Search

Designed ankyrin repeat proteins (DARPins) are artificial binding proteins that have found many uses in therapy, diagnostics and biochemical research. They substantially extend the scope of antibody-derived binders. Their high affinity and specificity, rigidity, extended paratope, and facile bacterial production make them attractive for structural biology. Complexes with simple DARPins have been crystallized for a long time, but particularly the rigid helix fusion strategy has opened new opportunities. Rigid DARPin fusions expand crystallization space, enable recruitment of targets in a host lattice and reduce the size limit for cryo-EM. Besides applications in structural biology, rigid DARPin fusions also serve as molecular probes in cells to investigate spatial restraints in targets.

Temperature influences the reaction kinetics and evolvability of all enzymes. To understand how evolution shapes the thermodynamic drivers of catalysis, we optimized the modest activity of a computationally designed enzyme for an elementary proton-transfer reaction by nearly 4 orders of magnitude over 9 rounds of mutagenesis and screening. As theorized for primordial enzymes, the catalytic effects of the original design were almost entirely enthalpic in origin, as were the rate enhancements achieved by laboratory evolution. However, the large reductions in ΔH⧧ were partially offset by a decrease in TΔS⧧ and unexpectedly accompanied by a negative activation heat capacity, signaling strong adaptation to the operating temperature. These findings echo reports of temperature-dependent activation parameters for highly evolved natural enzymes and are relevant to explanations of enzymatic catalysis and adaptation to changing thermal environments.

α-adrenergic receptors (αARs) are G protein-coupled receptors that regulate vital functions of the cardiovascular and nervous systems. The therapeutic potential of αARs, however, is largely unexploited and hampered by the scarcity of subtype-selective ligands. Moreover, several aminergic drugs either show off-target binding to αARs or fail to interact with the desired subtype. Here, we report the crystal structure of human α1BAR bound to the inverse agonist (+)-cyclazosin, enabled by the fusion to a DARPin crystallization chaperone. The α1BAR structure allows the identification of two unique secondary binding pockets. By structural comparison of α1BAR with α2ARs, and by constructing α1BAR-α2CAR chimeras, we identify residues 3.29 and 6.55 as key determinants of ligand selectivity. Our findings provide a basis for discovery of α1BAR-selective ligands and may guide the optimization of aminergic drugs to prevent off-target binding to αARs, or to elicit a selective interaction with the desired subtype., This study reports the X-ray structure of the α1B-adrenergic G protein-coupled receptor bound to an inverse agonist, and unveils key determinants of subtype-selective ligand binding that may help the design of aminergic drugs with fewer side-effects.

The structure of a designed ankyrin-repeat protein that selectively binds to domain 4 of HER3, an important driver of malignant growth in many tumors, has been determined. The structure helps to explain the selectivity against other members of the HER family, and binding to this epitope will interfere with its interactions in the tethered (inactive) and extended (active) conformations., The members of the human epidermal growth factor receptor (HER) family are among the most intensely studied oncological targets. HER3 (ErbB3), which had long been neglected, has emerged as a key oncogene, regulating the activity of other receptors and being involved in progression and tumor escape in multiple types of cancer. Designed ankyrin-repeat proteins (DARPins) serve as antibody mimetics that have proven to be useful in the clinic, in diagnostics and in research. DARPins have previously been selected against EGFR (HER1), HER2 and HER4. In particular, their combination into bivalent binders that separate or lock receptors in their inactive conformation has proved to be a promising strategy for the design of potent anticancer therapeutics. Here, the selection of DARPins targeting extracellular domain 4 of HER3 (HER3d4) is described. One of the selected DARPins, D5, in complex with HER3d4 crystallized in two closely related crystal forms that diffracted to 2.3 and 2.0 Å resolution, respectively. The DARPin D5 epitope comprises HER3d4 residues 568–577. These residues also contribute to interactions within the tethered (inactive) and extended (active) conformations of the extracellular domain of HER3.

Changing the primary metal coordination sphere is a powerful strategy for tuning metalloprotein properties. Here we used amber stop codon suppression with engineered pyrrolysyl-tRNA synthetases, including two newly evolved enzymes, to replace the proximal histidine in myoglobin with N δ -methylhistidine, 5-thiazoyl-alanine, 4-thiazoylalanine and 3-(3-thienyl)alanine. In addition to tuning the heme redox potential over a >200 mV range, these noncanonical ligands modulate the protein's carbene transfer activity with ethyl diazoacetate. Variants with increased reduction potential proved superior for cyclopropanation and N-H insertion, whereas variants with reduced E o values gave higher S-H insertion activity. Given the functional importance of histidine in many enzymes, these genetically encoded analogues could be valuable tools for probing mechanism and enabling new chemistries.

Crystal structures of NTSR1 bound to agonists, inverse agonists, and in the apo state enabled by a novel fusion to DARPin D12., Neurotensin receptor 1 (NTSR1) and related G protein–coupled receptors of the ghrelin family are clinically unexploited, and several mechanistic aspects of their activation and inactivation have remained unclear. Enabled by a new crystallization design, we present five new structures: apo-state NTSR1 as well as complexes with nonpeptide inverse agonists SR48692 and SR142948A, partial agonist RTI-3a, and the novel full agonist SRI-9829, providing structural rationales on how ligands modulate NTSR1. The inverse agonists favor a large extracellular opening of helices VI and VII, undescribed so far for NTSR1, causing a constriction of the intracellular portion. In contrast, the full and partial agonists induce a binding site contraction, and their efficacy correlates with the ability to mimic the binding mode of the endogenous agonist neurotensin. Providing evidence of helical and side-chain rearrangements modulating receptor activation, our structural and functional data expand the mechanistic understanding of NTSR1 and potentially other peptidergic receptors.

