SUMO E2/E3 Enzyme Co-Recognize Substrates and Confers High Substrates Specificity

Abstract

Although various technologies can determine protein-protein interaction affinity, Kd, current approaches require at least one interacting protein to be purified. Here, we present a development of high-throughput approach to determine protein interaction affinity for un-purified interacting proteins using quantitative FRET assay. We developed this approach first using SUMO E2 conjugating enzyme, Ubc9, and SUMO substrate, RanGap1c. The interaction affinities from two purified proteins and two unpurified proteins in the presence of BSA, bacterial extracts or two mixtures are all in excellent agreement with that obtained from the SPR measurement. We then applied this approach to determine the interaction affinities of SUMO E3 PIAS1 and Ubc9 with a SUMO substrate, influenza virus protein, NS1, and, for the first time, the SUMO E3 ligase-substrate interaction affinity is determined, which enables us to provide a kinetics explanation for the two-enzyme substrate recognition mode. In general, our studies allow high-throughput determination of protein interacting affinity without any purification, and both the approach and scientific discoveries can be applied to proteins that are difficult to be expressed in general. O_LIProtein interaction dissociation constant, Kd, determination without any protein purification; C_LIO_LICo-recognition of substrates by two enzymes in one reaction; C_LIO_LIPotential affinity improvement mechanism of substrate recognition revealed with kinetics parameters in vivo. C_LI