Designed ankyrin repeat proteins (DARPins) can recognize targets with specificities and affinities that equal or surpass those of antibodies, but because of their robustness and extreme stability, ...they allow a multitude of more advanced formats and applications. This review highlights recent advances in DARPin design, illustrates their properties, and gives some examples of their use. In research, they have been established as intracellular, real-time sensors of protein conformations and as crystallization chaperones. For future therapies, DARPins have been developed by advanced, structure-based protein engineering to selectively induce apoptosis in tumors by uncoupling surface receptors from their signaling cascades. They have also been used successfully for retargeting viruses. In ongoing clinical trials, DARPins have shown good safety and efficacy in macular degeneration diseases. These developments all ultimately exploit the high stability, solubility, and aggregation resistance of these molecules, permitting a wide range of conjugates and fusions to be produced and purified.
Intracellular protein-protein interactions in aberrant signaling pathways have emerged as a prime target in several diseases, particularly cancer. Since many protein-protein interactions are mediated ...by rather flat surfaces, they can typically not be interrupted by small molecules as they require cavities for binding. Therefore, protein drugs might be developed to compete with undesired interactions. However, proteins in general are not able to translocate from the extracellular side to the cytosolic target site by themselves, and thus an efficient protein translocation system, ideally combining efficient translocation with receptor specificity, is in high demand. Anthrax toxin, the tripartite holotoxin of Bacillus anthracis, is one of the best studied bacterial protein toxins and has proven to be a suitable candidate for cell-specific translocation of cargoes in vitro and in vivo. Our group recently developed a retargeted protective antigen (PA) variant fused to different Designed Ankyrin Repeat Proteins (DARPins) to achieve receptor specificity, and we incorporated a receptor domain to stabilize the prepore and prevent cell lysis. This strategy had been shown to deliver high amounts of cargo DARPins fused behind the N-terminal 254 amino acids of Lethal Factor (LF
). Here, we established a cytosolic binding assay, demonstrating the ability of DARPins to refold in the cytosol and bind their target after been translocated by PA.
Highlights ► Designed repeat protein scaffolds form a promising alternative to antibodies. ► These proteins lack disulfides, allowing soluble expression in the cytoplasm of E. coli . ► Novel ...molecular formats can be engineered in a convenient manner. ► DARPins, TPRs, αREPs and armadillos are examples of consensus designed scaffolds. ► Novel applications of these proteins, particularly DARPins, are highlighted.
Determining how proteins interact to form stable complexes is of crucial importance, for example in the development of novel therapeutics. Computational methods to determine the thermodynamically ...stable conformation of complexes from the structure of the binding partners, such as RosettaDock, might potentially emerge to become a promising alternative to traditional structure determination methods. However, while models virtually identical to the correct experimental structure can in some cases be generated, the main difficulty remains to discriminate correct or approximately correct models from decoys. This is due to the ruggedness of the free-energy landscape, the approximations intrinsic in the scoring functions, and the intrinsic flexibility of proteins. Here we show that molecular dynamics simulations performed starting from a number top-scoring models can not only discriminate decoys and identify the correct structure, but may also provide information on an initial map of the free energy landscape that elucidates the binding mechanism.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
G protein coupled receptors (GPCRs) are central players in recognizing a variety of stimuli to mediate diverse cellular responses. This myriad of functions is accomplished by their modular ...interactions with downstream intracellular transducers, such as heterotrimeric G proteins and arrestins. Assembling a specific GPCR-G protein pair as a purified complex for their structural and functional investigations remains a challenging task, however, because of the low affinity of the interaction. Here, we optimized fusion constructs of the Gα subunit of the heterotrimeric G protein and engineered versions of rat Neurotensin receptor 1 (NTR1), coexpressed and assembled in vivo with Gβ and Gγ. This was achieved by using the baculovirus-based MultiBac system. We thus generated a functional receptor-G protein fusion complex, which can be efficiently purified using ligand-based affinity chromatography on large scales. Additionally, we utilized a purification method based on a designed ankyrin repeat protein tightly binding to Green Fluorescent Protein (GFP-DARPin) that may be used as a generic approach for a large-scale purification of GPCR-G protein fusion complexes for which no ligands column can be generated. The purification methods described herein will support future studies that aim to understand the structural and functional framework of GPCR activation and signaling.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Parathyroid hormone 1 receptor (PTH1R) is a class B multidomain G-protein-coupled receptor (GPCR) that controls calcium homeostasis. Two endogenous peptide ligands, parathyroid hormone (PTH) and ...parathyroid hormone-related protein (PTHrP), activate the receptor, and their analogs teriparatide and abaloparatide are used in the clinic to increase bone formation as an effective yet costly treatment for osteoporosis. Activation of PTH1R involves binding of the peptide ligand to the receptor extracellular domain (ECD) and transmembrane domain (TMD), a hallmark of class B GPCRs. Here, we present the crystal structure of human PTH1R in complex with a peptide agonist at 2.5-Å resolution, allowing us to delineate the agonist binding mode for this receptor and revealing molecular details within conserved structural motifs that are critical for class B receptor function. Thus, this study provides structural insight into the function of PTH1R and extends our understanding of this therapeutically important class of GPCRs.
