Genetic Code Expansion Facilitates Programmable Ubiquitylation via UBE2W

Genetic Code Expansion Facilitates Programmable Ubiquitylation via UBE2W | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Genetic Code Expansion Facilitates Programmable Ubiquitylation via UBE2W View ORCID Profile Paul Schnacke , View ORCID Profile Maximilian Fottner , View ORCID Profile Denys Kvasha , View ORCID Profile Fabian Willenborg , View ORCID Profile Kathrin Lang doi: https://doi.org/10.1101/2025.09.22.676137 Paul Schnacke 1 Laboratory for Organic Chemistry (LOC), Department of Chemistry and Applied Biosciences (D-CHAB), ETH Zurich , Vladimir-Prelog-Weg 3, 8093 Zurich, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Paul Schnacke Maximilian Fottner 1 Laboratory for Organic Chemistry (LOC), Department of Chemistry and Applied Biosciences (D-CHAB), ETH Zurich , Vladimir-Prelog-Weg 3, 8093 Zurich, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Maximilian Fottner For correspondence: kathrin.lang{at}org.chem.ethz.ch mfottner{at}ethz.ch Denys Kvasha 1 Laboratory for Organic Chemistry (LOC), Department of Chemistry and Applied Biosciences (D-CHAB), ETH Zurich , Vladimir-Prelog-Weg 3, 8093 Zurich, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Denys Kvasha Fabian Willenborg 1 Laboratory for Organic Chemistry (LOC), Department of Chemistry and Applied Biosciences (D-CHAB), ETH Zurich , Vladimir-Prelog-Weg 3, 8093 Zurich, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Fabian Willenborg Kathrin Lang 1 Laboratory for Organic Chemistry (LOC), Department of Chemistry and Applied Biosciences (D-CHAB), ETH Zurich , Vladimir-Prelog-Weg 3, 8093 Zurich, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Kathrin Lang For correspondence: kathrin.lang{at}org.chem.ethz.ch mfottner{at}ethz.ch Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Deciphering the ubiquitin code requires homogenous, site-specifically ubiquitylated proteins, yet their access remains difficult. Current chemical and enzymatic methods are often limited by harsh conditions, low yields, or the introduction of non-native scars. Here, we present UbyW (Ubiquitylation by UBE2W), a chemoenzymatic platform that overcomes these hurdles. By repurposing the E2 enzyme UBE2W to target a genetically encoded non-canonical amino acid, a near-native Ub-protein conjugate is formed with high efficiency and site-specificity. The method is broadly applicable to diverse proteins, including those with structured domains, and can be performed in vitro or within a reconstituted cascade in living E. coli for streamlined production. Crucially, UbyW allows for the direct installation of bioorthogonal and photocrosslinking handles at the linkage site, providing powerful tools to probe the functional consequences of ubiquitylation and map ubiquitin-dependent interactomes. Introduction Post-translational modification (PTM) of proteins with Ubiquitin (Ub) governs nearly every aspect of a protein’s life, from its localization and function to its ultimate degradation. This functional diversity is orchestrated by a complex network, termed the “ubiquitin code”, where the modification site on a substrate protein, the modification type, and the length and linkage topology of Ub chains can collectively dictate specific downstream signaling outcomes. 1 , 2 To decipher this complexity, probes based on well-defined ubiquitin-protein of interest (Ub-POI) conjugates are essential, as they provide insight into how a particular ubiquitylation event influences a protein’s structure, function, and interactions. 3 However, accessing such Ub-POI probes has remained challenging. Consequently, the development of tools capable of generating these probes, especially those incorporating crosslinkers directly at the modification site to capture transient interactors, remains a critical need in the field. 4 Over the past decades, significant efforts have been directed towards the synthesis of ubiquitylated proteins using methods like Native Chemical Ligation (NCL) and click chemistry. 5 The required conjugation handles are either introduced through solid phase peptide synthesis (SPPS) or recombinantly, via genetic code expansion (GCE). 