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.

Carrier Protein Interaction with Competing Adenylation and Epimerization Domains in a Nonribosomal Peptide Synthetase Analyzed by FRET.

In multi-domain nonribosomal peptide synthetases (NRPSs) the order of domains and their catalytic specificities dictate the structure of the peptide product. Peptidyl-carrier proteins (PCPs) bind activated amino acids and channel elongating peptidyl intermediates along the protein template. To this end, fine-tuned interactions with the catalytic domains and large-scale PCP translocations are necessary. Despite crystal structure snapshots of several PCP-domain interactions, the conformational dynamics under catalytic conditions in solution remain poorly understood. We report a FRET reporter of gramicidin S synthetase 1 (GrsA; with A-PCP-E domains) to study for the first time the interaction between PCP and adenylation (A) domain in the presence of an epimerization (E) domain, a competing downstream partner for the PCP. Bulk FRET measurements showed that upon PCP aminoacylation a conformational shift towards PCP binding to the A domain occurs, indicating the E domain acts on its PCP substrate out of a disfavored conformational equilibrium. Furthermore, the A domain was found to preferably bind the D-Phe-S-Ppant-PCP stereoisomer, suggesting it helps in establishing the stereoisomeric mixture in favor of the D-aminoacyl moiety. These observations surprisingly show that the conformational logic can deviate from the order of domains and thus reveal new principles in the multi-domain interplay of NRPSs.

Subdomain dynamics enable chemical chain reactions in non-ribosomal peptide synthetases.

Many peptide-derived natural products are produced by non-ribosomal peptide synthetases (NRPSs) in an assembly-line fashion. Each amino acid is coupled to a designated peptidyl carrier protein (PCP) through two distinct reactions catalysed sequentially by the single active site of the adenylation domain (A-domain). Accumulating evidence suggests that large-amplitude structural changes occur in different NRPS states; yet how these molecular machines orchestrate such biochemical sequences has remained elusive. Here, using single-molecule Förster resonance energy transfer, we show that the A-domain of gramicidin S synthetase I adopts structurally extended and functionally obligatory conformations for alternating between adenylation and thioester-formation structures during enzymatic cycles. Complementary biochemical, computational and small-angle X-ray scattering studies reveal interconversion among these three conformations as intrinsic and hierarchical where intra-A-domain organizations propagate to remodel inter-A-PCP didomain configurations during catalysis. The tight kinetic coupling between structural transitions and enzymatic transformations is quantified, and how the gramicidin S synthetase I A-domain utilizes its inherent conformational dynamics to drive directional biosynthesis with a flexibly linked PCP domain is revealed.

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.

Conformational and Interface Variability in Multivalent SIM-SUMO Interaction.

SUMO targeted ubiqutin ligases (STUbLs) like RNF4 or Arkadia/RNF111 recognize SUMO chains through multiple SUMO interacting motifs (SIMs). Typically, these are contained in disordered regions of these enzymes and also the individual SUMO domains of SUMO chains move relatively freely. It is assumed that binding the SIM region significantly restricts the conformational freedom of SUMO chains. Here, we present the results of extensive molecular dynamics simulations on the complex formed by the SIM2-SIM3 region of RNF4 and diSUMO3. Though our simulations highlight the importance of typical SIM-SUMO interfaces also in the multivalent situation, we observe that frequently other regions of the peptide than the canonical SIMs establish this interface. This variability regarding the individual interfaces leads to a conformationally highly flexible complex. Comparison with previous experimental measurements clearly supports our findings and indicates that our observations can be extended to other multivalent SIM-SUMO complexes.

FRET Monitoring of a Nonribosomal Peptide Synthetase Elongation Module Reveals Carrier Protein Shuttling between Catalytic Domains.

Nonribosomal peptide synthetases (NRPSs) employ multiple domains, specifically arranged in modules, for the assembly-line biosynthesis of a plethora of bioactive peptides. It is poorly understood how catalysis is correlated with the domain interplay and associated conformational changes. We developed FRET sensors of an elongation module to study in solution the intramodular interactions of the peptidyl carrier protein (PCP) with adenylation (A) and condensation (C) domains. Backed by HDX-MS analysis, we discovered dynamic mixtures of conformations that undergo distinct population changes in favor of the PCP-A and PCP-C interactions upon completion of the adenylation and thiolation reactions, respectively. To probe this model we blocked PCP binding to the C domain by photocaging and triggered peptide bond formation with light. Changing intramodular domain affinities of the PCP appear to result in conformational shifts according to the logic of the templated assembly process.

A Photo-Crosslinking Approach to Identify Class II SUMO-1 Binders.