Changing the primary metal coordination sphere is a powerful strategy for modulating metalloprotein properties. Taking advantage of this approach, we have replaced the proximal histidine ligand in myoglobin with the histidine analogues Nd-methylhistidine (NMH), 5‑thiazoylalanine (5ThzA), 4-thiazoylalanine (4ThzA) and 3-(3-thienyl)alanine (3ThiA) by amber stop codon suppression using engineered pyrrolysyl-tRNA synthetases, including two newly evolved enzymes. In addition to tuning the heme redox potential over a >200 mV range, these noncanonical ligands modulate the protein’s promiscuous carbene transfer activity with ethyl diazoacetate. Myoglobin variants with increased reduction potentials (NMH and 5ThzA) proved superior for cyclopropanation and N-H insertion, especially under aerobic conditions, and could even promote these reactions in the absence of reducing agent. In contrast, the variants with the lowest Eo values (4ThzA and 3ThiA) exhibit comparatively high S-H insertion activity even though the respective histidine surrogates do not coordinate the heme iron. Given the important functional roles played by histidine in many enzymes, these genetically encoded histidine analogues represent valuable tools for probing mechanism and enabling new chemistries in metalloproteins.

Stimulation of regulatory T (Treg) cells holds great promise for the treatment of autoimmune, chronic inflammatory, and certain metabolic diseases. Recent clinical trials with low-dose interleukin-2 (IL-2) to expand Treg cells led to beneficial results in autoimmunity, but IL-2 immunotherapy can activate both Treg cells and pathogenic T cells. Use of IL-2 receptor α (IL-2Rα, CD25)-biased IL-2/anti-IL-2 antibody complexes improves IL-2 selectivity for Treg cells; however, the mechanism of action of such IL-2 complexes is incompletely understood, thus hampering their translation into clinical trials. Using a cell-based and dynamic IL-2R platform, we identified a particular anti-human IL-2 antibody, termed UFKA-20. When bound to UFKA-20, IL-2 failed to stimulate cells expressing IL-2Rβ (CD122) and IL-2Rγ (CD132), unless these cells also expressed high amounts of CD25. CD25 allowed IL-2/UFKA-20 complexes to bind, and binding to CD25 in the presence of CD122 and CD132 was followed by rapid dissociation of UFKA-20 from IL-2, delivery of IL-2 to CD122 and CD132, and intracellular signaling. IL-2/UFKA-20 complexes efficiently and preferentially stimulated CD4+ Treg cells in freshly isolated human T cells ex vivo and in mice and rhesus macaques in vivo. The crystal structure of the IL-2/UFKA-20 complex demonstrated that UFKA-20 interfered with IL-2 binding to CD122 and, to a lesser extent, also CD25. Together, we translated CD25-biased IL-2 complexes from mice to nonhuman primates and extended our mechanistic understanding of how CD25-biasing anti-human IL-2 antibodies work, which paves the way to clinical trials of CD25-biased IL-2 complexes.

To untangle the complex signaling of the c-Jun N-terminal kinase (JNK) isoforms, we need tools that can selectively detect and inhibit individual isoforms. Because of the high similarity between JNK1, JNK2 and JNK3, it is very difficult to generate small-molecule inhibitors with this discriminatory power. Thus, we have recently selected protein binders from the designed ankyrin repeat protein (DARPin) library which were indeed isoform-specific inhibitors of JNK1 with low nanomolar potency. Here we provide the structural basis for their isotype discrimination and their inhibitory action. All our previous attempts to generate crystal structures of complexes had failed. We have now made use of a technology we recently developed which consists of rigid fusion of an additional special DARPin, which acts as a crystallization enhancer. This can be rigidly fused with different geometries, thereby generating a range of alternative crystal packings. The structures reveal the molecular basis for isoform specificity of the DARPins and their ability to prevent JNK activation and may thus form the basis of further investigation of the JNK family as well as novel approaches to drug design.

Multivalent binding proteins can gain biological activities beyond what is inherent in the individual binders, by bringing together different target molecules, restricting their conformational flexibility or changing their subcellular localization. In this study, we demonstrate a method to build up rigid multivalent and multispecific scaffolds by exploiting the modular nature of a repeat protein scaffold and avoiding flexible linkers. We use DARPins (Designed Ankyrin Repeat Proteins), synthetic binding proteins based on the Ankyrin-repeat protein scaffold, as binding units. Their ease of in vitro selection, high production yield and stability make them ideal specificity-conferring building blocks for the design of more complex constructs. C- and N-terminal DARPin capping repeats were re-designed to be joined by a shared helix in such a way that rigid connector modules are formed. This allows us to join two or more DARPins in predefined geometries without compromising their binding affinities and specificities. Nine connector modules with distinct geometries were designed; for eight of these we were able to confirm the structure by X-ray crystallography, while only one did not crystallize. The bispecific constructs were all able to bind both target proteins simultaneously.

Designed armadillo repeat proteins (dArmRP) are α-helical solenoid repeat proteins with an extended peptide binding groove that were engineered to develop a generic modular technology for peptide recognition. In this context, the term "peptide" not only denotes a short unstructured chain of amino acids, but also an unstructured region of a protein, as they occur in termini, loops, or linkers between folded domains. Here we report two crystal structures of dArmRPs, in complex with peptides fused either to the N-terminus of Green Fluorescent Protein or to the C-terminus of a phage lambda protein D. These structures demonstrate that dArmRPs bind unfolded peptides in the intended conformation also when they constitute unstructured parts of folded proteins, which greatly expands possible applications of the dArmRP technology. Nonetheless, the structures do not fully reflect the binding behavior in solution, that is, some binding sites remain unoccupied in the crystal and even unexpected peptide residues appear to be bound. We show how these differences can be explained by restrictions of the crystal lattice or the composition of the crystallization solution. This illustrates that crystal structures have to be interpreted with caution when protein-peptide interactions are characterized, and should always be correlated with measurements in solution.

