The role of dynamics in E3 ligase function

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The project will explore the motions of a large cellular machine that labels proteins for destruction. The label is a small protein called ubiquitin (Ub), which is attached to the condemned protein to target it to the proteasome where it is degraded. The cellular machines (E3 ligases) that are responsible for tagging proteins with Ub are very large, nearly 300 kilodaltons in size, and they contain eight to ten protein subunits that all work together to put the Ub tag on the condemned protein. These machines function in all higher life forms, so it is essential to understand how they work. Structural and dynamic experiments will explore the mechanism of how these machines work in vitro using an assembled, purified, active machine. Two graduate students will work full-time on the project, and they will learn both protein biochemistry and biophysical experimental methods. High school students will work in the lab during the summer months. The high school students, some of whom are from disadvantaged backgrounds, will gain knowledge of cutting edge mass spectrometry methods used to understand protein dynamics. The project focuses on ASB-CUL5-RING E3s (ASB-CRL), a large class of E3 ligases. They consist of one of 18 Ankyrin and SOCS box proteins (ASBs) as substrate receptors, elongins B and C (ELOB/C), cullin 5 (CUL5), and the E2-binding protein (RBX2). Each ASB binds 10-15 substrates. No structures are available for ASB-CRLs. The project aims to understand the structure, dynamics, and function of the ASB9-CRL. Thus far, full-length ASB9-CRL has been purified following strategic co-expression in E. coli. The purified complex is active in vitro and ubiquitylation of two substrates, creatine kinase (CK) and histone octamers has been demonstrated. Structural models of CRLs predict a distance of over 60 angstroms between Ub and the substrate, and it remains unknown how Ub is transferred across such a long distance. Additionally, neddylation has been shown to regulate the ubiquitylation activity of CRLs. In the first part of the project, ubiquitylation assays will be used to quantitatively compare the rate of Ub-transfer to CK and to histone octamers by the neddylated and un-neddylated ligase. The Ub attachment sites and extent of poly-ubiquitylation will be measured. In the second part of the project, hydrogen/deuterium exchange mass spectrometry (HDXMS) will be used to probe the internal and global conformational dynamics that drive the long distance conformational changes required for Ub transfer by the ASB9-CRL. In the third part of the project, cryo-electron microscopy (cryoEM) will be used to obtain high-resolution structures of the full ASB-CRL complex and important sub-complexes. The populations and rates of interconversion of the conformational states will be investigated by single molecule FRET (smFRET). Together, the results will reveal the internal dynamics and global dynamics of the ASB9-CRL that are required to achieve processive ubiquitylation of protein substrates.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.