Structure of Staphylococcus aureus ClpP Bound to the Covalent Active-Site Inhibitor Cystargolide A.

The caseinolytic protease is a highly conserved serine protease, crucial to prokaryotic and eukaryotic protein homeostasis, and a promising antibacterial and anticancer drug target. Herein, we describe the potent cystargolides as the first natural ß-lactone inhibitors of the proteolytic core ClpP. Based on the discovery of two clpP genes next to the cystargolide biosynthetic gene cluster in Kitasatospora cystarginea, we explored ClpP as a potential cystargolide target. We show the inhibition of Staphylococcus aureus ClpP by cystargolide A and B by different biochemical methods in vitro. Synthesis of semisynthetic derivatives and probes with improved cell penetration allowed us to confirm ClpP as a specific target in S. aureus cells and to demonstrate the anti-virulence activity of this natural product class. Crystal structures show cystargolide A covalently bound to all 14 active sites of ClpP from S. aureus, Aquifex aeolicus, and Photorhabdus laumondii, and reveal the molecular mechanism of ClpP inhibition by ß-lactones, the predominant class of ClpP inhibitors.

Characterization of the cystargolide biosynthetic gene cluster and functional analysis of the methyltransferase CysG.

Cystargolides are natural products originally isolated from Kitasatospora cystarginea NRRL B16505 as inhibitors of the proteasome. They are composed of a dipeptide backbone linked to a ß-lactone warhead. Recently, we identified the cystargolide biosynthetic gene cluster, but systematic genetic analyses had not been carried out because of the lack of a heterologous expression system. Here, we report the discovery of a homologous cystargolide biosynthetic pathway in Streptomyces durhamensis NRRL-B3309 by genome mining. The gene cluster was cloned via transformation-associated recombination and heterologously expressed in Streptomyces coelicolor M512. We demonstrate that it contains all genes necessary for the production of cystargolide A and B. Single gene deletion experiments reveal that only five of the eight genes from the initially proposed gene cluster are essential for cystargolide synthesis. Additional insights into the cystargolide pathway could be obtained from in vitro assays with CysG and chemical complementation of the respective gene knockout. This could be further supported by the in vitro investigation of the CysG homolog BelI from the belactosin biosynthetic gene cluster. Thereby, we confirm that CysG and BelI catalyze a cryptic SAM-dependent transfer of a methyl group that is critical for the construction of the cystargolide and belactosin ß-lactone warheads.

Structural and Biochemical Insights into Post-Translational Arginine-to-Ornithine Peptide Modifications by an Atypical Arginase.

Landornamide A is a ribosomally synthesized and post-translationally modified peptide (RiPP) natural product with antiviral activity. Its biosynthetic gene cluster encodes─among other maturases─the peptide arginase OspR, which converts arginine to ornithine units in an unusual post-translational modification. Peptide arginases are a recently discovered RiPP maturase family with few characterized representatives. They show little sequence similarity to conventional arginases, a well-characterized enzyme family catalyzing the hydrolysis of free arginine to ornithine and urea. Peptide arginases are highly promiscuous and accept a variety of substrate sequences. The molecular basis for binding the large peptide substrate and for the high promiscuity of peptide arginases remains unclear. Here, we report the first crystal structure of a peptide arginase at a resolution of 2.6 Å. The three-dimensional structure reveals common features and differences between conventional arginases and the peptide arginase: the binuclear metal cluster and the active-site environment strongly resemble each other, while the quaternary structures diverge. Kinetic analyses of OspR with various substrates provide new insights into the order of biosynthetic reactions during the post-translational maturation of landornamide A. These results provide the basis for pathway engineering to generate derivatives of landornamide A and for the general application of peptide arginases as biosynthetic tools for peptide engineering.

Structural insights into cooperative DNA recognition by the CCAAT-binding complex and its bZIP transcription factor HapX.

The heterotrimeric CCAAT-binding complex (CBC) is a fundamental eukaryotic transcription factor recognizing the CCAAT box. In certain fungi, like Aspergilli, the CBC cooperates with the basic leucine zipper HapX to control iron metabolism. HapX functionally depends on the CBC, and the stable interaction of both requires DNA. To study this cooperative effect, X-ray structures of the CBC-HapX-DNA complex were determined. Downstream of the CCAAT box, occupied by the CBC, a HapX dimer binds to the major groove. The leash-like N terminus of the distal HapX subunit contacts the CBC, and via a flexible polyproline type II helix mediates minor groove interactions that stimulate sequence promiscuity. In vitro and in vivo mutagenesis suggest that the structural and functional plasticity of HapX results from local asymmetry and its ability to target major and minor grooves simultaneously. The latter feature may also apply to related transcription factors such as yeast Hap4 and distinct Yap family members.

