Allosteric network in Ube2T drives specificity for RING E3 catalysed ubiquitin signals

Abstract

10In eukaryotes, DNA damage repair is implemented by a host of proteins that are coordinated 11 by defined molecular signals. One such signal that transpires during the Fanconi Anemia 12 (FA) -interstrand crosslink (ICL) repair pathway is the site-specific monoubiquitination of 13 FANCD2 and FANCI proteins by a large, multi-protein FA core complex. The mechanics for 14 this exquisitely specific monoubiquitin signal has been elusive. Here we show FANCL, the 15 RING E3 module of the FA core complex, allosterically activates its cognate E2 Ube2T for 16 monoubiquitination by a mechanism distinct from the typical RING-based catalysis. FANCL 17 triggers intricate re-wiring of Ube2T's intra-residue network thus activating the E2 for 18 precision targeting. This network is intrinsically regulated by conserved gates and loops 19 which can be engineered to yield Ube2T variants that enhance FANCD2 ubiquitination by 20 ~30-fold without compromising on target specificity. Finally, we also uncover allosteric 21 networks in other ubiquitin E2s that can be leveraged by RING E3 ligases to drive specific 22 ubiquitination. 23 24 25 Keywords: DNA repair / E2 / Enzyme allostery / RING E3 / Ubiquitination 26 109 110 7 Results 111The E2 -E3 pair Ube2T -FANCL ubiquitinates its substrates via an atypical 112 mechanism 113 Previous in vitro studies using recombinant chicken (Alpi et al., 2008; Sato et al., 2012), frog 114 (Hodson et al., 2014) and human (Longerich et al., 2014) proteins report the isolated FANCL 115 enzyme with Ube2T directs FANCD2 monoubiquitination at its physiological target site. 116However, the underlying mechanism for the site-specific and strict mono-modification is 117 unclear. In order to understand how the Ube2T and FANCL enzyme pair catalyse this 118 specific signal we reconstituted a minimal E2 -E3 module with human proteins. Based on 119 our earlier work we designed and purified a FANCL URD-RING fragment (FANCL UR , 120 residues 109-375) that is stable, monomeric ( Supplementary Fig 1A) and comprises both the 121 substrate (UBC-RWD domain) and the E2 (RING domain) binding regions (Hodson et al., 122 2011; Hodson et al., 2014). We then tested the activity of the FANCL UR fragment in in vitro 123 FANCD2 ubiquitination assays using fluorescently labelled ubiquitin (Ub IR800 ). Previous 124 studies have shown the additional requirements of FANCI and DNA in complex with 125 FANCD2 for efficient monoubiquitination of this substrate. Consistent with this we observe 126Ube2T -FANCL UR mediated FANCD2 modification when present as a FANCD2-FANCI-127 dsDNA complex ( Fig 1A). Further, an arginine mutant of the physiological FANCD2 target 128 site (FANCD2 K561R) prevents ubiquitination thus confirming the minimal E2 -E3 module 129 is both active and site-specific. We wondered if the FANCL UR fragment could also 130 specifically ubiquitinate FANCI present in the FANCD2-FANCI-dsDNA complex. To test 131 this we titrated increasing amounts of the E2 -E3 module in reactions containing single 132 target site complexes ...

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

“…The homogeneous preparation of isolated hs FANCD2-Ub and hs FANCI-Ub is very challenging, as previous studies required a non-mammalian FANCD2–FANCI substrate complex, in addition to needing DNA and a six-protein E3 ligase complex (FANCC, FANCE, FANCF, FANCB, FANCL, and FAAP100), to stimulate the modification ( 32 ). To enhance the yield and purity of hs FANCD2-Ub and hs FANCI-Ub, we developed a method that requires only a FANCL fragment (FANCL ∆ELF ) and a hyperactive mutant form of the E2, Ube2Tv4 ( 33 ), to stimulate the reaction (see the Materials and Methods section). Using these enzymes ( Fig 2A ), we observed robust monoubiquitination of the isolated human FANCD2 and FANCI substrates within 60 min when monitored with either fluorescent ubiquitin (Ub 800 ) ( Fig S2A ) or GST-Ub ( Fig 2B ).…”

“…USP1 amino acids 1–60 (N-terminal) were amplified with BamH1 at either end for ligation into a pGex6bp1 vector containing the rat USP2 catalytic domain (GST-3C-USP1 1–60 -rUSP2 (271–618)). A mutant Ube2T with E54R, P93G, P94G, 1–152 (Ube2Tv4) is described in Chaugule et al, ( 33 ). The FANCL ΔELF (109–375) gene was cloned into a pET-SUMO (Invitrogen) vector using restriction-free cloning.…”

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

“…Full length USP1 FL with G670A/G671A (mutated autocleavage site 7 amplified with BamH1 at either end for ligation into a pGex6bp1 vector containing rat USP2 catalytic domain (GST-3C-USP1 1-60 -rUSP2 (271-618)). A mutant Ube2T with E54R, P93G, P94G, 1-152 (Ube2T V3 ) 33 . FANCL ΔELF (109-375) was cloned into a pET-SUMO (Invitrogen) vector using restriction free cloning.…”