Ubiquitin-specific protease 11 structure in complex with an engineered substrate mimetic reveals a molecular feature for deubiquitination selectivity
Sigrun K. Maurer, Matthias P. Mayer, Stephanie J. Ward, Sana Boudjema, Mohamed Reda Halawa, Jiatong Zhang, Simon G. Caulton, Jonas Emsley, Ingrid Dreveny
Abstract
Ubiquitin specific proteases (USPs) are crucial for controlling cellular proteostasis and signaling pathways but how deubiquitination is selective remains poorly understood, in particular between paralogues. Here, we developed a fusion tag method by mining the Protein Data Bank and trapped USP11, a key regulator of DNA double-strand break repair, in complex with a novel engineered substrate mimetic. Together, this enabled structure determination of USP11 as a Michaelis-like complex that revealed key S1 and S1' binding site interactions with a substrate. Combined mutational, enzymatic, and binding experiments identified Met 77 in linear di-ubiquitin as a significant residue that leads to substrate discrimination. We identified an aspartate ‘gatekeeper' residue in the S1' site of USP11 as a contributing feature for discriminating against linear di-ubiquitin. When mutated to a glycine, the corresponding residue in paralogue USP15, USP11 acquired elevated activity towards linear di-ubiquitin in gel shift assays, but not controls. The reverse mutation in USP15 confirmed that this position confers paralogue-specific differences impacting di-ubiquitin cleavage rates. The results advance our understanding of the molecular basis for the higher selectivity of USP11 compared to USP15 and may aid targeted inhibitor development. Moreover, the reported carrier-based crystallization strategy may be applicable to other challenging targets.