6 While powerful, these approaches have inherent limitations. SPPS is fundamentally restricted to smaller, readily refoldable protein domains. Furthermore, NCL itself requires harsh, denaturing conditions. Click chemistry-based approaches, conversely, introduce non-native chemical ligation scars, potentially perturbing the protein’s local environment and interactome. 5 To address this, the field has turned its attention towards chemoenzymatic strategies to form native-like linkages under physiological conditions (Supplementary Fig. 1a). Methods such as LACE 7 , 8 and SUE1 9 utilize E2 enzymes to attach Ub to a target protein. However, a constraint of these systems is the requirement of a recognition tag of four or more amino acids in length, thereby limiting the scope of accessible endogenous modification sites. Sortylation, relying on the transpeptidase Sortase 2A, addresses this limitation by selectively recognizing a non-canonical amino acid (ncAA) introduced by GCE. 10 , 11 Yet, this technique introduces its own set of considerations, including the requirement for mutations at the Ub C-terminus, and comparably low protein yields due to GCE inefficiencies. Additionally, none of these methods can incorporate warheads or photo-crosslinking handles into the conjugates. To address these challenges, we present UbyW (Ubiquitylation by UBE2W), a novel chemoenzymatic platform designed for efficient and facile site-specific ubiquitylation. Our method exploits the intrinsic, but underreported activity of the E2 conjugating enzyme UBE2W to selectively target neo-N-termini, introduced into the POI by incorporation of isopeptide-linked XisoK ncAAs via GCE. Thereby generated conjugates closely resemble their native counterparts and can be generated in high yield. UbyW operates not only in vitro but also within a reconstituted cascade directly in living E. coli . This allows for the straightforward production and single-step purification of complex, modified proteins. Crucially, this platform also provides access to the direct installation of bioorthogonal handles and photocrosslinkers close to the isopeptide inkage site, opening new avenues for studying the functional consequences of site-specific ubiquitylation (Supplementary Fig. 1b). Results Site-specific modification of G-XisoK-bearing target proteins by UBE2W Our work leverages the recently developed G-XisoK toolbox, a versatile platform enabling efficient active uptake of G-XisoK tripeptidic ncAAs into E. coli cells, facilitating genetic encoding of XisoKs at wild-type-like expression levels using low ncAA concentrations (Supplementary Fig. 2). 12 Intriguingly, recent work has shown that XisoKs naturally occur as PTMs through the aminoacylation of N-ε of lysine by aminoacyl-tRNA synthetases (aaRS). 13 Furthermore, proteomic studies have suggested that these XisoK PTMs are ubiquitylated by the atypical E2 conjugating enzyme UBE2W. 14 Inspired by these findings we envisioned to develop a method for facile, site-specific, high yield generation of ubiquitylated proteins based on the XisoK toolbox and UBE2W. For this, we first investigated the UBE2W-mediated ubiquitylation of a XisoK-bearing model protein. We expressed SUMO2 bearing LisoK (L-leucine linked via an isopeptide bond to lysine) at position 11 ( Fig. 1a ). As UBE2W is known to ubiquitylate SUMO2’s native N-terminus we introduced an N-terminal proline residue to prohibit its modification. Initial incubation of SUMO2(K11LisoK), UBA1, Ub and UBE2W in vitro resulted in the formation of the desired Ub-SUMO2(K11LisoK) conjugate, albeit with very low efficiency ( Fig. 1b ). We also observed significant UBE2W auto-ubiquitylation, a known characteristic of this enzyme. 15 Download figure Open in new tab Figure 1. Site-specific ubiquitylation of proteins via UBE2W and LisoK incorporation using genetic code expansion. a , SUMO2 endowed with non-canonical amino acid (ncAA) LisoK at K11 is modified site-specifically by UBE2W, resulting in a near-native like conjugate. b , Left: SDS-PAGE analysis of initial results for the incubation of SUMO2(K11LisoK) with UBE2W, UBA1, and Ub, Right: SDS-PAGE analysis of in vitro modification of SUMO2(K11LisoK) by UBE2W S/U under optimized reaction conditions, wtSUMO2 is not a substrate for UBE2W S/U . c , SDS-PAGE analysis of purified Ub-SUMO2(K11LisoK) conjugate d , LC-MS analysis confirms successful conjugation. d , MSMS of Ub-SUMO2(K11LisoK) confirms specific attachment of Ub onto LisoK. Full gels are found in Supplementary Fig. 4. Consistent results were obtained over