The Structure of Ca2+ Sensor Case16 Reveals the Mechanism of Reaction to Low Ca2+ Concentrations

Here we report the first crystal structure of a high-contrast genetically encoded circularly permuted green fluorescent protein (cpGFP)-based Ca2+ sensor, Case16, in the presence of a low Ca2+ concentration. The structure reveals the positioning of the chromophore within Case16 at the first stage of the Ca2+-dependent response when only two out of four Ca2+-binding pockets of calmodulin (CaM) are occupied with Ca2+ ions. In such a “half Ca2+-bound state”, Case16 is characterized by an incomplete interaction between its CaM-/M13-domains. We also report the crystal structure of the related Ca2+ sensor Case12 at saturating Ca2+ concentration. Based on this structure, we postulate that cpGFP-based Ca2+ sensors can form non-functional homodimers where the CaM-domain of one sensor molecule binds symmetrically to the M13-peptide of the partner sensor molecule. Case12 and Case16 behavior upon addition of high concentrations of free CaM or M13-peptide reveals that the latter effectively blocks the fluorescent response of the sensor. We speculate that the demonstrated intermolecular interaction with endogenous substrates and homodimerization can impede proper functioning of this type of Ca2+ sensors in living cells

New methods for efficient protein production in drug discovery.

The requirement for high levels of stable and functional proteins remains a bottleneck in many processes of modern drug discovery, including the high-throughput screening for novel active compounds or the determination of protein structures. Recently, numerous developments have been made to improve the production of soluble and active proteins in heterologous expression systems. These include versatile expression vectors, new methods for quick cloning, the introduction of novel and/or improved prokaryotic and eukaryotic expression systems, and more efficient and faster chromatographic procedures that result in highly pure proteins. In addition, several techniques allow the attachment of small molecular labels to proteins in a site-specific manner, which can be highly useful for various important experimental techniques in current drug discovery

Evaluation of two novel tag-based labelling technologies for site-specific modification of proteins.

Modern drug discovery strongly depends on the availability of target proteins in sufficient amounts and with desired properties. For some applications, proteins have to be produced with specific modifications such as tags for protein purification, fluorescent or radiometric labels for detection, glycosylation and phosphorylation for biological activity, and many more. It is well known that covalent modifications can have adverse effects on the biological activity of some target proteins. It is therefore one of the major challenges in protein chemistry to generate covalent modifications without affecting the biological activity of the target protein. Current procedures for modification mostly rely on non-specific labelling of lysine or cysteine residues on the protein of interest, but alternative approaches dedicated to site-specific protein modification are being developed and might replace most of the commonly used methodologies. In this study, we investigated two novel methods where target proteins can be expressed in E. coli with a fusion partner that allows protein modification in a covalent and highly selective manner. Firstly, we explored a method based on the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (hAGT) as a fusion tag for site-directed attachment of small molecules. The AGT-tag (SNAP-tag) can accept almost any chemical moiety when it is attached to the guanine base through a benzyl group. In our experiments we were able to label a target protein fused to the AGT-tag with various fluorophores coupled to O6-benzylguanine. Secondly, we tested in vivo and in vitro site-directed biotinylation with two different tags, consisting of either 15 (AviTag) or 72 amino acids (BioEase tag), which serve as a substrate for bacterial biotin ligase birA. When birA protein was co-expressed in E. coli biotin was incorporated almost completely into a model protein which carried these recognition tags at its C-terminus. The same findings were also obtained with in vitro biotinylation assays using pure birA independently over-expressed in E. coli and added to the biotinylation reaction in the test tube. For both biotinylation methods, peptide mapping and LC-MS proved the highly site-specific modification of the corresponding tags. Our results indicate that these novel site-specific labelling reactions work in a highly efficient manner, allow almost quantitative labelling of the target proteins, have no deleterious effect on the biological activity and are easy to perform in standard laboratories

HDL-like discs for assaying membrane proteins in drug discovery

To broaden the use of the recombinant high-density lipoprotein (rHDL) approach to the characterization of lead compounds, we investigated the pharmacology of the human beta-2-adrenoceptor in nanolipid bilayers (rHDL) with a broad set of beta-adrenoceptor antagonists. To that end, we developed a homogeneous copper-chelate scintillation proximity binding assay (SPA) in order to compare receptor-ligand binding affinities before and after reconstitution into rHDLs. Our results clearly suggest that the receptor in rHDLs display the same pharmacology than in cell membranes and that rHDLs can be used not only to measure affinities for a range of ligands but also to study binding kinetics

Structural basis of species-selective antagonist binding to the succinate receptor

The tricarboxylic acid cycle intermediate succinate is involved in metabolic processes and plays a crucial role in the homeostasis of mitochondrial reactive oxygen species. The receptor responsible for succinate signalling, SUCNR1 (also known as GPR91), is a member of the G-protein-coupled-receptor family and links succinate signalling to renin-induced hypertension, retinal angiogenesis and inflammation. Because SUCNR1 senses succinate as an immunological danger signal—which has relevance for diseases including ulcerative colitis, liver fibrosis, diabetes and rheumatoid arthritis—it is of interest as a therapeutic target. Here we report the high-resolution crystal structure of rat SUCNR1 in complex with an intracellular binding nanobody in the inactive conformation. Structure-based mutagenesis and radioligand-binding studies, in conjunction with molecular modelling, identified key residues for speciesselective antagonist binding and enabled the determination of the high-resolution crystal structure of a humanized rat SUCNR1 in complex with a high-affinity, humanselective antagonist denoted NF-56-EJ40. We anticipate that these structural insights into the architecture of the succinate receptor and its antagonist selectivity will enable structure-based drug discovery and will further help to elucidate the function of SUCNR1 in vitro and in vivo

Synthesis and characterization of fluorescent ubiquitin derivatives as highly sensitive substrates for the deubiquitinating enzymes UCH-L3 and USP-2.