To overcome the laborious identification of crystallisation conditions for protein X-ray crystallography, we developed a method where the examined protein is immobilised as a guest molecule in a universal host lattice. We applied crystal engineering to create a generic crystalline host lattice under reproducible, predefined conditions and analysed the structures of target guest molecules of different size, namely two 15-mer peptides and green fluorescent protein (sfGFP). A fusion protein with an N-terminal endo-α-N-acetylgalactosaminidase (EngBF) domain and a C-terminal designed ankyrin repeat protein (DARPin) domain establishes the crystal lattice. The target is recruited into the host lattice, always in the same crystal form, through binding to the DARPin. The target structures can be determined rapidly from difference Fourier maps, whose quality depends on the size of the target and the orientation of the DARPin.

Zugehöriger Artikel: https://digitalcollection.zhaw.ch/handle/11475/21751

English version available: "Drug Design Inspired by Nature: Crystallographic Detection of an Auto‐Tailored Protease Inhibitor Template", https://doi.org/10.1002/anie.201812348 Das De‐novo‐Design neuer Wirkstoffmoleküle ist nach wie vor eine anspruchsvolle Aufgabe bei der Suche nach wirksamen und selektiven Modulatoren für therapeutisch relevante Zielproteine. Hier berichten wir über die unerwartete Entdeckung eines peptidischen Liganden durch Röntgenkristallographie, der vom therapeutischen Zielprotein MMP‐13 durch partiellen Selbstabbau generiert wurde, und die anschließende strukturbasierte Optimierung dieses Peptids zu einem hochwirksamen und selektiven β‐Faltblatt‐Peptidmimetikum der endogenen Gewebeinhibitoren von Metalloproteinasen (TIMPs). Der Einbau von nicht‐proteinogenen Aminosäuren in Kombination mit einer Zyklisierungsstrategie erwies sich als entscheidend für das De‐novo‐Design der TIMP‐Peptidmimetika. Das optimierte zyklische Peptid (ZHAWOC7726) ist membrangängig, hat einen IC50‐Wert von 21 nm für MMP‐13 und ein vielversprechendes Selektivitätsprofil bezüglich eines polypharmakologischen Ansatzes mit den Anti‐Krebs‐Zielproteinen MMP‐2 (IC50: 170 nm) und MMP‐9 (IC50: 140 nm).

Evolution trains a from-scratch catalyst Metal-bound peptides can catalyze simple reactions such as ester hydrolysis and may have been the starting point for the evolution of modern enzymes. Studer et al. selected progressively more-proficient variants of a small protein derived from a computationally designed zinc-binding peptide. The resulting enzyme could perform the trained reaction at rates typical for naturally evolved enzymes and serendipitously developed a strong preference for a single enantiomer of the substrate. A structure of the final catalyst highlights how small, progressive changes can remodel both catalytic residues and protein architecture in unpredictable ways. Science , this issue p. 1285

De novo drug discovery is still a challenge in the search for potent and selective modulators of therapeutically relevant target proteins. Here, we disclose the unexpected discovery of a peptidic ligand 1 by X-ray crystallography, which was auto-tailored by the therapeutic target MMP-13 through partial self-degradation and subsequent structure-based optimization to a highly potent and selective β-sheet peptidomimetic inhibitor derived from the endogenous tissue inhibitors of metalloproteinases (TIMPs). The incorporation of non-proteinogenic amino acids in combination with a cyclization strategy proved to be key for the de novo design of TIMP peptidomimetics. The optimized cyclic peptide 4 (ZHAWOC7726) is membrane permeable with an IC50 of 21 nm for MMP-13 and an attractive selectivity profile with respect to a polypharmacology approach including the anticancer targets MMP-2 (IC50 : 170 nm) and MMP-9 (IC50 : 140 nm).

Current Microbiology, 63 (3), ISSN:0343-8651, ISSN:1432-0991

Designed armadillo repeat proteins (dArmRPs) were developed to create a modular peptide binding technology where each of the structural repeats binds two residues of the target peptide. An essential prerequisite for such a technology is a dArmRP geometry that matches the peptide bond length. To this end, we determined a large set (n=27) of dArmRP X-ray structures, of which 12 were previously unpublished, to calculate curvature parameters that define their geometry. Our analysis shows that consensus dArmRPs exhibit curvatures close to the optimal range for modular peptide recognition. Binding of peptide ligands can induce a curvature within the desired range, as confirmed by single-molecule FRET experiments in solution. On the other hand, computationally designed ArmRPs, where side chains have been chosen with the intention to optimally fit into a geometrically optimized backbone, turned out to be more divergent in reality, and thus not suitable for continuous peptide binding. Furthermore, we show that the formation of a crystal lattice can induce small but significant deviations from the curvature adopted in solution, which can interfere with the evaluation of repeat protein scaffolds when high accuracy is required. This study corroborates the suitability of consensus dArmRPs as a scaffold for the development of modular peptide binders.