three distinct replicate experiments. To enhance the reaction yield, we systematically optimized the reaction conditions. By adjusting the reaction buffer, the stoichiometric ratio of reaction components, and generating a Strep-SUMO2-Ub-UBE2W fusion construct, termed UBE2W S/U , we were able to reduce UBE2W autoubiquitylation and increase conversion more than five-fold. Importantly, no ubiquitylation was observed for wild-type SUMO2 as a substrate, confirming the reaction’s dependence on the neo-N-terminus within LisoK ( Fig. 1b , Supplementary Figs 3,4). The resulting Ub-SUMO2(K11LisoK) conjugate was readily purified to homogeneity using a combination of Ni-NTA affinity chromatography, TEV protease cleavage, and size-exclusion chromatography (SEC) ( Fig. 1c , Supplementary Fig. 4). The identity and site-specificity of the modification were confirmed by intact protein mass spectrometry (ESI-LC-MS) and tandem mass spectrometry (MS/MS), which identified a peptide fragment corresponding to Ub linked specifically to the LisoK residue ( Fig. 1d, e , Supplementary Fig. 4). We term this method UbyW (Ubiquitylation by UBE2W). Exploring UBE2Ws Donor substrates and XisoK permissiveness Next, we investigated the permissiveness of UbyW towards different Ub donor substrates. We found that linear (M1) di-, tri-, and tetraUb chains were conjugated to SUMO2(K11LisoK) with efficiencies comparable to that of monoUb, and the conjugation yield was independent of Ub chain length ( Fig. 2a , Supplementary Fig. 8a). Furthermore, we tested the UBE2W-mediated attachment of a K63-linked diUb to LisoK at position 11 within SUMO2. This conjugate was also formed successfully, albeit with slightly reduced efficiency compared to the linear chains ( Fig. 2b , Supplementary Fig. 8b). These results demonstrate the promiscuity of UBE2W to accept different Ub donor substrates, from monoUb to Ub chains of defined length and linkage type, which are involved in distinct signaling pathways. 16 Download figure Open in new tab Figure 2. Promiscuity of UbyW towards donors and XisoK acceptors. a , SDS-PAGE analysis of the attachment of linear (M1) Ub polymers onto SUMO2(K11LisoK). b , SDS-PAGE analysis of the attachment of K63-linked diUb onto SUMO2(K11LisoK). Consistent results were obtained over three distinct replicate experiments. c , SDS-PAGE analysis of UBE2W mediated modification of SUMO2-K11 endowed with different XisoKs and different unstructured N-termini. Full gels are found in Supplementary Fig. 8. Building on this, we then investigated the reaction’s dependency on the identity of the acceptor amino acid XisoK. A key advantage of the XisoK toolbox is the ability to incorporate a wide variety of ncAAs. 12 We generated a panel of SUMO2(K11XisoK) variants, including small (A, S), aliphatic (V, L), nucleophilic (C), and conformationally restricted (P) L-amino acids, as well as D-amino acids (a, s) and ncAAs bearing bioorthogonal handles (PrgG, ClA) and a photocrosslinker (pL) as X residue (Supplementary Figs 5,6). Incubating these SUMO2 XisoK variants under optimized conditions revealed a strong preference for primary L-amino acids as X residues, with high conversion rates observed for A, S, T, V, L, and C ( Fig. 2a , Supplementary Fig. 7). In contrast, PisoK was a poor substrate, and XisoKs bearing D-amino acids as X were modified to a very low degree, indicating a strict stereochemical requirement for the reaction. Notably, non-canonical amino acids PrgG, ClA, and pL were efficiently modified. Thereby generated probes may serve for subsequent bioorthogonal conjugation, trapping, or crosslinking studies. For comparison, we also tested SUMO2 constructs with different N-termini under identical reaction conditions. While SUMO2 with a native N-terminal alanine or an HA-tag (displaying a N-terminal methionine) have both been characterized as substrates of UBE2W, 15 , 17 we found them to be significantly poorer substrates compared to the L-XisoK variants, indicating a potential preference of UBE2W for aminoacylated lysines. Ubiquitylation of structured protein domains at endogenous modification sites With the broad scope of possible XisoK acceptors established, we next probed UBE2W permissiveness toward different acceptor proteins, focusing on more complex and biologically relevant targets. We found that both Histone H3 and the microtubule-associated protein