A Nut for Every Bolt: Subunit-Selective Inhibitors of the Immunoproteasome and Their Therapeutic Potential.

At the heart of the ubiquitin-proteasome system, the 20S proteasome core particle (CP) breaks down the majority of intracellular proteins tagged for destruction. Thereby, the CP controls many cellular processes including cell cycle progression and cell signalling. Inhibitors of the CP can suppress these essential biological pathways, resulting in cytotoxicity, an effect that is beneficial for the treatment of certain blood cancer patients. During the last decade, several preclinical studies demonstrated that selective inhibition of the immunoproteasome (iCP), one of several CP variants in mammals, suppresses autoimmune diseases without inducing toxic side effects. These promising findings led to the identification of natural and synthetic iCP inhibitors with distinct chemical structures, varying potency and subunit selectivity. This review presents the most prominent iCP inhibitors with respect to possible scientific and medicinal applications, and discloses recent trends towards pan-immunoproteasome reactive inhibitors that cumulated in phase II clinical trials of the lead compound KZR-616 for chronic inflammations.

Structural and Mechanistic Insights into C-S Bond Formation in Gliotoxin.

Glutathione-S-transferases (GSTs) usually detoxify xenobiotics. The human pathogenic fungus Aspergillus fumigatus however uses the exceptional GST GliG to incorporate two sulfur atoms into its virulence factor gliotoxin. Because these sulfurs are essential for biological activity, glutathionylation is a key step of gliotoxin biosynthesis. Yet, the mechanism of carbon-sulfur linkage formation from a bis-hydroxylated precursor is unresolved. Here, we report structures of GliG with glutathione (GSH) and its reaction product cyclo[-l-Phe-l-Ser]-bis-glutathione, which has been purified from a genetically modified A. fumigatus strain. The structures argue for stepwise processing of first the Phe and second the Ser moiety. Enzyme-mediated dehydration of the substrate activates GSH and a helix dipole stabilizes the resulting anion via a water molecule for the nucleophilic attack. Activity assays with mutants validate the interactions of GliG with the ligands and enrich our knowledge about enzymatic C-S bond formation in gliotoxin and epipolythiodioxopiperazine (ETP) natural compounds in general.

Activation, Structure, Biosynthesis and Bioactivity of Glidobactin-like Proteasome Inhibitors from Photorhabdus laumondii.

The glidobactin-like natural products (GLNPs) glidobactin A and cepafungin I have been reported to be potent proteasome inhibitors and are regarded as promising candidates for anticancer drug development. Their biosynthetic gene cluster (BGC) plu1881-1877 is present in entomopathogenic Photorhabdus laumondii but silent under standard laboratory conditions. Here we show the largest subset of GLNPs, which are produced and identified after activation of the silent BGC in the native host and following heterologous expression of the BGC in Escherichia coli. Their chemical diversity results from a relaxed substrate specificity and flexible product release in the assembly line of GLNPs. Crystal structure analysis of the yeast proteasome in complex with new GLNPs suggests that the degree of unsaturation and the length of the aliphatic tail are critical for their bioactivity. The results in this study provide the basis to engineer the BGC for the generation of new GLNPs and to optimize these natural products resulting in potential drugs for cancer therapy.

Legionella effector AnkX displaces the switch II region for Rab1b phosphocholination.

The causative agent of Legionnaires disease, Legionella pneumophila, translocates the phosphocholine transferase AnkX during infection and thereby posttranslationally modifies the small guanosine triphosphatase (GTPase) Rab1 with a phosphocholine moiety at S76 using cytidine diphosphate (CDP)-choline as a cosubstrate. The molecular basis for Rab1 binding and enzymatic modification have remained elusive because of lack of structural information of the low-affinity complex with AnkX. We combined thiol-reactive CDP-choline derivatives with recombinantly introduced cysteines in the AnkX active site to covalently capture the heterocomplex. The resulting crystal structure revealed that AnkX induces displacement of important regulatory elements of Rab1 by placing a ß sheet into a conserved hydrophobic pocket, thereby permitting phosphocholine transfer to the active and inactive states of the GTPase. Together, the combination of chemical biology and structural analysis reveals the enzymatic mechanism of AnkX and the family of filamentation induced by cyclic adenosine monophosphate (FIC) proteins.

Fatal amyloid formation in a patient's antibody light chain is caused by a single point mutation.