The small ubiquitin-like modifier (SUMO) is involved in various cellular processes and mediates known non-covalent protein-protein interactions by three distinct binding surfaces, whose interactions are termed class I to class III. While interactors for the class I interaction, which involves binding of a SUMO-interacting motif (SIM) to a hydrophobic groove in SUMO-1 and SUMO-2/3, are widely abundant, only a couple of examples have been reported for the other two types of interactions. Class II binding is conveyed by the E67 loop region on SUMO-1. Many previous studies to identify SUMO binders using pull-down or microarray approaches did not strategize on the SUMO binding mode. Identification of SUMO binding partners is further complicated due to the typically transient and low affinity interactions with the modifier. Here we aimed to identify SUMO-1 binders selectively enriched for class II binding. Using a genetically encoded photo-crosslinker approach, we have designed SUMO-1 probes to covalently capture class II SUMO-1 interactors by strategically positioning the photo-crosslinking moiety on the SUMO-1 surface. The probes were validated using known class II and class I binding partners. We utilized the probe with p-benzoyl-phenylalanine (BzF, also termed BpF or Bpa) at the position of Gln69 to identify binding proteins from mammalian cell extracts using mass spectrometry. By comparison with results obtained with a similarly designed SUMO-1 probe to target SIM-mediated binders of the class I type, we identified 192 and 96 proteins specifically enriched by either probe, respectively. The implicated preferential class I or class II binding modes of these proteins will further contribute to unveiling the complex interplay of SUMO-1-mediated interactions.

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.

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.

Design and Preparation of Photobodies: Light-Activated Single-Domain Antibody Fragments.

Nanobodies are single-domain antibody fragments that have found widespread use in basic research, therapy, and diagnostics. Like other antibody formats, nanobodies can be developed with high affinity and specificity for desired antigens. A photobody is a light-activatable nanobody, obtained by incorporating a photo-labile caging group into the paratope region. The caging group prevents antigen binding until removed with light (365 nm), thereby rendering the binding controllable with high temporal and spatial resolution. Thus far photocaged tyrosine residues have been used for this purpose, as tyrosine is a frequent residue at critical positions of nanobody paratopes. Nanobodies without a tyrosine residue at the antigen-binding interface may require a different strategy. In this chapter, we describe methods to design and prepare photobodies by recombinant expression in Escherichia coli in combination with genetic code expansion technology to incorporate ortho-nitrobenzyl-tyrosine residues. We use the conversion of the anti-green fluorescent protein enhancer nanobody into a photobody as an example. These protocols should be applicable to many other nanobodies.

Preparation of Bacterial Cell-Surface Displayed Semisynthetic Cyclic Peptides.

Semisynthetic cyclic peptides bearing both non-proteinogenic and genetically encoded amino acids are excellent ligands for peptide-based drug discovery. While semisynthesis expands the chemical space, genetic encoding allows access to a large library via randomization at the nucleic acid level. Selection of novel binders of such macrocyclic ligands requires linking their genotype to phenotype. In this chapter, we report a bacterial cell-surface display system to present cyclic peptides composed of synthetic and genetically encoded fragments. The synthetic fragment along with the split intein partner and an aminooxy moiety is ligated and cyclized with the recombinant backbone containing an unnatural amino acid by protein trans-splicing and intramolecular oxime ligation, respectively. A pH-shift protocol was applied to accelerate on surface cyclization. This method will enable generation of semisynthetic cyclic peptide libraries and their selection by fluorescence-activated cell sorting.

Intermediary conformations linked to the directionality of the aminoacylation pathway of nonribosomal peptide synthetases.

Nonribosomal peptide synthetases (NRPSs) are multifunctional megaenzymes that govern the stepwise biosynthesis of pharmaceutically important peptides. In an ATP-dependent assembly-line mechanism dedicated domains are responsible for each catalytic step. Crystal structures have provided insight into several conformations of interacting domains. However, the complete picture in solution of how domain dynamics and the timing of conformational changes effect a directional biosynthesis remains only poorly understood and will be important for the efficient reprogramming of NRPSs. Here we dissect the multiple conformational changes associated with the adenylation and thiolation reactions of the aminoacylation pathway under catalytic conditions. We used pyrophosphate (PP i ) to biochemically drive the conformational changes backward and forward while performing an online monitoring with a Förster resonance energy transfer (FRET) didomain sensor, consisting of adenylation (A) and peptidyl-carrier protein (PCP) domains. Notably, we found aminoacyl thioester formation to efficiently occur in the presence of PP i even at millimolar concentrations, despite the chemically and conformationally reversing effect of this metabolite and by-product. This finding settles conflicting reports and explains why intracellular PP i concentrations do not impair NRP biosynthesis. A conserved amino acid was identified to be important for the mechanism under these conditions. FRET time-course analyses revealed that the directionality of the aminoacylation catalysis is correlated with conformational kinetics. Complemented by equilibrium hydrogen-deuterium exchange (HDX) analyses, our data uncovered the existence of at least one new intermediary conformation that is associated with the rate-determining step. We propose an expanded model of conformational changes in the NRPS aminoacylation pathway.