Tau bearing LisoK at endogenous ubiquitylation sites (H3(K27) and Tau(K353)), can be ubiquitylated by UBE2W ( Fig. 3a, b , Supplementary Fig. 9). No modification was observed for wtH3 and wtTau. We also successfully ubiquitylated α-synuclein at position 10 and 21 ( Fig. 3c , Supplementary Fig. 9). Both positions are located in a well-defined α-helical domain. This demonstrates UbyW’s ability to modify positions placed in folded secondary structures without the need to modify the endogenous POI sequence. This is a capability not shared by other chemoenzymatic methods that rely on the use of peptide tags. Download figure Open in new tab Figure 3. Ubiquitylation of proteins in unstructured and structured folds. a , Left: Structure of Histone H3 with K27 highlighted in red (Protein Data Bank, 1kx5) 18 . Right: SDS-PAGE analysis of in vitro ubiquitylation of H3*(K27LisoK). wtH3* is not modified. * The H3 construct includes a N-terminally placed Ub monomer as solubility tag for efficient expression in E. coli . b , Left: Structure of Tau with K353 highlighted in red (Protein Data Bank, 6hrf) 19 . Right: SDS-PAGE analysis of in vitro modification of Tau(244-372, K353LisoK). Wt-Tau(244-372) is not modified. c , Left: Structure of α-synuclein with K10 and K21 in the α-helical fold highlighted in red (Protein Data Bank, 1xq8) 20 . Right: SDS-PAGE analysis of in vitro modification of α-synuclein(K10LisoK), and α-synuclein(K21LisoK), wt-α-synuclein is not modified. Full gels are found in Supplementary Fig. 9. Consistent results were obtained over three distinct replicate experiments. However, we faced challenges with modifying more complex proteins like homotrimeric PCNA and the small GTPase Ran in vitro . Ubiquitylation of PCNA resulted in low modification yields and in the case of Ran in significant protein degradation under the in vitro reaction conditions (Supplementary Fig. 10). Moreover, the in vitro approach is inherently laborious, requiring the separate purification of all protein components (UBA1, UBE2W S/U , Ub, and the target protein), followed by a second purification step to isolate the final conjugate from the reaction mixture. A reconstituted ubiquitylation cascade in E. coli for site-specific ubiquitylation To address the limitations of in vitro UbyW, we explored the possibility of reconstituting the entire ubiquitylation cascade, as well as the required genetic code expansion machinery, in living E. coli ( Fig. 4a , Supplementary Fig. 11a). To test this, we co-expressed the model protein SUMO2, bearing an amber codon at the defined modification site K11, along with the corresponding aaRS/tRNA pair (PylRS, PylT). Upon supplementation of the culture medium with G-XisoK, its active import and proteolytic processing, XisoK is efficiently incorporated into SUMO2. In parallel we co-expressed all the other needed components so that UBA1 can charge UBE2W with Ub to then ubiquitylate the XisoK-endowed target protein SUMO2. Download figure Open in new tab Figure 4. Reconstituted ubiquitylation cascade in living E. coli enables direct modification of proteins by UBE2W. a , Active uptake of G-XisoK into E. coli via Opp and subsequent proteolytic cleavage by pepN and pepA allows for high-yielding amber suppression. Transiently co-expressed UBE2W is charged with Ub by UBA1 and modifies XisoK-bearing SUMO2 directly in E. coli . b , SDS-PAGE analysis of Ni-NTA purification and size exclusion chromatography of Ub-SUMO2(K11LisoK) conjugate from E. coli expressing the reconstituted cascade. c , SDS-PAGE analysis of purified conjugates. Full gels are found in Supplementary Fig. 11. Consistent results were obtained over three distinct replicate experiments. Initial results demonstrated detectable, but low, yields of ubiquitylated SUMO2. (Supplementary Fig. 11b). We therefore undertook a systematic optimization of the cellular expression system. By consolidating the genetic components onto fewer plasmids and testing different promoters to balance the expression levels of the cascade components, we achieved a more than ten-fold improvement in the yield of the isolated conjugate (Supplementary Fig. 11c). With this optimized UbyW system, we could isolate and purify 3.1 mg/L of Ub-SUMO2(K11LisoK) conjugate directly from cultures supplemented with as little as 0.5 mM of the G-LisoK pro-peptide ( Fig. 4b ). The identity of the conjugate