In systemic light chain amyloidosis, an overexpressed antibody light chain (LC) forms fibrils which deposit in organs and cause their failure. While it is well-established that mutations in the LC's VL domain are important prerequisites, the mechanisms which render a patient LC amyloidogenic are ill-defined. In this study, we performed an in-depth analysis of the factors and mutations responsible for the pathogenic transformation of a patient-derived λ LC, by recombinantly expressing variants in E. coli. We show that proteolytic cleavage of the patient LC resulting in an isolated VL domain is essential for fibril formation. Out of 11 mutations in the patient VL, only one, a leucine to valine mutation, is responsible for fibril formation. It disrupts a hydrophobic network rendering the C-terminal segment of VL more dynamic and decreasing domain stability. Thus, the combination of proteolytic cleavage and the destabilizing mutation trigger conformational changes that turn the LC pathogenic.

Amyloid light chain amyloidosis, shortened to AL amyloidosis, is a rare and often fatal disease. It is caused by a disorder of the bone marrow. Usually, cells in the bone marrow produce Y-shaped proteins called antibodies to fight infections. In AL amyloidosis, these cells release too much of the short arm of the antibody, known as its light chain, and the light chains also carry mutations. The antibodies are no longer able to assemble properly, and instead misfold and form structures, known as amyloid fibrils. The fibrils build up outside the cells, gradually causing damage to tissues and organs that can lead to life-threatening organ failure. Due to the rareness of the disease, diagnosis is often overlooked and delayed. People experience widely varying symptoms, depending on the organs affected. Also, given the diversity of antibodies people make, every person with AL amyloidosis has a variety of mutations implicated in their disease. It is thought that mutations in the antibody light chain make it unstable and prone to misfolding, but it remains unclear which specific mutations trigger a cascade of amyloid fibril formation. Now, Kazman et al. have pinpointed the exact mechanism in one case of the disease. First, tissue biopsies from a woman with advanced AL amyloidosis were analyzed, and the defunct antibody light chain was isolated. Eleven mutations were identified in the antibody light chain, only one of which was found to be responsible for the formation of the harmful fibrils. The next step was to determine how this one small change was so damaging. The experiments showed that after the antibody light chain was cut in two, a process that happens naturally in the body, this single mutation transforms it into a protein capable of causing disease. In this 'bedside to lab bench' study, Kazman et al. have succeeded in determining the molecular origin of one case of AL amyloidosis. The results have also shown that the instability of antibodies due to mutation does not alone explain the formation of amyloid fibrils in this disease and that the cutting of this protein in two is also important. It is hoped that, in the long run, this work will lead to new diagnostics and treatment options for people with AL amyloidosis.

Structures in Tetrahydrofolate Methylation in Desulfitobacterial Glycine Betaine Metabolism at Atomic Resolution.

Enzymes orchestrating methylation between tetrahydrofolate (THF) and cobalamin (Cbl) are abundant among all domains of life. During energy production in Desulfitobacterium hafniense, MtgA catalyzes the methyl transfer from methylcobalamin (Cbl-CH3 ) to THF in the catabolism of glycine betaine (GB). Despite its lack of sequence identity with known structures, we could show that MtgA forms a homodimeric complex of two TIM barrels. Atomic crystallographic insights into the interplay of MtgA with THF as well as analysis of a trapped reaction intermediate (THF-CH3 )+ reveal conformational rearrangements during the transfer reaction. Whereas residues for THF methylation are conserved, the binding mode for the THF glutamyl-p-aminobenzoate moiety (THF tail) is unique. Apart from snapshots of individual reaction steps of MtgA, structure-based mutagenesis combined with enzymatic activity assays allowed a mechanistic description of the methyl transfer between Cbl-CH3 and THF. Altogether, the THF-tail-binding motion observed in MtgA is unique compared to other THF methyltransferases and therefore contributes to the general understanding of THF-mediated methyl transfer.

Structure-Based Design of Inhibitors Selective for Human Proteasome ß2c or ß2i Subunits.

Subunit-selective proteasome inhibitors are valuable tools to assess the biological and medicinal relevance of individual proteasome active sites. Whereas the inhibitors for the ß1c, ß1i, ß5c, and ß5i subunits exploit the differences in the substrate-binding channels identified by X-ray crystallography, compounds selectively targeting ß2c or ß2i could not yet be rationally designed because of the high structural similarity of these two subunits. Here, we report the development, chemical synthesis, and biological screening of a compound library that led to the identification of the ß2c- and ß2i-selective compounds LU-002c (4; IC50 ß2c: 8 nM, IC50 ß2i/ß2c: 40-fold) and LU-002i (5; IC50 ß2i: 220 nM, IC50 ß2c/ß2i: 45-fold), respectively. Co-crystal structures with ß2 humanized yeast proteasomes visualize protein-ligand interactions crucial for subunit specificity. Altogether, organic syntheses, activity-based protein profiling, yeast mutagenesis, and structural biology allowed us to decipher significant differences of ß2 substrate-binding channels and to complete the set of subunit-selective proteasome inhibitors.