Designed armadillo repeat proteins (dArmRP) are promising modular proteins for the engineering of binding molecules that recognize extended polypeptide chains. We determined the structure of a dArmRP containing five internal repeats and 3rd generation capping repeats in three different states by X-ray crystallography: without N-terminal His6-tag and in the presence of calcium (YM5A/Ca2+), without N-terminal His6-tag and in the absence of calcium (YM5A), and with N-terminal His6-tag and in the presence of calcium (His-YM5A/Ca2+). All structures show different quaternary structures and superhelical parameters. His-YM5A/Ca2+ forms a crystallographic dimer, which is bridged by the His6-tag, YM5A/Ca2+ forms a domain-swapped tetramer, and only in the absence of calcium and the His6-tag, YM5A forms a monomer. The changes of superhelical parameters are a consequence of calcium binding, because calcium ions interact with negatively charged residues, which can also participate in the modulation of helix dipole moments between adjacent repeats. These observations are important for further optimizations of dArmRPs and provide a general illustration of how construct design and crystallization conditions can influence the exact structure of the investigated protein.

TDP-43 is a primarily nuclear RNA-binding protein, whose abnormal phosphorylation and cytoplasmic aggregation characterizes affected neurons in patients with amyotrophic lateral sclerosis and frontotemporal dementia. Here, we report that physiological nuclear TDP-43 in mouse and human brain forms homo-oligomers that are resistant to cellular stress. Physiological TDP-43 oligomerization is mediated by its N-terminal domain, which can adopt dynamic, solenoid-like structures, as revealed by a 2.1 Å crystal structure in combination with nuclear magnetic resonance spectroscopy and electron microscopy. These head-to-tail TDP-43 oligomers are unique among known RNA-binding proteins and represent the functional form of the protein in vivo, since their destabilization results in loss of alternative splicing regulation of known neuronal RNA targets. Our findings indicate that N-terminal domain-driven oligomerization spatially separates the adjoining highly aggregation-prone, C-terminal low-complexity domains of consecutive TDP-43 monomers, thereby preventing low-complexity domain inter-molecular interactions and antagonizing the formation of pathologic aggregates., Nature Communications, 8, ISSN:2041-1723

Armadillo repeat proteins (ArmRPs) recognize their target peptide in extended conformation and bind, in a first approximation, two residues per repeat. Thus, they may form the basis for building a modular system, in which each repeat is complementary to a piece of the target peptide. Accordingly, preselected repeats could be assembled into specific binding proteins on demand and thereby avoid the traditional generation of every new binding molecule by an independent selection from a library. Stacked armadillo repeats, each consisting of 42 aa arranged in three α-helices, build an elongated superhelical structure. Here, we analyzed the curvature variations in natural ArmRPs and identified a repeat pair from yeast importin-α as having the optimal curvature geometry that is complementary to a peptide over its whole length. We employed a symmetric in silico design to obtain a uniform sequence for a stackable repeat while maintaining the desired curvature geometry. Computationally designed ArmRPs (dArmRPs) had to be stabilized by mutations to remove regions of higher flexibility, which were identified by molecular dynamics simulations in explicit solvent. Using an N-capping repeat from the consensus-design approach, two different crystal structures of dArmRP were determined. Although the experimental structures of dArmRP deviated from the designed curvature, the insertion of the most conserved binding pockets of natural ArmRPs onto the surface of dArmRPs resulted in binders against the expected peptide with low nanomolar affinities, similar to the binders from the consensus-design series.

Enzymes rely on complex interactions between precisely positioned active site residues as a mechanism to compensate for the limited functionality contained within the genetic code. Heme enzymes provide a striking example of this complexity, whereby the electronic properties of reactive ferryl intermediates are finely tuned through hydrogen bonding interactions between proximal ligands and neighboring amino acids. Here, we show that introduction of a chemically programmed proximal Nδ-methyl histidine (NMH) ligand into an engineered ascorbate peroxidase (APX2) overcomes the reliance on the conserved Asp-His hydrogen bonding interaction, leading to a catalytically modified enzyme (APX2 NMH), which is able to achieve a significantly higher number of turnovers compared with APX2 without compromising catalytic efficiency. Structural, spectroscopic and kinetic characterization of APX2 NMH and several active site variants provides valuable insights into the role of the Asp-His-Fe triad of heme peroxidases. More significantly, simplification of catalytic mechanisms through the incorporation of chemically optimized ligands may facilitate efforts to create and evolve new active site heme environments within proteins.

Natural armadillo repeat proteins (nArmRP) like importin-α or β-catenin bind their target peptides such that each repeat interacts with a dipeptide unit within the stretched target peptide. However, this modularity is imperfect and also restricted to short peptide stretches of usually four to six consecutive amino acids. Here we report the development and characterization of a regularized and truly modular peptide-specific binding protein, based on designed armadillo repeat proteins (dArmRP), binding to peptides of alternating lysine and arginine residues (KR)n. dArmRP were obtained from nArmRP through cycles of extensive protein engineering, which rendered them more uniform. This regularity is reflected in the consistent binding of dArmRP to (KR)-peptides, where affinities depend on the lengths of target peptides and the number of internal repeats in a very systematic manner, thus confirming the modularity of the interaction. This exponential dependency between affinity and recognition length suggests that each module adds a constant increment of binding energy to sequence-specific recognition. This relationship was confirmed by comprehensive mutagenesis studies that also reveal the importance of individual peptide side chains. The 1.83 A resolution crystal structure of a dArmRP with five identical internal repeats in complex with the cognate (KR)5 peptide proves a modular binding mode, where each dipeptide is recognized by one internal repeat. The confirmation of this true modularity over longer peptide stretches lays the ground for the design of binders with different specificities and tailored affinities by the assembly of dipeptide-specific modules based on armadillo repeats.