Neurokinins (or tachykinins) are peptides that modulate a wide variety of human physiology through the neurokinin G protein-coupled receptor family, implicated in a diverse array of pathological ...processes. Here we report high-resolution crystal structures of the human NK
receptor (NK
R) bound to two small-molecule antagonist therapeutics - aprepitant and netupitant and the progenitor antagonist CP-99,994. The structures reveal the detailed interactions between clinically approved antagonists and NK
R, which induce a distinct receptor conformation resulting in an interhelical hydrogen-bond network that cross-links the extracellular ends of helices V and VI. Furthermore, the high-resolution details of NK
R bound to netupitant establish a structural rationale for the lack of basal activity in NK
R. Taken together, these co-structures provide a comprehensive structural basis of NK
R antagonism and will facilitate the design of new therapeutics targeting the neurokinin receptor family.
Cell surface proteins are key regulators of fundamental cellular processes and, therefore, often at the root of human diseases. Thus, a large number of targeted drugs which are approved or under ...development act upon cell surface proteins. Although down-regulation of surface proteins by many natural ligands is well-established, the ability of drug candidates to cause internalization or degradation of the target is only recently moving into focus. This property is important both for the pharmacokinetics and pharmacodynamics of the drug but may also constitute a potential resistance mechanism. The enormous numbers of drug candidates targeting cell surface molecules, comprising small molecules, antibodies, or alternative protein scaffolds, necessitate methods for the investigation of internalization and degradation in high throughput. Here, we present a generic high-throughput assay protocol, which allows the simultaneous and independent quantification of internalization and degradation of surface proteins on a single-cell level. Because we fuse a HaloTag to the cell surface protein of interest and exploit the differential cell permeability of two fluorescent HaloTag ligands, no labeling of the molecules to be screened is required. In contrast to previously described approaches, our homogeneous assay is performed with adherent live cells in a 96-well format. Through channel rescaling, we are furthermore able to obtain true relative abundances of surface and internal protein. We demonstrate the applicability of our procedure to three major drug targets, EGFR, HER2, and EpCAM, examining a selection of well-investigated but also novel small molecule ligands and protein affinity reagents.
We recently developed a display method for the directed evolution of integral membrane proteins in the inner membrane of Escherichia coli for higher expression and stability. For the neurotensin ...receptor 1, a G-protein-coupled receptor (GPCR), we had evolved a mutant with a 10-fold increase in functional expression that largely retains wild-type binding and signaling properties and shows higher stability in detergent-solubilized form. We have now evolved three additional human GPCRs. Unmodified wild-type receptor cDNA was subjected to successive cycles of mutagenesis and fluorescence-activated cell sorting, and functional expression could be increased for all three GPCR targets. We also present a new stability screening method in a 96-well assay format to quickly identify evolved receptors showing increased thermal stability in detergent-solubilized form and rapidly evaluate them quantitatively. Combining the two methods turned out to be very powerful; even for the most challenging GPCR target—the tachykinin receptor NK1, which is hardly expressed in E. coli and cannot be functionally solubilized—receptor mutants that are functionally expressed at 1 mg/l levels in E. coli and are stable in detergent solution could be quickly evolved. The improvements result from cumulative small changes in the receptor sequence. This combinatorial approach does not require preconceived notions for designing mutations. Our results suggest that this method is generally applicable to GPCRs. Existing roadblocks in structural and biophysical studies can now be removed by providing sufficient quantities of correctly folded and stable receptor protein.
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► Poorly expressed, unstable GPCRs can be evolved to be well expressed and stable. ► Improvements arise from additive small changes in the receptor sequence. ► Evolved receptors mostly retain high affinity for ligands. ► Receptor stability is rapidly screened by a novel assay in a 96-well format.
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