was confirmed by mass spectrometry (Supplementary Fig. 11d). UbyW in E. coli is highly versatile: We successfully generated SUMO2 conjugates with linear (M1) di-, tri-, and tetraUb chains directly in E. coli and purified them in high yield (up to 4.8 mg/L, Supplementary Fig. 11e-g). Furthermore, we were able to conjugate a Ub variant with a L73P mutation in the C-terminal region to XisoK-bearing target proteins by UbyW. This renders the Ub-conjugates stable against DUBs both in vitro and in untreated HEK293T cell lysate, creating probes suitable for delivery into mammalian cells or downstream applications that are dependent on such conditions. (Supplementary Fig. 12a, c, d, 13). 21 Notably, the system was also compatible with other Ub-like modifiers (Ubls). 22 We demonstrated the efficient conjugation of Nedd8, a Ubl whose recombinant expression, purification and conjugation is often low-yielding and cumbersome ( Fig. 4c , Supplementary Fig. 12b, 13). Given the high degree of conservation of UBE2W across different species, we investigated whether homologs from other organisms could function in our E. coli cascade as well. 23 We tested a selection of these and found that the ubiquitylation activity was indeed conserved, although the yields varied between species (Supplementary Fig. 14a-c). Excitingly, two homologs ( D. rerio and C. elegans ) exhibited higher activity than the human UBE2W, both in vitro and within the E. coli system increasing yield of isolated Ub-POI conjugate (Supplementary Fig. 14d). With this reconstituted UbyW cascade in hand we were able to site-specifically ubiquitylate SUMO1 at position 7 and NEMO at positions 302 and 309. Both positions reside within the α-helical UBAN domain, a structural motif proposed to control the regulatory activity of NEMO in the NF-κB pathway (Supplementary Fig. 15). 24 With our efficient in cellulo UbyW cascade in hand, we revisited the complex protein targets that had failed in the in vitro setting (Supplementary Fig. 16). We first targeted PCNA, a homo-trimeric clamp essential for DNA replication, at its key modification site, K164 ( Fig. 5a , Supplementary Fig. 16). Using the E. coli cascade, we successfully generated and purified the Ub-PCNA(K164LisoK) conjugate in high yields (35 mg/mL, Fig. 5b ). The UbyW system was also capable of producing Nedd8ylated PCNA, a natively occurring modification whose influence on DNA repair remains understudied ( Fig. 5c ). Next, we targeted the small GTPase Ran at K71, a residue located in the vicinity of the GTP binding pocket ( Fig. 5d ). 26 Using in cellulo UbyW, we were able to produce and purify the Ub-Ran(K71LisoK) conjugate without any observable degradation and >50% conjugation yield, in stark contrast to the in vitro approach ( Fig. 5e , Supplementary Fig. 17). The successful conjugation for both PCNA and Ran was confirmed by LC-MS analysis ( Fig. 5f , Supplementary Fig. 17). These results underscore the significant advantages of the reconstituted cellular cascade, which provides a protective environment that maintains the stability and integrity of challenging proteins during the enzymatic modification process. Overall, UbyW in E. coli is a robust, easy-to-use platform that enables the high-yield generation of site-specifically ubiquitylated protein conjugates through a simple transformation and culturing procedure, followed by a single purification step. Download figure Open in new tab Fig 5. Direct ubiquitylation of complex proteins in E. coli . a , Structure of the PCNA homo-trimer with K164 highlighted in red (Protein Data Bank, 1axc) 25 . b , SDS-PAGE analysis of purification of Ub-PCNA(K164LisoK) conjugate generated in E. coli . c , SDS-PAGE analysis of purification of Nedd8-PCNA(K164LisoK) conjugate generated in E. coli . d , Structure of small GTPase Ran, K71 is in vicinity of the GTP binding pocket and highlighted in red, co-crystallized GTP and Mg 2+ are also shown (Protein Data Bank, 3gj0) 26 . E , SDS-PAGE analysis of purification of Ub-Ran(K71LisoK) conjugate generated in E. coli . F , LC-MS analysis confirms successful conjugation. Full gels are found in Supplementary Fig. 16,17. Consistent results were obtained over three distinct replicate experiments. Discussion In this study, we introduce the UbyW (Ubiquitylation by UBE2W) platform for site-specific ubiquitylation. UbyW has been demonstrated to efficiently