SUMMARY The b-aminopeptidase BapA from Sphingosinicella xenopeptidilytica belongs to the N-terminal nucleophile (Ntn) hydrolases of the DmpA-like family and has the unprecedented property of cleaving N-terminal b-amino acid residues from peptides. We determined the crystal structures of the native (ab)4 heterooctamer and of the 153 kDa precursor homotetramer at a resolution of 1.45 and 1.8 Au , respectively. These structures together with mutational analyses strongly support mechanisms for autoproteolysis and catalysis that involve residues Ser250, Ser288, and Glu290. The autoproteolytic mechanism is different from the one so far described for Ntn hydrolases. The structures together with functional data also provide insight into the discriminating features of the active site cleft that determine substrate specificity.

The armadillo domain is a right-handed super-helix of repeating units composed of three α-helices each. Armadillo repeat proteins (ArmRPs) are frequently involved in protein–protein interactions, and because of their modular recognition of extended peptide regions they can serve as templates for the design of artificial peptide binding scaffolds. On the basis of sequential and structural analyses, different consensus-designed ArmRPs were synthesized and show high thermodynamic stabilities, compared to naturally occurring ArmRPs. We determined the crystal structures of four full-consensus ArmRPs with three or four identical internal repeats and two different designs for the N- and C-caps. The crystal structures were refined at resolutions ranging from 1.80 to 2.50 A for the above mentioned designs. A redesign of our initial caps was required to obtain well diffracting crystals. However, the structures with the redesigned caps caused domain swapping events between the N-caps. To prevent this domain swap, 9 and 6 point mutations were introduced in the N- and C-caps, respectively. Structural and biophysical analysis showed that this subsequent redesign of the N-cap prevented domain swapping and improved the thermodynamic stability of the proteins. We systematically investigated the best cap combinations. We conclude that designed ArmRPs with optimized caps are intrinsically stable and well-expressed monomeric proteins and that the high-resolution structures provide excellent structural templates for the continuation of the design of sequence-specific modular peptide recognition units based on armadillo repeats.

A number of strategies and protocols for the expression, purification and kinetic characterization of human caspases are described in the literature. We have systematically revised these protocols and present comprehensive optimized expression and purification protocols for caspase-1 to -9 as well as improved assay conditions for their reproducible kinetic characterization. Our studies on active site titration revealed that the reproducibility is strongly affected by the presence of DTT in the assay buffer. Furthermore, we observed that not all caspases show a linear relationship between enzymatic activity and protein concentration, which explains the discrepancy between published values of specific activities from different laboratories. Our broad kinetic analysis allows the conclusion that the dependency of caspase activities on protein concentration is an effect of concentration-dependent dimerization, which can also be influenced by kosmotropic salts. The protocol recommendations as an outcome of this work will yield higher reproducibility regarding expression and purification of human caspases and contribute to standardization of enzyme kinetic data.

Helicobacter pylori infection increases the risk of cardiovascular diseases besides leading to duodenal and gastric peptic ulcerations. H. pylori cysteine-rich protein B (HcpB) is a disulfide-rich repeat protein that belongs to the family of Sel1-like repeat proteins. HcpB contains four pairs of anti-parallel alpha helices that fold into four repeats with disulfide bonds bridging the helices of each repeat. Recent in vitro oxidative refolding of HcpB identified that the formation and folding of the disulfide bond in the N-terminal repeat are the rate limiting step. Here we attempted to understand the disulfide formation of HcpB in the periplasm of Escherichia coli. The protein was expressed in wild type (possessed enzymes DsbA, B, C, and D) and knock out (Dsb enzymes deleted one at a time) E. coli strains. The soluble part of the periplasm when analyzed by SDS-PAGE and Western Blot showed that the wild type and DsbC/D knock out strains contained native oxidized HcpB while the protein was absent in the DsbA/B knock out strains. Hence the recombinant expression of HcpB in E. coli requires DsbA and DsbB for disulfide bond formation and it is independent of DsbC and DsbD. Prolonged cell growth resulted in the proteolytic degradation of the N-terminal repeat of HcpB. The delayed folding of the N-terminal repeat observed during in vitro oxidative refolding could be the reason for the enhanced susceptibility to proteolytic cleavage in the periplasm. In summary, a good correlation between in vivo and in vitro disulfide bond formation of HcpB is observed.

The inherited autoinflammatory syndrome familial Mediterranean fever (FMF) is characterized by recurrent episodes of fever, which are independent of any bacterial or viral infections. This disease is associated with point mutations in the mefv gene product pyrin. Although the precise molecular functions of pyrin are unknown, it seems to be involved in the maturation and secretion of interleukin-1beta. Approximately two thirds of all FMF-associated mutations cluster in the C-terminal B30.2 domain of pyrin. To investigate the molecular consequences of FMF-associated mutations, we determined the crystal structure of the pyrin B30.2 domain at 1.35-A resolution. The comparison with other B30.2/ligand complex structures revealed a shallow cavity, which seems to be involved in binding the pyrin ligand. The bottom of this cavity is covered mainly with hydrophobic amino acids, suggesting that pyrin recognizes its ligand by hydrophobic contacts and surface complementarities. FMF-associated mutations cluster around two sites on the B30.2 surface. Approximately two thirds, including those mutations with the most severe disease outcomes, are observed in the vicinity of the predicted peptide binding site, suggesting that they will have a direct impact on ligand binding. A second mutational hot spot was observed on the opposite side of the B30.2 domain in the neighbourhood of its artificial N-terminus. Although most FMF-associated mutations are solvent exposed, several will modify the main-chain conformation of loops. The experimental crystal structure of the pyrin B30.2 domain serves as a basis for an accurate modelling of these mutations.

