Genetically Directed Production of Recombinant, Isosteric and Nonhydrolysable Ubiquitin Conjugates

Abstract We describe the genetically directed incorporation of aminooxy functionality into recombinant proteins by using a mutant Methanosarcina barkeri pyrrolysyl‐tRNA synthetase/tRNACUA pair. This allows the general production of nonhydrolysable ubiquitin conjugates of recombinant origin by bioorthogonal oxime ligation. This was exemplified by the preparation of nonhydrolysable versions of diubiquitin, polymeric ubiquitin chains and ubiquitylated SUMO. The conjugates exhibited unrivalled isostery with the native isopeptide bond, as inferred from structural and biophysical characterisation. Furthermore, the conjugates functioned as nanomolar inhibitors of deubiquitylating enzymes and were recognised by linkage‐specific antibodies. This technology should provide a versatile platform for the development of powerful tools for studying deubiquitylating enzymes and for elucidating the cellular roles of diverse polyubiquitin linkages.


Introduction
Post-translational modification of proteins with ubiquitin (Ub) regulates variousc ellular processes, and defects within this pathway result in numerous pathologies. [1] Ubiquitylation is orchestrated by as eries of enzymes (E1s, E2s andE 3s) whose action culminates in the covalent attachment of the Ub carboxy terminus to Ne-amino groups of lysine residues by an isopeptideb ond. [2] Ub itself has seven lysines (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48 and Lys63) that can accept another Ub molecule. This resultsi nt he formation of seven different isopeptide-linked polyUb chains, whichh ave been proposed to serve as ac ellularc ode. [3] Despite the presenceo fa ll linkage types in cells, the cellular roles of the various linkages are poorly defined. Furthermore, Ub conjugation is reversible, as the isopeptide bond can be hydrolysed by deubiquitylating enzymes( DUBs),a round 100 of whicha re encoded in the human genome. [4] Studyingt he cellular roles of distinct Ub linkages poses an umber of challenges because of complicationsint he preparation of homogenously modified protein anda ni nability to genetically disrupt distinct linkagetypes in cells. Nonhydrolysable analogues of polyUbc hains of defined linkage type could serve as powerful probesbyi nhibiting linkage-specific processes. Assessment of their effects on biological function could help establish their roles. [5] DUBs are also key cellular regulators and are attractive therapeutic targets. However,t he overwhelmingn umber of proteins that associate with DUBs raises the question:w hich ones are bona fide substrates? [6] Nonhydrolysable analogues of ubiquitylated substrates could be used as affinity probes to address this question, as DUBs would be predicted to confer specificity for the modifiedf orm of the substrate, and the nonhydrolysablen ature would circumventc omplications associatedw ith DUB-mediated cleavage of the native counterpart. Nonhydrolysable Ub conjugates of recombinantp rotein would also facilitates tructure determination of substrate-DUB complexes, the raising substrate-specific antibodies (where native conjugates would be hydrolysed in vivo) andtarget validation of DUB inhibitors.
Current methods for the nonhydrolysable conjugation of Ub to recombinantp rotein use al inkaget hat typically has compromised isostery with the native isopeptideb ond ( Figure S1 in the Supporting Information). [5,7] This is often exacerbated by the steric and electrostatic properties of the amino acid scaffolds bearing the requisite reactiveh andles.D espite ar ecent refinement [8] of the cysteine chemistry first reported by Wilkinson andc o-workers, [7a] this approach still precludes the use of recombinant substrates containing more than one cysteine. Solid-phase peptide synthesis (SPSS) approaches for preparing nonhydrolysable Ub conjugates have also been described, but these require specialist expertise and are not generally applicable. [9] Although conjugates formed with triazole-based linkages have been employed for biological studies, [5, 7d, 10] the behaviour towardsr eceptors or DUBs that recognise the linkagei tself [11] or that specifically" sense" the inter-ubiquitin distance in ap articular linkage [12] is unknown. Furthermore, there is no experimental structure of an onhydrolysable conjugate, and ac omparisono ft he affinity towards DUBs and Ub receptors relative to their native counterparts has not been investigated.