modify a wide range of protein targets (8 proteins at 10 different sites) - including challenging substrates like PCNA and Ran - within both unstructured regions and folded domains. The platform is user-friendly, particularly the reconstituted cascade in E. coli , simplifying the production of modified proteins. It requires no specialized expertise in chemical synthesis, or protein semi-synthesis for the high-yield production of Ub-protein conjugates with various chemical handles as it relies on a simple co-transformation and subsequent standard protein purification. The resulting conjugates closely mimic the native counterparts, differing only by the presence of a single non-canonical ‘X’ residue resulting from the XisoK handle that functionally introduces a spacer between the Ub C-terminus and the POI. This design offers several advantages: The core structure of the attached Ub remains unperturbed, preserving its native interaction surfaces for binding partners. Indeed, it has been shown to be advantageous to place handles, like photocrosslinkers after Ub G76. 27 However, this near-native structure also makes the linkage susceptible to cleavage by endogenous deubiquitinating enzymes (DUBs), a challenge that can be overcome by using the DUB-resistant Ub(L73P) variant. Although the X-extension is minimal, it is possible that enzymes whose recognition motif spans both the Ub C-terminus, and the substrate surface may exhibit altered binding. Future work will focus on evolving the E1-E2 machinery to accept Ub(ΔG76), which would yield a truly scarless product as well as to expand the scope to include more Ubls. 28 The UbyW system is built upon the unique and still poorly understood catalytic properties of UBE2W. Our findings lend further proof to UBE2W’s reported activity of modifying XisoK derivatives within target proteins. Moreover, our work not only demonstrates a preference of UBE2W for α-over ϵ-amine substrate residues, but also, and more unexpectedly, a potential preference for isopeptidic neo-N-termini over native N-termini. Indeed, the model protein SUMO2 used in our screen had been previously reported as a UBE2W substrate for N-terminal modification, 15 , 17 but the installed XisoKs acted as superior substrates. Additionally, the acceptor scope for L-XisoKs is remarkably broad. Distinct specificity is only encountered against D-X amino acids, which suggests a well-defined geometry in the reaction’s transition state. Explaining UBE2W’s unique reactivity regarding XisoKs is challenging. The enzyme possesses several features that set it apart from canonical E2 enzymes. It lacks a conserved gatekeeper aspartate residue, which may facilitate its ability to differentiate between α- and ε-amines by their different pKa’s. Furthermore, unlike the conserved HPN motif typically found in E2s - a region often implicated in oxyanion hole stabilization - UBE2W instead possesses an HPH motif (Supplementary Fig. 18b). 29 However, neo-N-termini on XisoKs and N-termini are very similar. Substrate recognition is thought to be primarily mediated by its highly flexible C-terminal region, but how this region achieves its specificity remains poorly understood (Supplementary Fig. 18a). 17 Regarding the positioning of the XisoK, analysis of consensus amino acid frequencies among the tested substrates, did not reveal any dependence on amino acid context close to XisoK (Supplementary Fig. 18c, d). This feature is favorable for the UbyW platform as it will enable broad applicability to many targets. Further structural studies are required to better understand the recognition of XisoKs by UBE2W. In parallel, functional investigations should be pursued to explore this E3-independent, moonlighting activity of UBE2W, which may play a biologically significant role. This function is conserved throughout evolution and could, 23 in addition to maintaining the homeostasis of XisoK PTMs, 14 serve as a defense mechanism against pathogenic clippases that generate neo-N-termini. 