Deubiquitinating enzymes (DUBs) catalyze the removal of attached ubiquitin molecules from amino groups of target proteins. The large family of DUBs plays an important role in the regulation of the intracellular homeostasis of different proteins and influences therefore key events such as cell division, apoptosis, etc. The DUB family members UCH-L3 and USP2 are believed to inhibit the degradation of various tumor-growth-promoting proteins by removing the trigger for degradation. Inhibitors of these enzymes should therefore lead to enhanced degradation of oncoproteins and may thus stop tumor growth. To develop an enzymatic assay for the search of UCH-L3 and USP2 inhibitors, C-terminally labeled ubiquitin substrates were enzymatically synthesized. We have used the ubiquitin-activating enzyme E1 and one of the ubiquitin-conjugating enzymes E2 to attach a fluorescent lysine derivative to the C terminus of ubiquitin. Since only the epsilon-NH(2) group of the lysine derivatives was free and reactive, the conjugates closely mimic the isopeptide bond between the ubiquitin and the lysine side chains of the targeted proteins. Various substrates were synthesized by this approach and characterized enzymatically with the two DUBs. The variant consisting of the fusion protein between the large N-terminal NusA tag and the ubiquitin which was modified with alpha-NH(2)-tetramethylrhodamin-lysine, was found to give the highest dynamic range in a fluorescence polarization readout. Therefore we have chosen this substrate for the development of a miniaturized, fluorescence-polarization-based high-throughput screening assay

Structural determinants of MALT1 protease activity

The formation of the Carma1-BCL10-MALT1 complex is pivotal for T-cell receptor mediated activation of the transcription-factor NF-κB. Recently it was shown that in this complex MALT1 not only acts as a scaffolding protein but also possesses proteolytic activity mediated by its caspase-like domain. Here we show that, as described for caspases which play an essential role in apoptosis, proteolytic activity of MALT1 can be reconstituted by induced proximity. However, unlike caspases which cleave specifically C-terminal to an aspartate, MALT1 has an absolute specificity for arginine. We further determined the structure of the MALT1 domains responsible for proteolytic activity in the absence of ligand and in complex with a covalently bound substrate like inhibitor. The structures explain the specificity for cleavage after arginine, and show that the protease domain undergoes large conformational changes upon substrate binding that also affect the structure of the C-terminal Ig-like domain. This C-terminal Ig-like domain is required for MALT1 activity and likely important for the interaction of MALT1 with its binding partners

Gift from nature : cyclomarin s kills Mycobacteria and malaria parasites by distinct modes of action

Malaria continues to be one of the most devastating human diseases despite many efforts to limit its spread by prevention of infection or by pharmaceutical treatment of patients. We have conducted a screen for antiplasmodial compounds by using a natural product library. Here we report on cyclomarin A as a potent growth inhibitor of Plasmodium falciparum and the identification of its molecular target, diadenosine triphosphate hydrolase (PfAp3Aase), by chemical proteomics. Using a biochemical assay, we could show that cyclomarin A is a specific inhibitor of the plasmodial enzyme but not of the closest human homologue hFHIT. Co-crystallisation experiments demonstrate a unique binding mode of the inhibitor. One molecule of cyclomarin A binds a dimeric PfAp3Aase and prevents the formation of the enzyme⋅substrate complex. These results validate PfAp3Aase as a new drug target for the treatment of malaria. We have previously elucidated the structurally unrelated regulatory subunit ClpC1 of the ClpP protease as the molecular target of cyclomarin A in Mycobacterium tuberculosis. Thus, cyclomarin A is a rare example of a natural product with two distinct and specific modes of action

A novel HERC4-dependent glue degrader targeting STING

Stimulator of interferon genes (STING) is a central component of the pathway sensing the presence of cytosolic nucleic acids, having a key role in type I interferon innate immune response. Localized at the endoplasmic reticulum (ER), STING becomes activated by cGAMP, which is generated by the intracellular DNA sensor cyclic GMP-AMP synthase (cGAS). Due to its critical role in physiological function and its’ involvement in a variety of diseases, STING has been a notable focus for drug discovery. Recent advances in drug discovery allow the targeting of proteins previously considered “un-druggable” by novel mechanism of actions. Molecular glue degraders are defined as the compounds leading targeted protein degradation (TPD) by creating novel ligase-substrate interactions. Here, we identified AK59 as a novel molecular glue degrader for STING. A genome-wide, CRISPR/Cas9 knockout screen showed that the compound-mediated degradation of STING by AK59 is compromised by the loss of HECT and RLD domain containing E3 ubiquitin protein ligase 4 (HERC4), ubiquitin-like modifier activating enzyme 5 (UBA5) and ubiquitin like modifier activating enzyme 6 (UBA6). While UBA5 and UBA6 could be the auxiliary factors for AK59 activity, our results indicate that HERC4 is the main E3 ligase for the observed degradation mechanism. Validation by individual CRISPR knockouts, co-immunoprecipitations, as well as proximity mediated reporter assays suggested that AK59 functions as a glue degrader by forming a novel interaction between STING and HERC4. Furthermore, our data reveals that AK59 was effective on the most common pathological STING mutations that cause STING-associated vasculopathy with onset in infancy (SAVI), suggesting a potential clinical application of this mechanism. Thus, these findings not only reveal a novel mechanism for compound-induced degradation of STING but also utilize HERC4 as potential E3 ligase that for TPD, enabling novel therapeutic applications