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.

Amide-functionalized 1,2,4-Triazol-5-amines as Covalent Inhibitors of Blood Coagulation Factor XIIa and Thrombin.

To counteract thrombosis, new safe and efficient antithrombotics are required. We herein report the design, synthesis, and biological activity of a series of amide-functionalized acylated 1,2,4-triazol-5-amines as selective inhibitors of blood coagulation factor XIIa and thrombin. The introduction of an amide moiety into the main scaffold of 3-aryl aminotriazoles added certain three-dimensional properties to synthesized compounds and allowed them to reach binding sites in FXIIa and thrombin previously unaddressed by non-functionalized 1,2,4-triazol-5-amines. Among synthesized compounds, one quinoxaline-derived aminotriazole bearing N-butylamide moiety inhibited FXIIa with the IC50 value of 28 nM, whereas the N-phenylamide-derived aminotriazole inhibited thrombin with the IC50 value of 41 nM. Performed mass-shift experiments and molecular modeling studies proved the covalent mechanism of FXIIa and thrombin inhibition by synthesized compounds. In plasma coagulation tests, developed aminotriazoles showed anticoagulant properties mainly affecting the intrinsic blood coagulation pathway, activation of which is associated with thrombosis but is negligible for hemostasis.

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 synthetic covalent ligand of the C/EBPß transactivation domain inhibits acute myeloid leukemia cells.

C/EBPß has recently emerged as a pro-leukemogenic transcription factor that cooperates with oncoprotein MYB to maintain proliferation and differentiation block of AML cells, making C/EBPß an interesting drug target for AML. Here we have studied the inhibitory potential and biological effects of a synthetic analog of the natural product helenalin, a known inhibitor of C/EBPß. The synthetic compound inhibits C/EBPß by covalent binding to cysteine residues in the transactivation domain, thereby causing up-regulation of differentiation-associated genes, cell death and reduced self-renewal potential of AML cells. Suppression of these effects by ectopic expression of C/EBPß or MYB and gene expression profiling validate C/EBPß as a relevant target of the helenalin-mimic and highlight its role as a pro-leukemogenic factor. Overall, our work demonstrates that the synthetic helenalin mimic acts as a covalent inhibitor of C/EBPß and identifies the cysteine residues in the transactivation domain of C/EBPß as ligandable sites. The helenalin mimic can be considered a potential "lead molecule" but needs further development towards more effective C/EBPß inhibitors before being used as a therapeutic agent.

Computational design and experimental characterization of a photo-controlled mRNA-cap guanine-N7 methyltransferase.

The spatial and temporal control of gene expression at the post-transcriptional level is essential in eukaryotic cells and developing multicellular organisms. In recent years optochemical and optogenetic tools have enabled the manipulation and investigation of many steps in the involved processes. However, examples for light-mediated control of eukaryotic mRNA processing and the responsible enzymes are still rare. In particular, methylation of the 5' cap of mRNA is required for ribosome assembly, and the responsible guanine-N7 methyltransferase (MTase) from E. cuniculi (Ecm1) proved suitable for activating translation. Here, we report on a photoswitchable MTase obtained by bridging the substrate-binding cleft of Ecm1 with a tetra-ortho-methoxy-azobenzene. This azobenzene derivative is characterized by efficient trans-to-cis isomerization using red light at 615 nm. Starting from a cysteine-free Ecm1 variant (ΔCys), we used a computational approach to identify suitable conjugation sites for the azobenzene moiety. We created and characterized the four best-ranked variants, each featuring two appropriately positioned cysteines close to the substrate-binding cleft. Conjugating and crosslinking the azobenzene between C149/C155 in a designed Ecm1 variant (VAR3-Az) enabled light-dependent modulation of the MTase activity and showed a 50% higher activity for the cis form than the trans-form of the azobenzene conjugated to VAR3-Az.

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.

Identification of SUMO Binding Proteins Enriched after Covalent Photo-Cross-Linking.