30 , 31 Looking forward, the UbyW platform opens numerous avenues for research. While direct application in mammalian cells is not feasible, as UBE2W is an endogenous enzyme, the generated probes can be delivered into cells via electroporation or incubated in mammalian cell lysate to study the functional consequences of a specific ubiquitylation event. In this context the true power of the platform may lie in its application to proteomics. The ability to install photocrosslinking ncAAs directly at the ubiquitylation site provides an unprecedented tool for trapping the transient, low-affinity interactors that are often recruited by specific Ub signals. Applying this strategy to challenging and biologically critical targets like PCNA and Ran will allow for an unbiased mapping of their site-specific Ub-dependent interactomes, providing deep insights into the complex signaling networks governed by the “ubiquitin code”. Funder Information Declared European Research Council, https://ror.org/0472cxd90 , ERC under the European Union’s Horizon 2020 research and innovation program, grant agreement no. 101003289ɪ#9472;Ubl-tool to K.L References 1. ↵ Swatek , K. N. & Komander , D. Ubiquitin modifications . Cell Res . 26 , 399 – 422 ( 2016 ). OpenUrl CrossRef PubMed 2. ↵ Komander , D. & Rape , M. The Ubiquitin Code . ( 2012 ) doi: 10.1146/annurev-biochem-060310-170328 . OpenUrl CrossRef PubMed Web of Science 3. ↵ Hameed , D. S. , Sapmaz , A. & Ovaa , H. How Chemical Synthesis of Ubiquitin Conjugates Helps To Understand Ubiquitin Signal Transduction . Bioconjug. Chem . 28 , 805 – 815 ( 2017 ). OpenUrl PubMed 4. ↵ Wanka , V. , Fottner , M. , Cigler , M. & Lang , K. Genetic Code Expansion Approaches to Decipher the Ubiquitin Code . Chem. Rev . 124 , 11544 – 11584 ( 2024 ). OpenUrl PubMed 5. ↵ Mali , S. M. , Singh , S. K. , Eid , E. & Brik , A. Ubiquitin Signaling: Chemistry Comes to the Rescue . J. Am. Chem. Soc . 139 , 4971 – 4986 ( 2017 ). OpenUrl PubMed 6. ↵ Schultz , P. Expanding the genetic code . Protein Sci . 32 , e4488 ( 2023 ). OpenUrl PubMed 7. ↵ Akimoto , G. , Fernandes , A. P. & Bode , J. W. Site-Specific Protein Ubiquitylation Using an Engineered, Chimeric E1 Activating Enzyme and E2 SUMO Conjugating Enzyme Ubc9 . ACS Cent. Sci . 8 , 275 – 281 ( 2022 ). OpenUrl PubMed 8. ↵ Hofmann , R. , Akimoto , G. , Wucherpfennig , T. G. , Zeymer , C. & Bode , J. W. Lysine acylation using conjugating enzymes for site-specific modification and ubiquitination of recombinant proteins . Nat. Chem . 12 , 1008 – 1015 ( 2020 ). OpenUrl CrossRef PubMed 9. ↵ Wu , X. et al. Structure-guided engineering enables E3 ligase-free and versatile protein ubiquitination via UBE2E1 . Nat. Commun . 15 , 1266 ( 2024 ). OpenUrl PubMed 10. ↵ Fottner , M. et al. A modular toolbox to generate complex polymeric ubiquitin architectures using orthogonal sortase enzymes . Nat. Commun . 2021 121 12 , 1 – 15 ( 2021 ). OpenUrl CrossRef PubMed 11. ↵ Fottner , M. et al. Site-specific ubiquitylation and SUMOylation using genetic-code expansion and sortase . Nat. Chem. Biol . 2019 153 15 , 276 – 284 ( 2019 ). OpenUrl CrossRef PubMed 12. ↵ Iype , T. , Fottner , M. , Böhm , P. , Piedrafita , C. & Lang , K. Hijacking a bacterial membrane transporter for efficient genetic code expansion . 2025.03.06.641855 Preprint at doi: 10.1101/2025.03.06.641855 ( 2025 ). OpenUrl Abstract / FREE Full Text 13. ↵ He , X.-D. et al. Sensing and Transmitting Intracellular Amino Acid Signals through Reversible Lysine Aminoacylations . Cell Metab . 27 , 151 - 166 .e6 ( 2018 ). OpenUrl CrossRef PubMed 14. ↵ Zang , J. et al. Genetic code expansion reveals aminoacylated lysine ubiquitination mediated by UBE2W . Nat. Struct. Mol. Biol . 30 , 62 – 71 ( 2023 ). OpenUrl CrossRef PubMed 15. ↵ Tatham , M. H. , Plechanovová , A. , Jaffray , E. G. , Salmen , H. & Hay , R. T. Ube2W conjugates ubiquitin to α-amino groups of protein N-termini . Biochem. J . 453 , 137 – 145 ( 2013 ). OpenUrl Abstract / FREE Full Text 16. ↵ Cappadocia , L. & Lima , C. D. Ubiquitin-like Protein Conjugation: Structures, Chemistry, and Mechanism . Chem. Rev . 118 , 889 – 918 ( 2018 ). OpenUrl CrossRef PubMed 17. ↵ Vittal , V. et al. Intrinsic disorder drives N-terminal ubiquitination by Ube2w . Nat. Chem. Biol . 11 , 83 – 89 ( 2015 ). OpenUrl CrossRef PubMed 18. ↵ Davey , C. A. , Sargent , D. F. , Luger , K. , Maeder , A. W. & Richmond , T. J. Solvent Mediated Interactions in the Structure of the Nucleosome Core Particle at 1.9 Å Resolution† . J. Mol. Biol . 319 , 1097 – 1113 ( 2002 ). OpenUrl CrossRef PubMed Web of Science 19. ↵ Falcon , B. et al. Tau filaments from multiple cases of sporadic and inherited Alzheimer’s disease adopt a common fold . Acta Neuropathol. (Berl .) 