Here we report am ethodf or genetically incorporating an aminooxy functionality into recombinant proteins by the incor-We describe the genetically directed incorporation of aminooxy functionality into recombinant proteins by using am utant Methanosarcina barkeri pyrrolysyl-tRNA synthetase/tRNA CUA pair.T his allows the general production of nonhydrolysable ubiquitinc onjugates of recombinanto rigin by bioorthogonal oxime ligation. This was exemplified by the preparation of nonhydrolysable versions of diubiquitin, polymericu biquitin chains and ubiquitylated SUMO. The conjugates exhibited un-rivalled isostery with the native isopeptide bond, as inferred from structurala nd biophysical characterisation. Furthermore, the conjugates functioned as nanomolar inhibitors of deubiquitylating enzymes and were recognised by linkage-specific antibodies. This technology should provide av ersatile platform for the development of powerful tools for studying deubiquitylating enzymes and for elucidating the cellular roles of diverse polyubiquitin linkages.
poration of the unnatural amino acid aminooxy-l-lysine (1, Scheme 1A)b yu sing an evolved Methanosarcina barkeri (Mb) pyrrolysyl-tRNA synthetase (PylS)/tRNA CUA pair.T hise nables the site-specific, nonhydrolysableu biquitylationo fp roteinsb yb ioorthogonal oxime ligation. [13] We demonstrate the generality of this approach by preparing diubiquitin (diUb) of distinct linkage types and SUMO2 (small ubiquitin-like modifier 2) modified with Ub at ap hysiologically relevant site. Structural, biochemicala nd biophysical characterisationo ft hese conjugates revealed that they accurately reflect the topologyo ftheir native counterparts. They also serve as potent (nanomolar) DUB inhibitors and provide insighti nto how the substrate specificity of au biquitin carboxy terminal hydrolase (UCH) family DUB towards its substrates is achieved. We also describe ah ybrid strategy thati nvolves genetic code expansion and intein chemistry to produce extended nonhydrolysable Ub polymers. This technology should be valuable for the identification of proteins that confer specificity for topologically distinct Ub polymers and ubiquitylateds ubstrates, and also for probingt he cellular roles of Ub linkages.

Results and Discussion
Genetically encoded e-aminooxy-l-lysine for the production of nonhydrolysable ubiquitin conjugates Simple synthetic peptides containing an aminooxy functionality in place of the e-aminof unctionality of al ysine residue can undergo bioorthogonal oxime ligationw ith Ub carrying aCterminal aldehyde group. [14] This furnishes as table nonhydrolysable oxime-linkedm imic that has high isostery with the isopeptideb ond. However,n ot only is this approach restricted to synthetic peptides, it also introduces ap otentially perturbing unnatural amide linkagew ithin the lysine side chain andd isrupts the electronic properties of the Ub Cterminus (Figure S1). [14] Incorporating 1 (Scheme 1A)bygeneticcode expansion based on the Mb PylS/tRNA CUA pair [15] would extend this technology to recombinantp rotein substrates, thereby enabling the production of nonhydrolysable conjugates that have unprecedented isostery with the isopeptide bond (Scheme 1B, Figure S1). However,w ea nticipated that it would be challenging to evolve am utant Mb PylS/tRNA CUA pair that could selectively recognise 1 (that differs from native lysine by conservative replacement of the e-methyleneg roup with an e-oxygen atom) yet exclude structurally similara nd cellularly abundant lysine. Furthermore, af ree aminooxy group in the cell could potentially undergo oxime formation with cellular keto compounds such as pyruvate.
We considered al atent Ne-protected form of 1 that has previously been employed for structurally similar lysine analogues. [16] The Ne-protecting group would also serve as arecognition elementf or an Mb PylS/tRNA CUA pair.T he protecting group could then be removed post-translationally by chemical methods. [17] Thus we synthesised Ne-(tert-butyloxycarbonyl)protected aminooxy-l-lysine (2;S cheme 1A). Deprotectiono f the Boc group by acid treatment would furnish 1.The Boc-protected derivative was initially chosen, as Ne-(tert-butyloxycarbonyl)-l-lysine (4;S cheme 1A)i sahighly efficient substrate of the wild-type PylS/tRNA CUA pair. [18] Derivatives of 4 modified at the neighbouring d-position can be incorporated with an evolved SHKRS/tRNA CUA pair that contains aY 349W mutation in the PylS gene. [19] We therefore tested the abilities of both systemstod irect the incorporation of 1mm 2 into C-terminally His-tagged Ub with aT AG codon at position6. [17] We found that 2 was not incorporated by the wild-type PylS/tRNA CUA pair despite, as expected, efficient incorporation of 4 ( Figure 1A). However,t he SHKRS/tRNA CUA pair incorporated 2 with efficiency comparable to that of the PylS/tRNA CUA pair with 4 ( Figure 1A). These results verify the first route to genetically direct the incorporation of aminooxy functionality into recombinant proteins,a nd demonstrate that in the contexto ft he wild-type PylS/tRNA CUA pair, the Y349W mutationp ermits incorporation of lysine derivatives augmented at the e-position as well as the d-position. [19] We next designed and synthesised ap hotocaged variant of aminooxy-l-lysine, 3 (Scheme 1A). This would allow mild photo-deprotection of the aminooxy group (Scheme 1B), [20] as has been demonstrated in live cells with the analogous lysine derivative. [21] Not only would this broaden the scope for forming nonhydrolysable Ub conjugates of recombinant proteins in vitro, but it also paves the way for photoactivated bioorthogonal labellingo fp roteins in live cells. As an evolved PylS/ tRNA CUA pair (PCKRS/tRNA CUA )h as been shown to direct the incorporation of photocaged lysine, [21] we tested the incorporation of 3 into superfolder green fluorescent protein (sfGFP) with aT AG stop codon at position 150 and aC -terminal hexahistidinet ag, [22] by using the PCKRS/tRNA CUA pair and aP CKRS/ tRNA CUA pair combined with the Y349W mutation (PCKRS*/ tRNA CUA ). Immunoblotting against the His 6 tag revealed that in-Scheme1.Genetic incorporation of e-aminooxy-l-lysine via acid-and photolabile precursors. A) 1,aminooxy-l-lysine (aminooxylysine); 2, Ne-(tert-butyloxycarbonyl)aminooxylysine; 3, Ne-photocaged aminooxylysine; 4, Ne-(tertbutyloxycarbonyl)-l-lysine. B) Geneticallydirecting isosteric and nonhydrolysable ubiquitin conjugation. Incorporation of 2 or 3 by an evolved MbPylS/ tRNA CUA pair enablesthe incorporation of the latent aminooxyfunctionality. Acidolysis or photolysis provides af acile route to the site-specific incorporation of 1.F acile oximeligation with ubiquitin aldehyde (Ub-CHO)f urnishes an onhydrolysablei sostericm imetic of the isopeptide bond. The geometry of the oximeconjugatel argely reflects that of the native isopeptide counterpart.
ChemBioChem 2016, 17,1472 -1480 www.chembiochem.org corporation of 3 wasp ossible albeit inefficient, and, surprisingly,e fficiency wash igherw ith PCKRS ( Figure S2). These results indicatet hat the Y349W mutant only facilitates incorporation of e-augmentationso fl ysine derivatives in certain contexts, and alternative mutations can have similar effect,p resumably by allosteric restructuring of the active site.