Post-translational modification with the small ubiquitin-like modifier (SUMO) affects thousands of proteins in the human proteome and is implicated in numerous cellular processes. The main outcome of SUMO conjugation is a rewiring of protein-protein interactions through recognition of the modifier's surface by SUMO binding proteins. The SUMO-interacting motif (SIM) mediates binding to a groove on SUMO; however, the low affinity of this interaction and the poor conservation of SIM sequences complicates the isolation and identification of SIM proteins. To address these challenges, we have designed and biochemically characterized monomeric and multimeric SUMO-2 probes with a genetically encoded photo-cross-linker positioned next to the SIM binding groove. Following photoinduced covalent capture, even weak SUMO binders are not washed away during the enrichment procedure, and very stringent washing conditions can be applied to remove nonspecifically binding proteins. A total of 329 proteins were isolated from nuclear HeLa cell extracts and identified using mass spectrometry. We found the molecular design of our probes was corroborated by the presence of many established SUMO interacting proteins and the high percentage (>90%) of hits containing a potential SIM sequence, as predicted by bioinformatic analyses. Notably, 266 of the 329 proteins have not been previously reported as SUMO binders using traditional noncovalent enrichment procedures. We confirmed SUMO binding with purified proteins and mapped the position of the covalent cross-links for selected cases. We postulate a new SIM in MRE11, involved in DNA repair. The identified SUMO binding candidates will help to reveal the complex SUMO-mediated protein network.

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.

Photobodies: Light-Activatable Single-Domain Antibody Fragments.

Photocaged antibody fragments, termed photobodies, have been developed that are impaired in their antigen-binding capacity and can be activated by irradiation with UV light (365 nm). This rational design concept builds on the selective photocaging of a single tyrosine in a nanobody (a single-domain antibody fragment). Tyrosine is a frequently occurring residue in central positions of the paratope region. o-Nitrobenzyl-protected tyrosine variants were incorporated into four nanobodies, including examples directed against EGFR and HER2, and photodeprotection restores the native sequence. An anti-GFP photobody exhibited an at least 10 000-fold impaired binding affinity before photodeprotection compared with the parent nanobody. A bispecific nanobody-photobody fusion protein was generated to trigger protein heterodimerization by light. Photoactivatable antibodies are expected to become versatile protein reagents and to enable novel approaches in diagnostic and therapeutic applications.

Expanding the genetic code with a lysine derivative bearing an enzymatically removable phenylacetyl group.

We report the genetically encoded incorporation of phenylacetyl protected lysine (PacK) into proteins in Escherichia coli. This unnatural side-chain modification can be enzymatically removed using either penicillin G acylase (PGA) or, surprisingly, the sirtuin SrtN from Bacillus subtilis. Our approach expands the toolbox to reversibly control protein structure and function under very mild and non-denaturing conditions, as demonstrated by triggering the activity of the nonribosomal peptide synthetase GrsA.

Light-control of the ultra-fast Gp41-1 split intein with preserved stability of a genetically encoded photo-caged amino acid in bacterial cells.

Inteins change the structure and function of their host protein in a unique way and the Gp41-1 split intein is the fastest protein trans-splicing intein known to date. To design a photo-activatable variant, we have incorporated ortho-nitrobenzyl-tyrosine (ONBY) at the position of a structurally conserved phenylalanine in the Gp41-1-N fragment. Using irradiation at 365 nm, the splicing reaction was triggered with virtually unchanged rates. The partial cellular reduction of the nitro group in ONBY, previously observed during bacterial protein expression for several photo-caged amino acids, was overcome by periplasmatic expression and by using an E. coli K12(DE3) strain instead of BL21(DE3). Together, our findings provide new tools for the artificial photo-control of proteins.

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.

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.

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.

Mapping of the Communication-Mediating Interface in Nonribosomal Peptide Synthetases Using a Genetically Encoded Photocrosslinker Supports an Upside-Down Helix-Hand Motif.