Full-consensus designed ankyrin repeat proteins (DARPins), in which randomized positions of the previously described DARPin library have been fixed, are characterized. They show exceptionally high thermodynamic stabilities, even when compared to members of consensus DARPin libraries and even more so when compared to naturally occurring ankyrin repeat proteins. We determined the crystal structure of a full-consensus DARPin, containing an N-capping repeat, three identical internal repeats and a C-capping repeat at 2.05 A resolution, and compared its structure with that of the related DARPin library members E3_5 and E3_19. This structural comparison suggests that primarily salt bridges on the surface, which arrange in a network with almost crystal-like regularity, increase thermostability in the full-consensus NI(3)C DARPin to make it resistant to boiling. In the crystal structure, three sulfate ions complement this network. Thermal denaturation experiments in guanidine hydrochloride directly indicate a contribution of sulfate binding to the stability, providing further evidence for the stabilizing effect of surface-exposed electrostatic interactions and regular charge networks. The charged residues at the place of randomized residues in the DARPin libraries were selected based on sequence statistics and suggested that the charge interaction network is a hidden design feature of this protein family. Ankyrins can therefore use design principles from proteins of thermophilic organisms and reach at least similar stabilities.

Designing catalysts that achieve the rates and selectivities of natural enzymes is a long-standing goal in protein chemistry. Here, we show that an ultrahigh-throughput droplet-based microfluidic screening platform can be used to improve a previously optimized artificial aldolase by an additional factor of 30 to give a >109 rate enhancement that rivals the efficiency of class I aldolases. The resulting enzyme catalyses a reversible aldol reaction with high stereoselectivity and tolerates a broad range of substrates. Biochemical and structural studies show that catalysis depends on a Lys-Tyr-Asn-Tyr tetrad that emerged adjacent to a computationally designed hydrophobic pocket during directed evolution. This constellation of residues is poised to activate the substrate by Schiff base formation, promote mechanistically important proton transfers and stabilize multiple transition states along a complex reaction coordinate. The emergence of such a sophisticated catalytic centre shows that there is nothing magical about the catalytic activities or mechanisms of naturally occurring enzymes, or the evolutionary process that gave rise to them.

Designed armadillo-repeat proteins are promising scaffolds for modular peptide-recognition systems. The crystal structures of His-YIIIM4AII, His-YIIIM5AII and YIIIM5AII highlight structural heterogeneity in full-consensus designs and aid the improvement of future constructs., The armadillo repeat serves as a scaffold for the development of modular peptide-recognition modules. In order to develop such a system, three crystal structures of designed armadillo-repeat proteins with third-generation N-caps (YIII-type), four or five internal repeats (M-type) and second-generation C-caps (AII-type) were determined at 1.8 Å (His-YIIIM4AII), 2.0 Å (His-YIIIM5AII) and 1.95 Å (YIIIM5AII) resolution and compared with those of variants with third-generation C-caps. All constructs are full consensus designs in which the internal repeats have exactly the same sequence, and hence identical conformations of the internal repeats are expected. The N-cap and internal repeats M1 to M3 are indeed extremely similar, but the comparison reveals structural differences in internal repeats M4 and M5 and the C-cap. These differences are caused by long-range effects of the C-cap, contacting molecules in the crystal, and the intrinsic design of the repeat. Unfortunately, the rigid-body movement of the C-terminal part impairs the regular arrangement of internal repeats that forms the putative peptide-binding site. The second-generation C-cap improves the packing of buried residues and thereby the stability of the protein. These considerations are useful for future improvements of an armadillo-repeat-based peptide-recognition system.

Solenoid proteins, which are distinguished from general globular proteins by their modular architectures, are frequently involved in signal transduction pathways. Proteins from the tetratricopeptide repeat (TPR) and Sel1-like repeat (SLR) families share similar alpha-helical conformations but different consensus sequence lengths and superhelical topologies. Both families are characterized by low sequence similarity levels, rendering the identification of functional homologous difficult. Therefore current knowledge of the molecular and cellular functions of the SLR proteins Sel1, Hrd3, Chs4, Nif1, PodJ, ExoR, AlgK, HcpA, Hsp12, EnhC, LpnE, MotX, and MerG has been reviewed. Although SLR proteins possess different cellular functions they all seem to serve as adaptor proteins for the assembly of macromolecular complexes. Sel1, Hrd3, Hsp12 and LpnE are activated under cellular stress. The eukaryotic Sel1 and Hrd3 proteins are involved in the ER-associated protein degradation, whereas the bacterial LpnE, EnhC, HcpA, ExoR, and AlgK proteins mediate the interactions between bacterial and eukaryotic host cells. LpnE and EnhC are responsible for the entry of L. pneumophila into epithelial cells and macrophages. ExoR from the symbiotic microorganism S. melioti and AlgK from the pathogen P. aeruginosa regulate exopolysaccaride synthesis. Nif1 and Chs4 from yeast are responsible for the regulation of mitosis and septum formation during cell division, respectively, and PodJ guides the cellular differentiation during the cell cycle of the bacterium C. crescentus. Taken together the SLR motif establishes a link between signal transduction pathways from eukaryotes and bacteria. The SLR motif is so far absent from archaea. Therefore the SLR could have developed in the last common ancestor between eukaryotes and bacteria.