Discovery of the First-in-Class Dual Histone Deacetylase-Proteasome Inhibitor.

Dual- or multitarget drugs have emerged as a promising alternative to combination therapies. Proteasome inhibitors (PIs) possess synergistic activity with histone deacetylase (HDAC) inhibitors due to the simultaneous blockage of the ubiquitin degradation and aggresome pathways. Here, we present the design, synthesis, binding modes, and anticancer properties of RTS-V5 as the first-in-class dual HDAC-proteasome ligand. The inhibition of both targets was confirmed by biochemical and cellular assays as well as X-ray crystal structures of the 20S proteasome and HDAC6 complexed with RTS-V5. Cytotoxicity assays with leukemia and multiple myeloma cell lines as well as therapy refractory primary patient-derived leukemia cells demonstrated that RTS-V5 possesses potent and selective anticancer activity. Our results will thus guide the structure-based optimization of dual HDAC-proteasome inhibitors for the treatment of hematological malignancies.

Iron Scavenging in Aspergillus Species: Structural and Biochemical Insights into Fungal Siderophore Esterases.

Fungi utilize high-affinity chelators termed siderophores with chemically diverse structures to scavenge the essential nutrient iron from their surroundings. Since they are among the strongest known Fe3+ binding agents, intracellular release of the heavy metal atom is facilitated by the activity of specific hydrolases. In this work, we report the characterization and X-ray crystal structures of four siderophore esterases: AfEstB and AfSidJ from Aspergillus fumigatus, as well as AnEstB and AnEstA from Aspergillus nidulans. Even though they all display the conserved α/ß-hydrolase fold, we found significant structural and enzymatic discrepancies in their adaption to both related and chemically diverse substrates. A structure of AfEstB in complex with its substrate triacetylfusarinine C gives insight into the active enzyme and shows tetrahedral coordination between the catalytic serine and the scissile ester bond.

Design, synthesis, and evaluation of cystargolide-based ß-lactones as potent proteasome inhibitors.

The peptidic ß-lactone proteasome inhibitors (PIs) cystargolides A and B were used to conduct structure-activity relationship (SAR) studies in order to assess their anticancer potential. A total of 24 different analogs were designed, synthesized and evaluated for proteasome inhibition, for cytotoxicity towards several cancer cell lines, and for their ability to enter intact cells. X-ray crystallographic analysis and subunit selectivity was used to determine the specific subunit binding associated with the structural modification of the ß-lactone (P1), peptidic core, (Px and Py), and end-cap (Pz) of our scaffold. The cystargolide derivative 5k, structurally unique at both Py and P1, exhibited the most promising inhibitory activity for the ß5 subunit of human proteasomes (IC50 = 3.1 nM) and significant cytotoxicity towards MCF-7 (IC50 = 416 nM), MDA-MB-231 (IC50 = 74 nM) and RPMI 8226 (IC50 = 41 nM) cancer cell lines. Cellular infiltration assays revealed that minor structural modifications have significant effects on the ability of our PIs to inhibit intracellular proteasomes, and we identified 5k as a promising candidate for continued therapeutic studies. Our novel drug lead 5k is a more potent proteasome inhibitor than carfilzomib with mid-to-low nanomolar IC50 measurements and it is cytotoxic against multiple cancer cell lines at levels approaching those of carfilzomib.

(-)-Homosalinosporamide A and Its Mode of Proteasome Inhibition: An X-ray Crystallographic Study.

Upon acylation of the proteasome by the ß-lactone inhibitor salinosporamide A (SalA), tetrahydrofuran formation occurs by intramolecular alkylation of the incipient alkoxide onto the choroethyl sidechain and irreversibly blocks the active site. Our previously described synthetic approach to SalA, utilizing a bioinspired, late-stage, aldol-ß-lactonization strategy to construct the bicyclic ß-lactone core, enabled synthesis of (⁻)-homosalinosporamide A (homoSalA). This homolog was targeted to determine whether an intramolecular tetrahydropyran is formed in a similar manner to SalA. Herein, we report the X-ray structure of the yeast 20S proteasome:homoSalA-complex which reveals that tetrahydropyran ring formation does not occur despite comparable potency at the chymotrypsin-like active site in a luminogenic enzyme assay. Thus, the natural product derivative homoSalA blocks the proteasome by a covalent reversible mode of action, opening the door for further fine-tuning of proteasome inhibition.

