Combining poly-epitope MoonTags and labeled nanobodies for signal amplification in cell-specific PET imaging in vivo.

Immunorecognition provides an excellent basis for targeted imaging techniques covering a wide range from basic research to diagnostics and from single cells to whole organisms. Fluorescence- or radioisotope-labeled antibodies, antibody fragments or nanobodies enable a direct signal readout upon binding and allow for versatile imaging from microscopy to whole-body imaging. However, as the signal intensity directly correlates with the number of labeled antibodies bound to their epitopes (1:1 binding), sensitivity for low-expressing epitopes can be limiting for visualization. For the first time, we developed poly-epitope tags with multiple copies (1 to 7) of a short peptide epitope, specifically the MoonTag, that are recognized by a labeled nanobody and aimed at signal amplification in microscopy and cell-specific PET imaging. In transiently transfected HeLa cells or stably transduced A4573 cells we characterized complex formation and in vitro signal amplification. Indeed, using fluorescently and radioactively labeled nanobodies we found an approximately linear signal amplification with increasing numbers of epitope copies in vitro. To test the poly-epitope approach in vivo, A4573 tumor cells were injected subcutaneously into the shoulder of NSG mice, with A4573 tumor cells expressing a poly-epitope of 7 MoonTags on one side and WT cells on the other side. Using a [68Ga]-labeled NODAGA-conjugated MoonTag nanobody, we performed PET/CT imaging at day 8-9 after tumor implantation. Specific binding of a [68Ga]-labeled NODAGA-conjugated MoonTag nanobody was observed in 7xMoonTag tumors (1.7 ± 0.5%ID/mL) by PET imaging, showing significantly higher radiotracer accumulation compared to the WT tumors (1.1 ± 0.3%ID/mL; p < 0.01). Ex vivo gamma counter measurements confirmed significantly higher uptake in 7xMoonTag tumors compared to WT tumors (p < 0.001). In addition, MoonTag nanobody binding was detected by autoradiography which was spatially matched with histological analysis of the tumor tissues. In conclusion, we expect nanobody-based poly-epitope tag strategies to be widely applicable for multimodal imaging techniques given the advantageous properties of nanobodies and their amenability to genetic and chemical engineering.

Towards targeted Cas9 (CRISPR-Cas) delivery: Preparation of IgG antibody-Cas9 conjugates using a split intein.

The CRISPR-Cas9 system has revolutionized the field of genetic engineering, but targeted cellular delivery remains a central problem. The delivery of the preformed ribonuclease-protein (RNP) complex has the advantages of fewer side effects and avoidance of potential permanent effects. We reasoned that an internalizing IgG antibody as a targeting device could address the delivery of Cas9-RNP. We opted for protein trans-splicing mediated by a split intein to facilitate posttranslational conjugation of the two large protein entities. We recently described the cysteine-less CL split intein that efficiently performs under oxidizing conditions and does not interfere with disulfide bonds or thiol bioconjugation chemistries. Using the CL split intein, we report for the first time the ligation of monoclonal IgG antibody precursors, expressed in mammalian cells, and a Cas9 precursor, obtained from bacterial expression. A purified IgG-Cas9 conjugate was loaded with sgRNA to form the active RNP complex and introduced a double-strand break in its target DNA in vitro. Furthermore, a synthetic peptide variant of the short N-terminal split intein precursor proved useful for chemical modification of Cas9. The split intein ligation procedure reported here for IgG-Cas9 provides the first step towards a novel CRISPR-Cas9 targeting approach involving the preformed RNP complex.

Unraveling Structural Information of Multi-Domain Nonribosomal Peptide Synthetases by Using Photo-Cross-Linking Analysis with Genetic Code Expansion.

Nonribosomal peptide synthetases (NRPSs) are large, multifunctional enzymes that facilitate the stepwise synthesis of modified peptides, many of which serve as important pharmaceutical products. Typically, NRPSs contain one module for the incorporation of one amino acid into the growing peptide chain. A module consists of the domains required for activation, covalent binding, condensation, termination, and optionally modification of the aminoacyl or peptidyl moiety. We here describe a protocol using genetically encoded photo-cross-linking amino acids to probe the 3D architecture of NRPSs by determining spatial proximity constraints. p-benzoyl-L-phenylalanine (BpF) is incorporated at positions of presumed contact interfaces between domains. The covalent cross-link products are visualized by SDS-PAGE-based methods and precisely mapped by tandem mass spectrometry. Originally intended to study the communication (COM) domains, a special pair of docking domains of unknown structure between two interacting subunits of one NRPS system, this cross-linking approach was also found to be useful to interrogate the spatial proximity of domains that are not connected on the level of the primary structure. The presented photo-cross-linking technique thus provides structural insights complementary to those obtained by protein crystallography and reports on the protein in solution.

Structural and biochemical analysis of a novel atypically split intein reveals a conserved histidine specific to cysteine-less inteins.

Protein trans-splicing mediated by a split intein reconstitutes a protein backbone from two parts. This virtually traceless autoprocessive reaction provides the basis for numerous protein engineering applications. Protein splicing typically proceeds through two thioester or oxyester intermediates involving the side chains of cysteine or serine/threonine residues. A cysteine-less split intein has recently attracted particular interest as it can splice under oxidizing conditions and is orthogonal to disulfide or thiol bioconjugation chemistries. Here, we report the split PolB16 OarG intein, a second such cysteine-independent intein. As a unique trait, it is atypically split with a short intein-N precursor fragment of only 15 amino acids, the shortest characterized to date, which was chemically synthesized to enable protein semi-synthesis. By rational engineering we obtained a high-yielding, improved split intein mutant. Structural and mutational analysis revealed the dispensability of the usually crucial conserved motif N3 (block B) histidine as an obvious peculiar property. Unexpectedly, we identified a previously unnoticed histidine in hydrogen-bond forming distance to the catalytic serine 1 as critical for splicing. This histidine has been overlooked so far in multiple sequence alignments and is highly conserved only in cysteine-independent inteins as a part of a newly discovered motif NX. The motif NX histidine is thus likely of general importance to the specialized environment in the active site required in this intein subgroup. Together, our study advances the toolbox as well as the structural and mechanistic understanding of cysteine-less inteins.

Microarray screening reveals two non-conventional SUMO-binding modules linked to DNA repair by non-homologous end-joining.

SUMOylation is critical for numerous cellular signalling pathways, including the maintenance of genome integrity via the repair of DNA double-strand breaks (DSBs). If misrepaired, DSBs can lead to cancer, neurodegeneration, immunodeficiency and premature ageing. Using systematic human proteome microarray screening combined with widely applicable carbene footprinting, genetic code expansion and high-resolution structural profiling, we define two non-conventional and topology-selective SUMO2-binding regions on XRCC4, a DNA repair protein important for DSB repair by non-homologous end-joining (NHEJ). Mechanistically, the interaction of SUMO2 and XRCC4 is incompatible with XRCC4 binding to three other proteins important for NHEJ-mediated DSB repair. These findings are consistent with SUMO2 forming a redundant NHEJ layer with the potential to regulate different NHEJ complexes at distinct levels including, but not limited to, XRCC4 interactions with XLF, LIG4 and IFFO1. Regulation of NHEJ is not only relevant for carcinogenesis, but also for the design of precision anti-cancer medicines and the optimisation of CRISPR/Cas9-based gene editing. In addition to providing molecular insights into NHEJ, this work uncovers a conserved SUMO-binding module and provides a rich resource on direct SUMO binders exploitable towards uncovering SUMOylation pathways in a wide array of cellular processes.

A Light-Activatable Photocaged Variant of the Ultra-High Affinity ALFA-Tag Nanobody.

Nanobodies against short linear peptide-epitopes are widely used to detect and bind proteins of interest (POI) in fusion constructs. Engineered nanobodies that can be controlled by light have found very recent attention for various extra- and intracellular applications. We here report the design of a photocaged variant of the ultra-high affinity ALFA-tag nanobody, also termed ALFA-tag photobody. ortho-Nitrobenzyl tyrosine was incorporated into the paratope region of the nanobody by genetic code expansion technology and resulted in a ≥9,200 to 100,000-fold impairment of the binding affinity. Irradiation with light (365 nm) leads to decaging and reconstitutes the native nanobody. We show the light-dependent binding of the ALFA-tag photobody to HeLa cells presenting the ALFA-tag. The generation of the first photobody directed against a short peptide epitope underlines the generality of our photobody design concept. We envision that this photobody will be useful for the spatiotemporal control of proteins in many applications using cultured cells.