Production of nonhydrolysable analogues of diUb
We expressed and purified 3.5 mg of His-tagged Ub containing 2 at position6 (Ub-BocONH 2 K6;F igure 1B), and characterised it by ESI-MS ( Figure 1C). The C-terminalH is-tagw as removed by treatment with the DUB UCH-L3, [17] then Ub-BocONH 2 K6 was purified by reversed-phase (RP)-HPLC ( Figure 1D). The Boc protecting group was subsequently removed by treatment with 60 %T FA to yield Ub-ONH 2 K6, and the polypeptide was recovered by ether precipitation [17] (Figure 1E). In parallel, we prepared Ub aldehyde (Ub-CHO)a sd escribed previously. [23] Incubation of Ub-ONH 2 K6 with at wofold excess of Ub-CHOi n denaturing buffer for 1h at pH 6r esulted in formation of the site-specifically conjugated diUb product (UbK6 2 -ox);t his was purified by RP-HPLC, refolded and characterisedb yE SI-MS and SDS-PAGE (Figures 2A,Da nd S3). Resistance to DUB hydrolysis was confirmed by treatment with increasingc oncentrations of the DUB USP21 [24] ( Figure 2B). We observed no hydrolysis of UbK6 2 -ox at 37 8Cf or 1h(even with 800 nm enzyme), whereas native K6-linked diUb exhibited near complete hydrolysis in the presence of 800 nm USP21 after 1h ( Figure 2B). Prepara-tion of K48-linked diUb was also carried out by expression of Ub containing aT AG codon atp osition 48 ( Figures 2D and S3).

Production of nonhydrolysable analogues of ubiquitylated SUMO
In order to explore the generality of preparing nonhydrolysable Ub conjugates, we prepared an onhydrolysable isostere of native SUMO ubiquitylateda tL ys11( Ub-SUMO2K11). As SUMO2 has an ative cysteine, this would test the compatibility of our strategy with cysteine-containing proteins. Arsenic-induced formation of Ub-SUMO2K11 on promyelocytic leukaemia protein (PML) leads to resolution of acute promyelocytic leukaemia (APL). [25] The physiological DUB that reverses this conjugation is unknown. Intriguingly,D UBs belonging to the Ub Cterminalh ydrolase (UCH) family, [4] which have historically been considered inactive towards ubiquitylated proteins, have recently been shown to have high activity towards Ub-SUMO2K11. [26] As the mechanism of this activity is unknown, an isosteric yet nonhydrolysable analogue of Ub-SUMO2K11 would be valuable for identifying DUBs that remove Ub from PML conjugates, as well as as tructuralt oolf or determining the activity requirements of UCH-family DUBs. We explored the possibility of carrying out the production of oxime-linked Ub-SUMO2K11 (Ub-SUMO-ox) on folded protein, withoutchaotropic salts, by using ao ne-pot deprotection-ligations trategy.H istagged SUMO2 bearing 2 at position1 1( SUMO-BocONH 2 K11) was expressed in good yield (3 mg L À1 culture medium; Figures S4 and S5). After purification,a cid deprotection of the Boc group was carriedout on the folded protein. [27] Quantitative removal of the Boc group was confirmed by LC-MS ( Figure S6). The pH was then raised to pH 7, and at wofold excess of Ub-CHO was added. Aniline-catalysed oxime ligation [28] at 37 8C was then monitored by LC-MS.The reactionw ent to near completion after 15 h( Figure 2C), and the oxime-linked conjugate (Ub-SUMO-ox) was purified under native conditions by sizeexclusion chromatography and characterised by SDS-PAGE and ESI-MS ( Figures 2D,Eand S3).