Nonribosomal peptide synthetases (NRPSs) are large modular protein templates that assemble bioactive peptides, many of which possess therapeutic importance. Protein-protein interactions between subunits of bacterial NRPSs are essential for proper template formation. The structural basis of the typical subunit interface between epimerization (E) and condensation domains is only poorly understood. Conflicting helix-helix and helix-hand models were previously proposed. Here, the genetically encoded photocrosslinker p-benzoylphenylalanine (BpF) was incorporated into the C-terminal communication-mediating domain (COM) of GrsA. Using the partner elongation module TycB1 to form a dipeptide product, we could correlate the ability to form covalent crosslinks with the functional module interaction. Perturbation of the module interaction with the large side chain of BpF in a scan at 19 positions demonstrated the importance of three hydrophobic residues in an α-helical arrangement. Mapping of covalent crosslinks using tandem mass spectrometry revealed the residues from the interior of the condensation domain as part of the protein interface; a finding not predicted by the helix-helix model. The epimerization domain of GrsA was found to be important for the interaction. Together with multiple sequence analyses and structural modeling, our results suggest an upside-down helix-hand model in which the C-terminal COM-helix is embedded in a hand motif with a hydrophobic core in a reversed orientation compared to a previous proposal. Our results provide a more detailed and the first direct structural understanding of the COM domain interaction and will contribute to successful biocombinatorial engineering attempts in the design of artificial NRPS templates.

Measuring interference of drug-like molecules with the respiratory chain: toward the early identification of mitochondrial uncouplers in lead finding.

The electron transport chain (ETC) couples electron transfer between donors and acceptors with proton transport across the inner mitochondrial membrane. The resulting electrochemical proton gradient is used to generate chemical energy in the form of adenosine triphosphate (ATP). Proton transfer is based on the activity of complex I-V proteins in the ETC. The overall electrical activity of these proteins can be measured by proton transfer using Solid Supported Membrane technology. We tested the activity of complexes I, III, and V in a combined assay, called oxidative phosphorylation assay (oxphos assay), by activating each complex with the corresponding substrate. The oxphos assay was used to test in-house substances from different projects and several drugs currently available on the market that have reported effects on mitochondrial functions. The resulting data were compared to the influence of the respective compounds on mitochondria as determined by oxygen consumption and to data generated with an ATP depletion assay. The comparison shows that the oxidative phosphorylation assay provides both a rapid approach for detecting interaction of compounds with respiratory chain proteins and information on their mode of interaction. Therefore, the oxphos assay is a useful tool to support structure activity relationship studies by allowing early identification of mitotoxicity and for analyzing the outcome of phenotypic screens that are susceptible to the generation of mitotoxicity-related artifacts.

An automatic electrophysiological assay for the neuronal glutamate transporter mEAAC1.

A rapid and robust electrophysiological assay based on solid supported membranes (SSM) for the murine neuronal glutamate transporter mEAAC1 is presented. Measurements at different concentrations revealed the EAAC1 specific affinities for l-glutamate (K(m)=24microM), l-aspartate (K(m)=5microM) and Na(+) (K(m)=33mM) and an inhibition constant K(i) for dl-threo-beta-benzyloxyaspartic acid (TBOA) of 1microM. Inhibition by 3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid (HIP-B) was not purely competitive with an IC(50) of 13microM. Experiments using SCN(-) concentration jumps yielded large transient currents in the presence of l-glutamate showing the characteristics of the glutamate-gated anion conductance of EAAC1. Thus, SSM-based electrophysiology allows the analysis of all relevant transport modes of the glutamate transporter on the same sample. K(+) and Na(+) gradients could be applied to the transporter. Experiments in the presence and absence of Na(+) and K(+) gradients demonstrated that the protein is still able to produce a charge translocation when no internal K(+) is present. In this case, the signal amplitude is smaller and a lower apparent affinity for l-glutamate of 144microM is found. Finally the assay was adapted to a commercial fully automatic system for SSM-based electrophysiology and was validated by determining the substrate affinities and inhibition constants as for the laboratory setup. The combination of automatic function and its ability to monitor all transport modes of EAAC1 make this system an universal tool for industrial drug discovery.

Transporter assays using solid supported membranes: a novel screening platform for drug discovery.

Transporters are important targets in drug discovery. However, high throughput-capable assays for this class of membrane proteins are still missing. Here we present a novel drug discovery platform technology based on solid supported membranes. The functional principles of the technology are described, and a sample selection of transporter assays is discussed: the H(+)-dependent peptide transporter PepT1, the gastric proton pump, and the Na(+)/Ca(2+) exchanger. This technology promises to have an important impact on the drug discovery process.