously demonstrated to be capable of decarboxylating L-ornithine. However, the active site of this enzyme contains a key amino acid substitution (Glu for Asp) of a residue that interacts with the ␦-amino group of ornithine analogs in the x-ray structures of ODC. To determine whether this active-site change affects substrate specificity, kinetic analysis of the PBCV-1 decarboxylase (PBCV-1 DC) on three basic amino acids was undertaken. The k cat /K m for L-arginine is 550-fold higher than for either L-ornithine or L-lysine, which were decarboxylated with similar efficiency. In addition, ␣-difluoromethylarginine was a more potent inhibitor of the enzyme than ␣-difluoromethylornithine. Mass spectrometric analysis demonstrated that inactivation was consistent with the formation of a covalent adduct at Cys 347 . These data demonstrate that PBCV-1 DC should be reclassified as an arginine decarboxylase. The eukaryotic ODCs, as well as PBCV-1 DC, are only distantly related to the bacterial and plant arginine decarboxylases from their common ␤/␣-fold class; thus, the finding that PBCV-1 DC prefers L-arginine to L-ornithine was unexpected based on evolutionary analysis. Mutational analysis was carried out to determine whether the Asp-toGlu substitution at position 296 (position 332 in Trypanosoma brucei ODC) conferred the change in substrate specificity. This residue was found to be an important determinant of substrate binding for both Larginine and L-ornithine, but it is not sufficient to encode the change in substrate preference.