Structural characterisation of oxime-linked diUb
In order to unequivocally demonstrate that the oxime conjugates accurately mirrored the structure of native isopeptidelinked conjugates we solved ac rystal structure of UbK6 2 -ox. The crystal structure of native K6-linked diUb (UbK6 2 )h as been determined, and this served as ar eference to assess the isostery of the oxime-linkedc ounterpart. [17] UbK6 2 -ox readily formed cubic crystals under identical conditions to those employed for UbK6 2 ,t hus enabling determination of a3 .5 c rystal structure ( Figure 3A and Table S1). The topology of UbK6 2 -ox accurately mirrored that of native UbK6 2 (backboneR MSD 1.1 ). An important consideration with our strategy is that the oxime linkage could potentially form am ixture of cis and trans regioisomers, thereby giving rise to structuralh eterogeneity. [29] However,u nambiguous electron density for the carboxy terminal residues of the distal Ub molecule and the oxime linkage with incorporated 1 was consistentw ith the trans regioisomer  Figure 3B). We cannot exclude the possibility that af raction of the cis isomer was present, and that the trans speciess elec-tively crystallised under the conditions tested. However, we suspectt hat the steric bulk of the protein reactants ensures that the favoured regioisomer upon oxime ligation is the trans species. These findings established that the topology of oximelinked conjugates is homogenous and near identical to that of the native counterpart.
Nonhydrolysable oxime-linked Ub conjugates are potent DUB inhibitors and bind with affinity comparable to that of native conjugates We next determined if the oxime-linked conjugates recapitulated the biochemical properties of the native isopeptide-linked conjugates,b ym easuring their capacity to inhibitD UBs. For this we determined IC 50 values against hydrolysis of the fluorogenic substrate Ub-Rhodamine. [30] The conjugates Ub-ox-SUMO and  www.chembiochem.org nm,r espectively;F igure 4A). As both conjugates werep otent inhibitors of UCH-L3 but only Ub-SUMO2K11i sasubstrate, UCH-L3 activity is not dictated by K m ,b ut rather by the significantly enhanced catalytic efficiency (k cat )t owards Ub-SUMO2K11. We also tested the inhibitory capacity of UbK48 2ox against the USP family DUB, USP2 ( Figure 4B): UbK48 2 -ox inhibitedU SP2 with an IC 50 of 120.1 (60.1-237.0) nm.I nt he inhibitory assays, the DUB concentrations weree xtremelyl ow (< 2.4 nm); therefore, in order to unequivocally confirm that inhibition was achievedb yt he oxime-linked conjugates( rather than trace contamination with Ub-CHO, ak nown DUB inhibitor), [31] we determined the dissociation constant (K d )b etween USP2 andU bK48 2 -ox by isothermal titration calorimetry (ITC; Figure 4C). K d was 98 nm (comparable to IC 50 ) , and the binding stoichiometry was closet ou nity (0.86), thus confirming that UbK48 2 -ox was indeed the inhibitory species in the IC 50 assay. In order to demonstrate that the binding affinity of UbK48 2 -ox recapitulated that of the native conjugate, we characterised the binding between UbK48 2 and the catalytically inactive USP2 mutant,C 223A ( Figure 4D). The dissociation constants were comparable (62 and 98 nm). Althought he thermodynamic signature was distinct, it is commonf or minor structural perturbationst og ive rise to significant enthalpy-entropy compensation effectsw ithout any gross change in binding mode. [32] Immunoblottingo fwild-type ubiquitin dimers and oxime conjugates Recognition of the oxime conjugates by Ub linkage-specific antibodies against the native counterparts would provide further validation of the physiological integrity of our conjugates. Furthermore, this would validate using the nonhydrolysable analoguesa sa ntigens( thereby capitalising on their enhanced in vivo half-life) for raising Ub linkage-specific antibodies that could be used in ar eciprocal manner to specifically recognise the native isopeptide-linked conjugate. Linkage-specific antibodiesfor K6, K27, K29 and K33 linkages are currently unavailable but would be powerful tools for elucidating the cellular roles of these linkage types. The diUb oxime conjugates (UbK48 2 -ox and UbK6 2 -ox) were analysed by immunoblotting with linkage-specific antibodies along with Ub dimers with native isopeptide linkages (UbK6 2 ,U bK11 2 ,U bK63 2 and UbK48 2 ;F igure 5). The K48 linkage-specific antibody [33] recognised UbK48 2 -ox, albeit with as lightly weaker signal than for the native counterpart( Figure 5). Furthermore, as expected, anti-K63 [33] and anti-Met1 [34] linkage-specific immunoblotting did not cross-react with either of the oxime-linked Ub conjugates ( Figure 5). Additionally,t otal Ub immunoblotting indicated that UbK6 2 -ox and UbK48 2 -ox were recognised to the same degree as their respective native isopeptide conjugates, thus . Native ubiquitylated SUMO is as ubstrate of UCH-L3, whereas UbK6 2 is not. This suggests that discrimination of these substrates by UCH-L3 is achieved only by differences in k cat .RF: relative fluorescence units.B )The ubiquitin-specific protease (USP)D UB USP2 is inhibitedbyU bK48 2 -ox. C) ITC demonstrates that K d is comparable to IC 50 .B inding stoichiometry is also close to unity,thus indicating thatt he oxime-linked conjugate confers inhibition in theI C 50 assays. D) K d of binding between native UbK48 2 and ac atalytically inactive USP2 mutant (USP2 C223A) is comparabletot hat of UbK48 2 -ox binding to wild-typeUSP2. Figure 5. Immunoblottinganalysis of native isopeptide linkedu biquitin dimers compared to oxime-linked ubiquitin conjugates. Ac omparison of oxime-conjugated ubiquitin dimers (UbK48 2 -ox and UbK6 2 -ox) to wild-type ubiquitin dimers (UbK6 2 ,U bK11 2 ,U bK63 2 ,U bK48 2 )i ndicates that oxime-conjugatedu biquitin dimersa re recognised similarly by an a-Ub (total Ub) antibody.Immunoblotting with linkage-selective a-Ub antibodies (a-K48, a-K63 and a-Met1) indicates that UbK48 2 -ox was successfully recognised by the linkagespecific a-K48 antibody. No cross-reactivity with a-K63 or a-Met1 was observed for UbK6 2 -ox or UbK48 2 -ox;only the relevant wild-typeU b conjugates were recognised. Silver stainingw as used as aloading control because of inconsistent immunoreactivity of total Ub antibodies across different linkages types. indicating that the oxime linkaged id not give rise to aberrant maskingo ff unctional epitopes on the Ub surfaces or to alteration in the topology of the Ub conjugate ( Figure 5).
These nonhydrolysable Ub polymers would be valuable tools for affinity purification of linkage-specific DUBs and ubiquitin-binding proteins from cell extracts. Thus, we conjugated polyUb-oxt ob iotin, thereby enabling immobilisation on streptavidin resin, by site-specific C-terminal thiazolidine formation with cysteine-functionalised biotin (Cys-biotin; Figure S9). [37] Conclusion We describeap owerful toolkit based on oxime ligation to advance the study of substrate-specific DUBs and the cellular roles of polyUb linkages, based on the first genetically directed incorporation of aminooxy functionality into recombinant proteins. This was achieved by the incorporation of either acid-or photo-labile protected precursors of aminooxy-l-lysine (1). Amino acid 1 has extremelyh igh isostery with native lysine as it differs by conservative substitution of am ethyleneg roup by an oxygen atom at e-position. We demonstrated that 1 could be site-specifically incorporated into both Ub and SUMO2v ia ap rotected precursor.T his enabled the chemoselective conjugation of Ub with Ub-CHO by oxime ligation. The dimeric Ub conjugates were resistant to DUBs that are highly active against the native isopeptide-linked counterparts. The oxime linkage exhibits unprecedentedi sostery with the native isopeptideb ond, thus making it ap referred strategy for preparing nonhydrolysable versions of Ub conjugates. This was demonstrated by structuralc haracterisation of aK 6-linked Ub dimer by X-ray crystallography.T he structure of the oximelinked speciesa ccurately mirroredt hat of the native counterpart. Furthermore, unambiguous electron density confirmed that the trans regioisomer was the predominant, if not exclusive, product upon oxime ligationb etween proteins. The non- www.chembiochem.org hydrolysable oxime-linkedU bc onjugates also proved to be nanomolar DUB inhibitors. This high isostery with native conjugates, combinedw ith hydrolytic stability, should allow ubiquitin conjugates prepared by this approacht ob eu sed as inhibitors of linkages pecific processes. Such experiments could be conducted with cell extracts or in intact cells by microinjection. As functionalisation of Ub-like (Ubl) proteins [38] with an aldehyde group is possible, [23] it should also be possible to prepare nonhydrolysable variantso fU b-like conjugates (e.g.,N EDD8, ISG15, SUMO).