Fluorescent probes constitute a valuable toolbox to address a variety of biological questions and they have become irreplaceable for imaging methods. Commonly, such probes consist of fluorescent proteins or small organic fluorophores coupled to biological molecules of interest. Recently, a novel class of fluorescence-based probes, fluorogen-activating proteins (FAPs), has been reported. These binding proteins are based on antibody single-chain variable fragments and activate fluorogenic dyes, which only become fluorescent upon activation and do not fluoresce when free in solution. Here we present a novel class of fluorogen activators, termed FADAs, based on the very robust designed ankyrin repeat protein scaffold, which also readily folds in the reducing environment of the cytoplasm. The FADA generated in this study was obtained by combined selections with ribosome display and yeast surface display. It enhances the fluorescence of malachite green (MG) dyes by a factor of more than 11,000 and thus activates MG to a similar extent as FAPs based on single-chain variable fragments. As shown by structure determination and in vitro measurements, this FADA was evolved to form a homodimer for the activation of MG dyes. Exploiting the favorable properties of the designed ankyrin repeat protein scaffold, we created a FADA biosensor suitable for imaging of proteins on the cell surface, as well as in the cytosol. Moreover, based on the requirement of dimerization for strong fluorogen activation, a prototype FADA biosensor for in situ detection of a target protein and protein-protein interactions was developed. Therefore, FADAs are versatile fluorescent probes that are easily produced and suitable for diverse applications and thus extend the FAP technology.

Many tripartite motif-containing (TRIM) proteins, comprising RING-finger, B-Box and coiled-coil domains, carry additional B30.2 domains on the C-terminus of the TRIM motif and are considered to be pattern recognition receptors involved in the detection of higher order oligomers (e.g. viral capsid proteins). To investigate the spatial architecture of domains in TRIM proteins we determined the crystal structure of the TRIM20Δ413 fragment at 2.4 Å resolution. This structure comprises the central helical scaffold (CHS) and C-terminal B30.2 domains and reveals an anti-parallel arrangement of CHS domains placing the B-box domains 170 Å apart from each other. Small-angle X-ray scattering confirmed that the linker between CHS and B30.2 domains is flexible in solution. The crystal structure suggests an interaction between the B30.2 domain and an extended stretch in the CHS domain, which involves residues that are mutated in the inherited disease Familial Mediterranean Fever. Dimerization of B30.2 domains by means of the CHS domain is crucial for TRIM20 to bind pro-IL-1β in vitro. To exemplify how TRIM proteins could be involved in binding higher order oligomers we discuss three possible models for the TRIM5α/HIV-1 capsid interaction assuming different conformations of B30.2 domains.

Caspases are cysteine proteases involved in the signalling cascades of programmed cell death in which caspase-3 plays a central role, since it propagates death signals from intrinsic and extrinsic stimuli to downstream targets. The atomic resolution (1.06 Angstroms) crystal structure of the caspase-3 DEVD-cmk complex reveals the structural basis for substrate selectivity in the S4 pocket. A low-barrier hydrogen bond is observed between the side-chains of the P4 inhibitor aspartic acid and Asp179 of the N-terminal tail of the symmetry related p12 subunit. Site-directed mutagenesis of Asp179 confirmed the significance of this residue in substrate recognition. In the 1.06 Angstroms crystal structure, a radiation damage induced rearrangement of the inhibitor methylketone moiety was observed. The carbon atom that in a substrate would represent the scissile peptide bond carbonyl carbon clearly shows a tetrahedral coordination and resembles the postulated tetrahedral intermediate of the acylation reaction.

Predominant T-helper 1 (Th1) responses with increased gamma interferon (IFN-γ) levels have been proposed to play an important role inHelicobacter pylori-induced gastritis and peptic ulceration. However, bacterial factors contributing to the initiation of Th1 polarization ofH. pylori-specific immune responses have not been characterized in detail thus far. We report here on the identification ofHelicobactercysteine-rich protein A (HcpA) as a novel proinflammatory and Th1-promoting protein. The capacity of HcpA to induce immune activation was studied in splenocyte cultures of naiveH. pylori-negative mice. HcpA stimulated the release of high concentrations of the proinflammatory and Th1-promoting cytokines interleukin-6 (IL-6) and IFN-γ, in addition to significant levels of IL-12, tumor necrosis factor alpha, and IL-10. The observed cytokine profile was comparable to that induced by lipopolysaccharide but differed in the kinetics and maximum levels of cytokine production. In addition, HcpA-induced cytokine release resembled that observed upon incubation withH. pyloriexcept for IL-10, which was only moderately released upon HcpA stimulation. Both HcpA- andH. pylori-mediated IFN-γ production was drastically reduced by a neutralizing antibody against IL-12 but not by an anti-IL-2 antibody. Thus, HcpA seems to represent a novel bacterial virulence factor triggering the release of a concerted set of cytokines to instruct the adaptive immune system for the initiation of proinflammatory and Th1-biased immunity.

Helicobacter pylori is a Gram-negative human pathogen that infects the gastric mucosa and causes an inflammatory process leading to gastritis, ulceration and cancer. Bacterial cell-surface and secreted proteins often play an important role in pathogen-host interactions and are thought to be selective mediators for the pathology of the infection. The Helicobacter cysteine-rich proteins (Hcp) represent a large family of secreted proteins that seem to be specific for microorganisms from the epsilon-subfamily of proteobacteria. Although significantly elevated levels of anti-Hcp antibodies were observed in many patients infected with H.pylori, details on the biological functions of Hcp proteins are sparse. Hcps belong to a large family of Sel1-like multi-repeat proteins. The crystal structure of HcpC was refined at 2.0 A resolution and revealed a super-helical topology composed of seven disulfide bridged alpha/alpha-repeats, an N-terminal capping helix and an extended C-terminal coil consisting of alternating hydrophobic and hydrophilic residues. In the crystal packing, the C-terminal coil interacts with the concave surface of a symmetry-related HcpC super-helix. A hydrophobic pocket and a cluster of negatively charged residues recognize the side-chains of Val290 and Lys287 from the C-terminal coil, respectively. The peptide nitrogen atom of His291 forms a short hydrogen bond with the side-chain of Asn66. The interactions seen in this crystal contact are strikingly similar to the peptide-binding modes of the Hsp70/Hsp90 organizing protein and the PEX5 receptor. The conservation of the peptide-binding mode suggests that HcpC might recognize its binding partner in a similar way.