C/EBPß is a MYB- and p300-cooperating pro-leukemogenic factor and promising drug target in acute myeloid leukemia.

Transcription factor MYB has recently emerged as a promising drug target for the treatment of acute myeloid leukemia (AML). Here, we have characterized a group of natural sesquiterpene lactones (STLs), previously shown to suppress MYB activity, for their potential to decrease AML cell proliferation. Unlike what was initially thought, these compounds inhibit MYB indirectly via its cooperation partner C/EBPß. C/EBPß-inhibitory STLs affect the expression of a large number of MYB-regulated genes, suggesting that the cooperation of MYB and C/EBPß broadly shapes the transcriptional program of AML cells. We show that expression of GFI1, a direct MYB target gene, is controlled cooperatively by MYB, C/EBPß, and co-activator p300, and is down-regulated by C/EBPß-inhibitory STLs, exemplifying that they target the activity of composite MYB-C/EBPß-p300 transcriptional modules. Ectopic expression of GFI1, a zinc-finger protein that is required for the maintenance of hematopoietic stem and progenitor cells, partially abrogated STL-induced myelomonocytic differentiation, implicating GFI1 as a relevant target of C/EBPß-inhibitory STLs. Overall, our data identify C/EBPß as a pro-leukemogenic factor in AML and suggest that targeting of C/EBPß may have therapeutic potential against AML.

Genetic Encoding and Enzymatic Deprotection of a Latent Thiol Side Chain to Enable New Protein Bioconjugation Applications.

The thiol group of the cysteine side chain is arguably the most versatile chemical handle in proteins. To expand the scope of established and commercially available thiol bioconjugation reagents, we genetically encoded a second such functional moiety in form of a latent thiol group that can be unmasked under mild physiological conditions. Phenylacetamidomethyl (Phacm) protected homocysteine (HcP) was incorporated and its latent thiol group unmasked on purified proteins using penicillin G acylase (PGA). The enzymatic deprotection depends on steric accessibility, but can occur efficiently within minutes on exposed positions in flexible sequences. The freshly liberated thiol group does not require treatment with reducing agents. We demonstrate the potential of this approach for protein modification with conceptually new schemes for regioselective dual labeling, thiol bioconjugation in presence of a preserved disulfide bond and formation of a novel intramolecular thioether crosslink.

Biochemical and Structural Characterization of an Unusual and Naturally Split Class 3 Intein.

Split inteins are indispensable tools for protein engineering because their ligation and cleavage reactions enable unique modifications of the polypeptide backbone. Three different classes of inteins have been identified according to the nature of the covalent intermediates resulting from the acyl rearrangements in the multistep protein-splicing pathway. Class 3 inteins employ a characteristic internal cysteine for a branched thioester intermediate. A bioinformatic database search of non-redundant protein sequences revealed the absence of split variants in 1701 class 3 inteins. We have discovered the first reported split class 3 intein in a metagenomics data set and report its biochemical, mechanistic and structural analysis. The AceL NrdHF intein exhibits low sequence conservation with other inteins and marked deviations in residues at conserved key positions, including a variation of the typical class-3 WCT triplet motif. Nevertheless, functional analysis confirmed the class 3 mechanism of the intein and revealed excellent splicing yields within a few minutes over a wide range of conditions and with barely detectable cleavage side reactions. A high-resolution crystal structure of the AceL NrdHF precursor and a mutagenesis study explained the importance and roles of several residues at the key positions. Tolerated substitutions in the flanking extein residues and a high affinity between the split intein fragments further underline the intein's future potential as a ligation tool.

Characterization of the zinc finger proteins ZMYM2 and ZMYM4 as novel B-MYB binding proteins.

B-MYB, a highly conserved member of the MYB transcription factor family, is expressed ubiquitously in proliferating cells and plays key roles in important cell cycle-related processes, such as control of G2/M-phase transcription, cytokinesis, G1/S-phase progression and DNA-damage reponse. Deregulation of B-MYB function is characteristic of several types of tumor cells, underlining its oncogenic potential. To gain a better understanding of the functions of B-MYB we have employed affinity purification coupled to mass spectrometry to discover novel B-MYB interacting proteins. Here we have identified the zinc-finger proteins ZMYM2 and ZMYM4 as novel B-MYB binding proteins. ZMYM4 is a poorly studied protein whose initial characterization reported here shows that it is highly SUMOylated and that its interaction with B-MYB is stimulated upon induction of DNA damage. Unlike knockdown of B-MYB, which causes G2/M arrest and defective cytokinesis in HEK293 cells, knockdown of ZMYM2 or ZMYM4 have no obvious effects on the cell cycle of these cells. By contrast, knockdown of ZMYM2 strongly impaired the G1/S-phase progression of HepG2 cells, suggesting that ZMYM2, like B-MYB, is required for entry into S-phase in these cells. Overall, our work identifies two novel B-MYB binding partners with possible functions in the DNA-damage response and the G1/S-transition.

Photo-crosslink analysis in nonribosomal peptide synthetases reveals aberrant gel migration of branched crosslink isomers and spatial proximity between non-neighboring domains.

Nonribosomal peptide synthetases (NRPSs) are large, multi-modular enzyme templates for the biosynthesis of important peptide natural products. Modules are composed of a set of semi-autonomous domains that facilitate the individual reaction steps. Only little is known about the existence and relevance of a higher-order architecture in these mega-enzymes, for which contacts between non-neighboring domains in three-dimensional space would be characteristic. Similarly poorly understood is the structure of communication-mediating (COM) domains that facilitate NRPS subunit docking at the boundaries between epimerization and condensation domains. We investigated a COM domain pair in a minimal two module NRPS using genetically encoded photo-crosslinking moieties in the N-terminal acceptor COM domain. Crosslinks into the C-terminal donor COM domain of the partner module resulted in protein products with the expected migration behavior on SDS-PAGE gels corresponding to the added molecular weight of the proteins. Additionally, an unexpected apparent high-molecular weight crosslink product was revealed by mass spectrometric analysis to represent a T-form isomer with branched connectivity of the two polypeptide chains. Synthesis of the linear L-form and branched T-form isomers by click chemistry confirmed this designation. Our data revealed a surprising spatial proximity between the acceptor COM domain and the functionally unrelated small subdomain of the preceding adenylation domain. These findings provide an insight into three-dimensional domain arrangements in NRPSs in solution and suggest the described photo-crosslinking approach as a promising tool for the systematic investigation of their higher-order architecture.

A mesophilic cysteine-less split intein for protein trans-splicing applications under oxidizing conditions.

Split intein-mediated protein trans-splicing has found extensive applications in chemical biology, protein chemistry, and biotechnology. However, an enduring limitation of all well-established split inteins has been the requirement to carry out the reaction in a reducing environment due to the presence of 1 or 2 catalytic cysteines that need to be in a reduced state for splicing to occur. The concomitant exposure of the fused proteins to reducing agents severely limits the scope of protein trans-splicing by excluding proteins sensitive to reducing conditions, such as those containing critical disulfide bonds. Here we report the discovery, characterization, and engineering of a completely cysteine-less split intein (CL intein) that is capable of efficient trans-splicing at ambient temperatures, without a denaturation step, and in the absence of reducing agents. We demonstrate its utility for the site-specific chemical modification of nanobodies and an antibody Fc fragment by N- and C-terminal trans-splicing with short peptide tags (CysTag) that consist of only a few amino acids and have been prelabeled on a single cysteine using classical cysteine bioconjugation. We also synthesized the short N-terminal fragment of the atypically split CL intein by solid-phase peptide synthesis. Furthermore, using the CL intein in combination with a nanobody-epitope pair as a high-affinity mediator, we showed chemical labeling of the extracellular domain of a cell surface receptor on living mammalian cells with a short CysTag containing a synthetic fluorophore. The CL intein thus greatly expands the scope of applications for protein trans-splicing.

Standard Binding Free Energy of a SIM-SUMO Complex.

Interaction of the small ubiquitin-related modifier (SUMO) and peptides containing a SUMO-interacting motif (SIM) has attracted a lot of interest in recent years, yet their structural properties and relationships between the composition of the peptide and binding free energy are not completely understood. We perform molecular dynamics simulations of the complex formed by SUMO and a peptide containing the tight-binding SIM of the protein inhibitor of activated STAT. The calculated standard binding free energy of -5.06 kcal/mol is in reasonable agreement with the experimental value of -6.54 kcal/mol. Experimental results for complexes formed by SUMO and SIM dimers indicate the existence of a parallel and an antiparallel binding mode for similar SIM peptides. We find that the parallel binding mode is highly favored in the present case. Furthermore, the simulations show that residues neighboring the SIM core motif contribute strongly to the binding energy. Structurally, the complex shows differences from the picture in which the SIM core motif lies deep in the SUMO binding groove. This also supports the idea that neighboring residues play an important role in binding.