Furthermore, we describe the use of oxime chemistry in polymerisation reactions with bifunctionalised Ubs, in order to generatep olyUbc onjugates linked by oxime isopeptide isosteres. The expedient synthesis of such conjugates, in conjunction with their resistance to proteolytic hydrolysis, makes these new conjugatesimportant probesfor studying cellularprocesses that are regulated by polyUb chains.
Finally,w ed escribed the incorporation of photocaged aminooxy-l-lysine (3). This should broaden the utility by enabling conjugation to acid-sensitiver ecombinant proteins.A lthough incorporation efficiency was low,amore efficient PylS/tRNA CUA should be obtainable by directed evolution. [39] Furthermore, recent reports have demonstrated that the aminooxy group can undergo rapid biocompatible oxime ligation with dialdehyde moieties [40] and in boronic-acid-mediated oxime ligations. [41] These reactions are ultra-fast, rivallings tate-of-the-art inverse electron-demand Diels-Alder bioconjugation between tetrazines and strained enes. [42] This would enable ultra-fast photoactivated protein labelling, thereby overcoming the diffusion limit associated with constitutively reactive bioorthogonal handles. Incorporation of the photocaged amino acid into proteins in live mammalian cells should be achievable, as this has been shown for structurally similar, photocaged lysine derivatives. [21,43] Furthermore, aldehyde functionality can be genetically encoded by using orthogonal aldehyde tag technology (compatible with prokaryotic and mammalian hosts). [44] Excitingly,this could provide astrategy for site-specific photoactivated covalent protein-protein tethering in live cells.
Crystallisation, structure determination and refinement: UbK6 2ox crystallised with cubic morphology at 0.8 mg mL À1 ,u nder previously reported conditions. [17] The best crystal diffracted to 3.5 (Beamline I02, Diamond Light Source, Harwell, UK). Initial phases were obtained by molecular replacement by using one ubiquitin moiety from the K6-diubiquitin structure (PDB ID:2 XK5). [17] Indexing and integration was carried out with XDS. [45] Structure refinement was carried out with PHENIX [46] and model building was carried out within COOT. [47] Data collection and refinement statistics are in Ta ble S1 (PDB ID:5KHY).
Isothermal titration calorimetry: ITC was conducted on an iTC200 Microcalorimeter (GE Healthcare). All proteins were dialysed against argon-purged ITC buffer (20 mm HEPES, 100 mm NaCl, 0.25 mm TCEP,p H7 .5). The sample cell contained either UbK48 2 WT (9.2 mm) or UbK48 2 -ox (8.4 mm); the concentrations were determined by using as tandard curve of UbK48 2 (absorbance at 214 nm). The syringe contained USP2 WT (93 mm)o rU SP2 C223A (99 mm); the concentrations were determined by absorbance at 280 nm (for USP2; e 280 = 41 370 Lmol À1 cm À1 ). In all, 13 injections (0.4 mL) were delivered (0.8 sa ddition time, interval 120 s). The stirrer speed was set to 750 rpm, and each binding experiment was carried out at 25 8C. Water was used in the reference cell, and titrations into buffer were carried out to assess the enthalpies of dilution. The data was smoothed with the simple arithmetic function in the Origin 7d ata analysis software (v.7.0552;OriginLab, Northampton, MA).