The receptor subunit Tom20 is dynamically associated with the TOM complex in mitochondria of human cells.

The outer membrane translocase (TOM) is the import channel for nuclear-encoded mitochondrial proteins. The general import pore contains Tom40, Tom22, Tom5, Tom6, and Tom7. Precursor proteins are bound by the (peripheral) receptor proteins Tom20, Tom22, and Tom70 before being imported by the TOM complex. Here we investigated the association of the receptor Tom20 with the TOM complex. Tom20 was found in the TOM complex, but not in a smaller subcomplex. In addition, a subcomplex was found without Tom40 and Tom7 but with Tom20. Using single particle tracking of labeled Tom20 in overexpressing human cells, we show that Tom20 has, on average, higher lateral mobility in the membrane than Tom7/TOM. After ligation of Tom20 with the TOM complex by post-tranlational protein trans-splicing using the traceless, ultrafast cleaved Gp41-1 integrin system, a significant decrease in the mean diffusion coefficient of Tom20 was observed in the resulting Tom20-Tom7 fusion protein. Exposure of Tom20 to high substrate loading also resulted in reduced mobility. Taken together, our data show that the receptor subunit Tom20 interacts dynamically with the TOM core complex. We suggest that the TOM complex containing Tom20 is the active import pore and that Tom20 is associated when substrate is available.

Light-control of cap methylation and mRNA translation via genetic code expansion of Ecm1.

Gene expression is tightly regulated in all domains of life, with post-transcriptional regulation being more pronounced in higher eukaryotes. Optochemical and optogenetic approaches enable the actuation of many underlying processes by light, which is an excellent tool to exert spatio-temporal control. However, light-mediated control of eukaryotic mRNA processing and the respective enzymes has not been reported. We used genetic code expansion to install a photo-caged tyrosine (Y) in the active site of the cap methyltransferase Ecm1. This enzyme is responsible for guanine N7 methylation of the 5' cap, which is required for translation. Substituting Y284 with the photocaged ortho-nitrobenzyl-tyrosine (ONBY) almost completely abrogated the methylation activity of Ecm1. Irradiation with light removed the ONB group, restoring the native tyrosine and Ecm1 activity, yielding up to 97% conversion of the minimal substrate GpppA within 60 min after activation. Using luciferase- and eGFP-mRNAs as reporters, we could show that light actuates translation by inducing activation of Ecm1 ONBY284 in a eukaryotic in vitro translation system.

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.

Semisynthetic head-to-tail cyclized peptides obtained by combining protein trans-splicing and intramolecular expressed protein ligation.

A dual-intein approach for the preparation of head-to-tail macrocyclic peptides is reported, where synthetic and genetically encoded fragments are ligated by two native peptide bonds. A split intein ligates the synthetic and genetically encoded peptides via protein trans-splicing and is followed by intramolecular cyclization through an expressed protein ligation step mediated with a cis-intein. We identified a suitable pair of orthogonal inteins and optimized the conditions for a one-pot cyclization protocol. We report the semisynthesis of model macrocyles with various ring sizes and of the natural product sunflower trypsin inhibitor (SFTI) along with its ornithine analog.

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.

Insights into the Microscopic Structure of RNF4-SIM-SUMO Complexes from MD Simulations.

Post-translational modification with one of the isoforms of the small ubiquitin-like modifier (SUMO) affects thousands of proteins in the human proteome. The binding of SUMO to SUMO interacting motifs (SIMs) can translate the SUMOylation event into functional consequences. The E3 ubiquitin ligase RNF4 contains multiple SIMs and connects SUMOylation to the ubiquitin pathway. SIM2 and SIM3 of RNF4 were shown to be the most important motifs to recognize SUMO chains. However, the study of SIM-SUMO complexes is complicated by their typically low affinity and variable binding of the SIMs in parallel and antiparallel orientations. We investigated properties of complexes formed by SUMO3 with peptides containing either SIM2 or SIM3 using molecular dynamics simulations. The affinities of the complexes were determined using a state-of-the-art free energy protocol and were found to be in good agreement with experimental data, thus corroborating our method. Long unrestrained simulations allowed a new interpretation of experimental results regarding the structure of the SIM-SUMO interface. We show that both SIM2 and SIM3 bind SUMO3 in parallel and antiparallel orientations and identified main interaction sites for acidic residues flanking the SIM. We noticed unusual SIM-SUMO interfaces in a previously reported NMR structure (PDB: 2mp2) of a complex formed by a SUMO3 dimer with the bivalent SIM2-SIM3 peptide. Computational determination of the individual SIM-SUMO affinities based on these structural arrangements yielded significantly higher dissociation constants. To our knowledge, our approach adds new opportunities to characterize individual SIM-SUMO complexes and suggests that further studies will be necessary to understand these interactions when occurring in multivalent form.