The protease GtgE from Salmonella exclusively targets inactive Rab GTPases.

Salmonella infections require the delivery of bacterial effectors into the host cell that alter the regulation of host defense mechanisms. The secreted cysteine protease GtgE from S. Typhimurium manipulates vesicular trafficking by modifying the Rab32 subfamily via cleaving the regulatory switch I region. Here we present a comprehensive biochemical, structural, and computational characterization of GtgE in complex with Rab32. Interestingly, GtgE solely processes the inactive GDP-bound GTPase. The crystal structure of the Rab32:GDP substrate in complex with the inactive mutant GtgEC45A reveals the molecular basis of substrate recognition. In combination with atomistic molecular dynamics simulations, the structural determinants for protein and activity-state specificity are identified. Mutations in a central interaction hub lead to loss of the strict GDP specificity. Our findings shed light on the sequence of host cell manipulation events during Salmonella infection and provide an explanation for the dependence on the co-secreted GTPase activating protein SopD2.

A Predictive Approach for the Optical Control of Carbonic Anhydrase II Activity.

Optogenetics and photopharmacology are powerful approaches to investigating biochemical systems. While the former is based on genetically encoded photoreceptors that utilize abundant chromophores, the latter relies on synthetic photoswitches that are either freely diffusible or covalently attached to specific bioconjugation sites, which are often native or engineered cysteines. The identification of suitable cysteine sites and appropriate linkers for attachment is generally a lengthy and cumbersome process. Herein, we describe an in silico screening approach that is designed to propose a small number of optimal combinations. By applying this computational approach to human carbonic anhydrase and a set of three photochromic tethered ligands, the number of potential site-ligand combinations was narrowed from over 750 down to 6, which we then evaluated experimentally. Two of these six combinations resulted in light-responsive human Carbonic Anhydrases (LihCAs), which were characterized with enzymatic activity assays, mass spectrometry, and X-ray crystallography. Our study also provides insights into the reactivity of cysteines toward maleimides and the hydrolytic stability of the adducts obtained.

Common Fibril Structures Imply Systemically Conserved Protein Misfolding Pathways In†Vivo.

Systemic amyloidosis is caused by the misfolding of a circulating amyloid precursor protein and the deposition of amyloid fibrils in multiple organs. Chemical and biophysical analysis of amyloid fibrils from human AL and murine AA amyloidosis reveal the same fibril morphologies in different tissues or organs of one patient or diseased animal. The observed structural similarities concerned the fibril morphology, the fibril protein primary and secondary structures, the presence of post-translational modifications and, in case of the AL fibrils, the partially folded characteristics of the polypeptide chain within the fibril. Our data imply for both analyzed forms of amyloidosis that the pathways of protein misfolding are systemically conserved; that is, they follow the same rules irrespective of where inside one body fibrils are formed or accumulated.

Structural Elucidation of a Nonpeptidic Inhibitor Specific for the Human Immunoproteasome.

Selective inhibition of the immunoproteasome is a promising approach towards the development of immunomodulatory drugs. Recently, a class of substituted thiazole compounds that combine a nonpeptidic scaffold with the absence of an electrophile was reported in a patent. Here, we investigated the mode of action of the lead compound by using a sophisticated chimeric yeast model of the human immunoproteasome for structural studies. The inhibitor adopts a unique orientation perpendicular to the ß5i substrate-binding channel. Distinct interactions between the inhibitor and the subpockets of the human immunoproteasome account for its isotype selectivity.

The CCAAT-binding complex (CBC) in Aspergillus species.

BACKGROUND: The CCAAT binding complex (CBC), consisting of a heterotrimeric core structure, is highly conserved in eukaryotes and constitutes an important general transcriptional regulator. Scope of the review. In this review we discuss the scientific history and the current state of knowledge of the multiple gene regulatory functions, protein motifs and structure of the CBC in fungi with a special focus on Aspergillus species. Major conclusions and general significance. Initially identified as a transcriptional activator of respiration in Saccharomyces cerevisiae, in other fungal species the CBC was found to be involved in highly diverse pathways, but a general rationale for its involvement was missing. Subsequently, the CBC was found to sense reactive oxygen species through oxidative modifications of cysteine residues in order to mediate redox regulation. Moreover, via interaction with the iron-sensing bZIP transcription factor HapX, the CBC was shown to mediate adaptation to both iron starvation and iron excess. Due to the control of various pathways in primary and secondary metabolism the CBC is of crucial importance for fungal virulence in both animal and plant hosts as well as antifungal resistance. Consequently, CBC-mediated control affects biological processes that are of high interest in biotechnology, agriculture and infection medicine. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani.