Allosteric Activation of Ubiquitin-Specific Proteases by β-Propeller Proteins UAF1 and WDR20

Abstract

SUMMARY Ubiquitin-specific proteases (USPs) constitute the largest family of deubiquitinating enzymes, whose catalytic competency is often modulated by their binding partners through unknown mechanisms. Here we report a series of crystallographic and biochemical analyses of an evolutionarily conserved deubiquitinase, USP12, which is activated by two β-propeller proteins, UAF1 and WDR20. Our structures reveal that UAF1 and WDR20 interact with USP12 at two distinct sites far away from its catalytic center. Without increasing substrate affinity of USP12, the two β-propeller proteins potentiate the enzyme through different allosteric mechanisms. UAF1 docks at the distal end of the USP12 Fingers domain and induces a cascade of structural changes that reach to a critical ubiquitin-contacting loop adjacent to the catalytic cleft. By contrast, WDR20 anchors at the base of this loop and remotely modulates the catalytic center of the enzyme. Our results provide a mechanistic example for allosteric activation of USPs by their regulatory partners.

A conserved two-step binding for the UAF1 regulator to the USP12 deubiquitinating enzyme

“…During revision of this manuscript, a structure for the USP12/UAF1/WDR20 complex was published (Li et al, 2016). In this structure WDR20 binds to a number of structural elements at the palm domain of USP12 with an affinity of 7 nM.…”

Specificity for deubiquitination of monoubiquitinated FANCD2 is driven by the N-terminus of USP1

“…USP1 has little DUB activity on its own, but is regulated by and forms a stoichiometric complex with UAF1. UAF1 also binds and activates two other DUBs, USP12 and USP46 19 , and studies that reveal how UAF1 binds and activates USP12 and USP46 suggest that UAF1 will bind to USP1 in an analogous manner 20,21 . UAF1 acts to stabilise its USP partners and increase catalytic activity 22 , and UAF1 knockout in mice is embryonic lethal, while USP1 knockouts result in a FA-like phenotype, reflecting the additional functions of UAF1 9,23 .…”

The WD40-repeat protein WDR-48 promotes the stability of the deubiquitinating enzyme USP-46 by inhibiting its ubiquitination and degradation

“…WDR-48, WDR-20 and USP-46 form a complex in vitro (24,28,29,36), and recently, the crystal structures of mammalian WDR-48/USP-46, WDR-48/USP-12 and WDR-48/WDR-20/USP-12 were recently solved (33)(34)(35). C. elegans USP-46 is the sole homolog of both mammalian USP-46 and USP-12, which share 90% sequence similarity with each other (12).…”

“…The mechanisms underlying the ability of the WDR proteins to activate USP-12 and USP-46 were recently revealed by their crystal structures. The crystal structures of both DUBs were solved in complex with WDR-48 (33)(34)(35), and USP-12 was additionally solved in a ternary complex with WDR-48 and WDR-20 (34). Together, these structural studies revealed that the WDR proteins interact at a site distal to the active site, suggesting an allosteric mechanism underlies the ability of the WDR proteins to stimulate catalytic activity of the DUBs.…”

“…Direct binding of WDR-48 to USP-46 is required to stabilize the DUB. A, Partial protein sequence alignment illustrating the similarity (grey shading) or identity (black shading) of amino acids between H. sapiens and C. elegans WDR48 in the region that interacts with USP46(34). Three point mutations in H. sapiens WDR48 (K214E, W256A, and R272D) that disrupt binding to the DUB are marked by arrows.Thepositions of the corresponding amino acids in C. elegans WDR-48 that were mutated in Myc-WDR-48(R224E, W266A, and K282D), also referred to as Myc-WDR-48(3Xmut), are marked by asterisks.…”