Rational design of an improved photo-activatable intein for the production of head-to-tail cyclized peptides.

Head-to-tail cyclization of genetically encoded peptides and proteins can be achieved with the split intein circular ligation of peptides and proteins (SICLOPPS) method by inserting the desired polypeptide between the C- and N-terminal fragments of a split intein. To prevent the intramolecular protein splicing reaction from spontaneously occurring upon folding of the intein domain, we have previously rendered this process light-dependent in a photo-controllable variant of the M86 intein, using genetically encoded ortho-nitrobenzyltyrosine at a structurally important position. Here, we report improvements on this photo-intein with regard to expression yields and rate of cyclic peptide formation. The temporally defined photo-activation of the purified stable intein precursor enabled a kinetic analysis that identified the final resolution of the branched intermediate as the rate-determining individual reaction of the three steps catalyzed by the intein. With this knowledge, we prepared an R143H mutant with a block F histidine residue. This histidine is conserved in most inteins and helps catalyze the third step of succinimide formation. The engineered intein formed the cyclic peptide product up to 3-fold faster within the first 15 min after irradiation, underlining the potential of protein splicing pathway engineering. The broader utility of the intein was also shown by formation of the 14-mer sunflower trypsin inhibitor 1.

Bridging of membrane surfaces by annexin A2.

The protein-mediated formation of membrane contacts is a crucial event in many cellular processes ranging from the establishment of organelle contacts to the docking of vesicles to a target membrane. Annexins are Ca2+ regulated membrane-binding proteins implicated in providing such membrane contacts; however, the molecular basis of membrane bridging by annexins is not fully understood. We addressed this central question using annexin A2 (AnxA2) that functions in secretory vesicle exocytosis possibly by providing membrane bridges. By quantitatively analyzing membrane contact formation using a novel assay based on quartz crystal microbalance recordings, we show that monomeric AnxA2 can bridge membrane surfaces Ca2+ dependently. However, this activity depends on an oxidative crosslink involving a cysteine residue in the N-terminal domain and thus formation of disulfide-linked dimers. Alkylated AnxA2 in which this cysteine residue has been modified and AnxA2 mutants lacking the N-terminal domain are not capable of bridging membrane surfaces. In contrast, a heterotetrameric complex comprising two membrane binding AnxA2 subunits linked by a S100A10 dimer can provide membrane contacts irrespective of oxidation status. Thus, monomeric AnxA2 only contains one lipid binding site and AnxA2-mediated linking of membrane surfaces under non-oxidative intracellular conditions most likely requires AnxA2-S100 complex formation.

Generation of Potent Anti-HER1/2 Immunotoxins by Protein Ligation Using Split Inteins.

Cell targeting protein toxins have gained increasing interest for cancer therapy aimed at increasing the therapeutic window and reducing systemic toxicity. Because recombinant expression of immunotoxins consisting of a receptor-binding and a cell-killing moiety is hampered by their high toxicity in a eukaryotic production host, most applications rely on recombinant production of fusion proteins consisting of an antibody fragment and a protein toxin in bacterial hosts such as Escherichia coli ( E. coli). These fusions often lack beneficial properties of whole antibodies like extended serum half-life or efficient endocytic uptake via receptor clustering. Here, we describe the production of full-length antibody immunotoxins using self-splicing split inteins. To this end, the short (11 amino acids) N-terminal intein part of the artificially designed split intein M86, a derivative of the Ssp DnaB intein, was recombinantly fused to the heavy chain of trastuzumab, a human epidermal growth factor receptor 2 (HER2) receptor targeting antibody and to a nanobody-Fc fusion targeting the HER1 receptor, respectively. Both antibodies were produced in Expi293F cells. The longer C-terminal counterpart of the intein was genetically fused to the protein toxins gelonin or Pseudomonas Exotoxin A, respectively, and expressed in E. coli via fusion to maltose binding protein. Using optimized in vitro splicing conditions, we were able to generate a set of specific and potent immunotoxins with IC50 values in the mid- to subpicomolar range.

