Eligible Student Poster 49th Lorne Conference on Protein Structure and Function 2024

The mechanism of USP16’s interaction with the H2AK119 ubiquitinated nucleosome  (#306)

Jack JA Alexandrovics 1 , Nicholas NK Kirk 1 , Jeff JJB Babon 1 , David DK Komander 1
  1. Walter & Eliza Hall Institute of Medical Research, Parkville, VIC, Australia

Ubiquitin (Ub) exists in a dynamic equilibrium between its conjugation to substrates and its free mono form in the Ub pool. Proteostasis adapts swiftly to changes in these Ub pool dynamics [1][2], with evidence suggesting sensors exist that can read the Ub pool levels via the unconjugated C-terminus of Ub and regulate proteostasis accordingly [3][4][5][6]. USP16 is a histone H2A K119 deubiquitinase and, through this substrate, modulates the expression of multiple genes [7][8]. USP16 also contains a regulatory Zinc-Finger (Znf) UBP domain that binds the Ub C-terminus. We hypothesise that the interaction of USP16 and the nucleosome is regulated by free Ub via this Znf-UBP domain. This may serve as a mechanism of gene expression regulation directly by the Ub pool. 

A crystal structure revealed the mechanism of binding of the Znf-UBP domain with mono Ub. Fluorescence-based kinetics assays of full-length USP16 variants with Znf-UBP mutations showed significantly reduced activity, suggesting the domain assists in catalytic turnover. Using SPR, USP16 variants catalytically blocked by a Ub probe showed the Znf-UBP domain is capable of binding Ub independently. Therefore, free Ub may regulate this catalytic turnover. Mass Photometry showed tight complex formation between USP16 and the Ubiquitinated nucleosome. Using Cryo-EM we have determined moderate resolution structures of full-length USP16 in complex with the ubiquitinated nucleosome during screening, and further experiments are optimized and ready for imminent high-resolution data collection. 

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