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Assay Method Information

Assay Name:  Binding Assay
Description:  The binding of SPI-01 to the enzyme-substrate complex was investigated. CSP analysis was carried out on the 40 kDa complex of 15N-labeled full length precursor SUMO-1-GGHSTV (SUMO-1-FL) with unlabeled SENP1-C603S. An equimolar amount of SPI-01 was added to the 1:1 enzyme-substrate complex. The only observed CSP on the 15N-labeled precursor SUMO-1-FL was on the C-terminal residues S99 and V101 (FIGS. 7 and 8) (Song et al., PNAS 101:14373-8 (2004)). This result indicates that SPI-01 binds the enzyme-substrate complex at the interface between SENP and the C-terminal tails of precursor SUMO-FL. X-ray crystal structures showed that the C-terminal tail of precursor SUMO sits in and projects out of the catalytic tunnel of SENPs (Shen et al., Nat. Struct. Mol. Biol. 13:1069-77 (2006)). In the case of SENP1, the region that interacts with the projected C-terminus is predominantly acidic and favors the C-terminus of SUMO-1, which is polar and positively charged, over that of SUMO-2, whose C-terminus is mainly hydrophobic (Shen et al., Nat. Struct. Mol. Biol. 13:1069-77 (2006); and Shen et al., The Biochemical Journal 397:279-88 (2006)).
Affinity data for this assay
 

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Last update November 1, 2007
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