Split2 Protein-Ligation Generates Active IL-6-Type Hyper-Cytokines from Inactive Precursors.

Trans-signaling of the major pro- and anti-inflammatory cytokines Interleukin (IL)-6 and IL-11 has the unique feature to virtually activate all cells of the body and is critically involved in chronic inflammation and regeneration. Hyper-IL-6 and Hyper-IL-11 are single chain designer trans-signaling cytokines, in which the cytokine and soluble receptor units are trapped in one complex via a flexible peptide linker. Albeit, Hyper-cytokines are essential tools to study trans-signaling in vitro and in vivo, the superior potency of these designer cytokines are accompanied by undesirable stress responses. To enable tailor-made generation of Hyper-cytokines, we developed inactive split-cytokine-precursors adapted for posttranslational reassembly by split-intein mediated protein trans-splicing (PTS). We identified cutting sites within IL-6 (E134/S135) and IL-11 (G116/S117) and obtained inactive split-Hyper-IL-6 and split-Hyper-IL-11 cytokine precursors. After fusion with split-inteins, PTS resulted in reconstitution of active Hyper-cytokines, which were efficiently secreted from transfected cells. Our strategy comprises the development of a background-free cytokine signaling system from reversibly inactivated precursor cytokines.

Withaferin A, a natural compound with anti-tumor activity, is a potent inhibitor of transcription factor C/EBPß.

Recent work has shown that deregulation of the transcription factor Myb contributes to the development of leukemia and several other human cancers, making Myb and its cooperation partners attractive targets for drug development. By employing a myeloid Myb-reporter cell line we have identified Withaferin A (WFA), a natural compound that exhibits anti-tumor activities, as an inhibitor of Myb-dependent transcription. Analysis of the inhibitory mechanism of WFA showed that WFA is a significantly more potent inhibitor of C/EBPß, a transcription factor cooperating with Myb in myeloid cells, than of Myb itself. We show that WFA covalently modifies specific cysteine residues of C/EBPß, resulting in the disruption of the interaction of C/EBPß with the co-activator p300. Our work identifies C/EBPß as a novel direct target of WFA and highlights the role of p300 as a crucial co-activator of C/EBPß. The finding that WFA is a potent inhibitor of C/EBPß suggests that inhibition of C/EBPß might contribute to the biological activities of WFA.

N-terminal chemical protein labeling using the naturally split GOS-TerL intein.

Chemoselective and regioselective chemical protein labeling is of great importance, yet no current technique is sufficiently general and simple to perform. Protein trans-splicing by split inteins can be used to ligate short tags with chemical labels to either the N or the C terminus of a protein. The CysTag approach exploits split intein fragments without a cysteine fused with such a short tag containing a single cysteine that is easily amenable to selective modification using classical cysteine bioconjugation. Labeling of the protein of interest is achieved through transfer of the pre-labeled tag by protein trans-splicing. This protocol keeps other cysteines unmodified. While split inteins for C-terminal CysTag labeling were previously reported, no high-yielding and naturally split intein for N-terminal labeling has been available. In this work, the recently discovered GOS-TerL intein was explored as the only known naturally split intein that both lacks a cysteine in its N-terminal fragment and is active under ambient conditions. Thioredoxin as a model protein and a camelid nanobody were labeled with a synthetic fluorophore by transferring the pre-labeling CysTag in the protein trans-splicing reaction with yields of about 50 to 90%. The short N-terminal intein fragment was also chemically synthesized with a tag to enable protein labeling by semi-synthetic protein trans-splicing. Our results expand the scope of the CysTag labeling strategy, which achieves selective chemical modification without the requirement for sophisticated biorthogonal functional groups and rather builds on the plethora of commercially available reagents directed at the thiol side chain of cysteine. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Covalent Capturing of Transient SUMO-SIM Interactions Using Unnatural Amino Acid Mutagenesis and Photocrosslinking.

The weak interaction between the post-translational modifier SUMO (small ubiquitin-like modifier) and proteins containing the SUMO-interacting motif (SIM) poses limitations to the identification of interaction partners of SUMOylated proteins and to the mapping of the interfaces. To overcome these limitations, genetically encoded photocrosslinker amino acids were incorporated close to the SIM-interaction groove in human SUMO1. UV irradiation resulted in the desired covalent crosslinks both in a purified protein environment and in cell extracts.