136 , 699 – 708 ( 2018 ). OpenUrl CrossRef PubMed 20. ↵ Ulmer , T. S. , Bax , A. , Cole , N. B. & Nussbaum , R. L. Structure and Dynamics of Micelle-bound Human α-Synuclein* . J. Biol. Chem . 280 , 9595 – 9603 ( 2005 ). OpenUrl Abstract / FREE Full Text 21. ↵ Békés , M. et al. DUB-Resistant Ubiquitin to Survey Ubiquitination Switches in Mammalian Cells . Cell Rep . 5 , 826 – 838 ( 2013 ). OpenUrl CrossRef PubMed 22. ↵ Kerscher , O. , Felberbaum , R. & Hochstrasser , M. Modification of Proteins by Ubiquitin and Ubiquitin-Like Proteins . Annu. Rev. Cell Dev. Biol . 22 , 159 – 180 ( 2006 ). OpenUrl CrossRef PubMed Web of Science 23. ↵ Qi , C. et al. Biochemical and structural characterization of a novel ubiquitin-conjugating enzyme E2 from Agrocybe aegeria reveals Ube2w family-specific properties . Sci. Rep . 5 , 16056 ( 2015 ). OpenUrl PubMed 24. ↵ Rahighi , S. et al. Specific Recognition of Linear Ubiquitin Chains by NEMO Is Important for NF-κB Activation . Cell 136 , 1098 – 1109 ( 2009 ). OpenUrl CrossRef PubMed Web of Science 25. ↵ Gulbis , J. M. , Kelman , Z. , Hurwitz , J. , O’Donnell , M. & Kuriyan , J. Structure of the C-Terminal Region of p21WAF1/CIP1 Complexed with Human PCNA . Cell 87 , 297 – 306 ( 1996 ). OpenUrl CrossRef PubMed Web of Science 26. ↵ Partridge , J. R. & Schwartz , T. U. Crystallographic and Biochemical Analysis of the Ran-binding Zinc Finger Domain . J. Mol. Biol . 391 , 375 – 389 ( 2009 ). OpenUrl CrossRef PubMed Web of Science 27. ↵ Shu , X. et al. Detecting Active Deconjugating Enzymes with Genetically Encoded Activity-Based Ubiquitin and Ubiquitin-like Protein Probes . Anal. Chem . 95 , 846 – 853 ( 2023 ). OpenUrl 28. ↵ Guntas , G. & Kuhlman , B. Redesigning the NEDD8 Pathway with a Bacterial Genetic Screen for Ubiquitin-Like Molecule Transfer . J. Mol. Biol . 418 , 161 – 166 ( 2012 ). OpenUrl PubMed 29. ↵ Scaglione , K. M. et al. The Ubiquitin-conjugating Enzyme (E2) Ube2w Ubiquitinates the N Terminus of Substrates . J. Biol. Chem . 288 , 18784 – 18788 ( 2013 ). OpenUrl Abstract / FREE Full Text 30. ↵ Hermanns , T. et al. A family of bacterial Josephin-like deubiquitinases with an irreversible cleavage mode . Mol. Cell 85 , 1202 - 1215 .e5 ( 2025 ). OpenUrl PubMed 31. ↵ Swatek , K. N. et al. Insights into ubiquitin chain architecture using Ub-clipping . Nature 572 , 533 – 537 ( 2019 ). OpenUrl CrossRef PubMed 32. Chang , C.-C. et al. Structural and Functional Roles of Daxx SIM Phosphorylation in SUMO Paralog-Selective Binding and Apoptosis Modulation . Mol. Cell 42 , 62 – 74 ( 2011 ). OpenUrl CrossRef PubMed Web of Science 33. Rahighi , S. , Iyer , M. , Oveisi , H. , Nasser , S. & Duong , V. Structural basis for the simultaneous recognition of NEMO and acceptor ubiquitin by the HOIP NZF1 domain . Sci. Rep . 12 , 12241 ( 2022 ). OpenUrl CrossRef PubMed 34. Crooks , G. E. , Hon , G. , Chandonia , J.-M. & Brenner , S. E. WebLogo: A Sequence Logo Generator . Genome Res . 14 , 1188 – 1190 ( 2004 ). OpenUrl Abstract / FREE Full Text View the discussion thread. Back to top Previous Next Posted September 22, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. 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Share Genetic Code Expansion Facilitates Programmable Ubiquitylation via UBE2W Paul Schnacke , Maximilian Fottner , Denys Kvasha , Fabian Willenborg , Kathrin Lang bioRxiv 2025.09.22.676137; doi: https://doi.org/10.1101/2025.09.22.676137 Share This Article: Copy Citation Tools Genetic Code Expansion Facilitates Programmable Ubiquitylation via UBE2W Paul Schnacke , Maximilian Fottner , Denys Kvasha , Fabian Willenborg , Kathrin Lang bioRxiv 2025.09.22.676137; doi: https://doi.org/10.1101/2025.09.22.676137 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Biochemistry Subject Areas All Articles Animal Behavior and Cognition (7633) Biochemistry (17680) Bioengineering (13889) Bioinformatics (41928) Biophysics (21445) Cancer Biology (18585) Cell Biology (25491) Clinical Trials (138) Developmental Biology (13373) Ecology (19897) Epidemiology (2067) Evolutionary Biology (24308) Genetics (15606) Genomics (22496) Immunology (17736) Microbiology (40385) Molecular Biology (17175) Neuroscience (88583) Paleontology (666) Pathology (2830) Pharmacology and Toxicology (4822) Physiology (7641) Plant Biology (15149) Scientific Communication and Education (2045) Synthetic Biology (4293) Systems Biology (9822) Zoology (2271)