S100 proteins are involved in metal-dependent intracellular signalling. Metal-free S100A3, a cysteine-rich Ca(2+)- and Zn(2+)-binding protein, has been crystallized by vapour diffusion under the strict exclusion of oxygen and in the absence of divalent metal ions. Metal binding induces large conformational changes, rendering the apo-S100A3 crystals very sensitive to various metal compounds. Therefore, the structure was solved by MIRAS phasing using potassium iodide and xenon derivatives. Iodide replaces a water molecule at the surface of the S100A3 protein, whereas xenon binds in a hydrophobic cavity at the dimer interface. Despite significant non-isomorphism, the combination of both derivatives was sufficient for structure determination. The overall apo-S100A3 structure resembles the structures of metal-free S100B and S100A6 solution structures. In contrast to the NMR structures, the EF-hand loops are well ordered in the apo-S100A3 crystal structure. In the N-terminal pseudo-EF-hand loop a water molecule occupies the position of the Ca(2+) ion. The C-terminal canonical EF-hand loop shows an extended conformation and a different helix arrangement to other S100/metal complex crystal structures.

Manganese superoxide dismutase (MnSOD) of Aspergillus fumigatus, a fungus involved in many pulmonary complications, has been identified as IgE-binding protein. It has been shown also that MnSODs from other organisms, including human, are recognized by IgE Abs from individuals sensitized to A. fumigatus MnSOD. Comparison of the fungal and the human crystal structure should allow the identification of structural similarities responsible for IgE-mediated cross-reactivity. The three-dimensional structure of A. fumigatus MnSOD has been determined at 2-Å resolution by x-ray diffraction analysis. Crystals belonged to space group P212121 with unit cell dimensions of a = 65.88 Å, b = 98.7 Å, and c = 139.28 Å. The structure was solved by molecular replacement using the structure of the human MnSOD as a search model. The final refined model included four chains of 199–200 amino acids, four manganese ions, and 745 water molecules, with a crystallographic R-factor of 19.4% and a free R-factor of 23.3%. Like MnSODs of other eukaryotic organisms, A. fumigatus MnSOD forms a homotetramer with the manganese ions coordinated by three histidines, one aspartic acid, and one water molecule. The fungal and the human MnSOD share high similarity on the level of both primary and tertiary structure. We identified conserved amino acids that are solvent exposed in the fungal and the human crystal structure and are therefore potentially involved in IgE-mediated cross-reactivity.

We have selected cyclin-dependent kinase 1 (CDK1), an enzyme participating in the regulation of the cell cycle, as a target in our efforts to discover new antitumor agents. By exploiting available structural information, we designed an ATP-site directed ligand scaffold that allowed us to identify 4-(3-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-ylamino)-benzenesulfonamide as a new potent inhibitor of CDK1 in a subsequent database search. The synthesis and testing of some analogues confirmed the interest of this class of compounds as novel CDK1 inhibitors.

The question of whether a protein whose natural sequence is inverted adopts a stable fold is still under debate. We have determined the 2.1-Å crystal structure of the retro -GCN4 leucine zipper. In contrast to the two-stranded helical coiled-coil GCN4 leucine zipper, the retro -leucine zipper formed a very stable, parallel four-helix bundle, which now lends itself to further structural and functional studies.

The beta-strand 326-333 is a key structural element in the formation of p53 tetramers. To investigate the contribution of its amino acid residues, an alanine scan was performed. The oligomerisation and DNA-binding properties of the mutant proteins were compared with those of wild-type proteins in vitro and analysed on the basis of the crystal structure of the p53 tetramerisation domain at 1.5 A resolution. Two categories of mutant proteins were identified. Phe328Ala, Leu330Ala and Ile332Ala mutant proteins are inactive for DNA binding and oligomerisation, while the Glu326Ala, Tyr327Ala, Thr329Ala, Gln331Ala and Arg333Ala mutant proteins have properties similar to those of wild-type proteins. These results suggest that single mutations within the p53 tetramerisation domain destabilise the structure of the whole protein, inhibiting its DNA-binding activity. Furthermore, the mutation of leucine 330 to alanine within the tetramerisation domain of the Arg175His protein abolishes the dominant negative effect of this mutant. This shows that the beta-strand 326-333 is a key structural element that mediates the dominant negative effect of p53 mutants.

Background: Hirustasin belongs to a class of serine protease inhibitors characterized by a well conserved pattern of cysteine residues. Unlike the closely related inhibitors, antistasin/ghilanten and guamerin, which are selective for coagulation factor Xa or neutrophil elastase, hirustasin binds specifically to tissue kallikrein. The conservation of the pattern of cysteine residues and the significant sequence homology suggest that these related inhibitors possess a similar three-dimensional structure to hirustasin. Results: The crystal structure of the complex between tissue kallikrein and hirustasin was analyzed at 2.4 A resolution. Hirustasin folds into a brick-like structure that is dominated by five disulfide bridges and is sparse in secondary structural elements. The cysteine residues are connected in an abab cdecde pattern that causes the polypeptide chain to fold into two similar motifs. As a hydrophobic core is absent from hirustasin the disulfide bridges maintain the tertiary structure and present the primary binding loop to the active site of the protease. The general structural topography and disulfide connectivity of hirustasin has not previously been described. Conclusions: The crystal structure of the kallikrein–hirustasin complex reveals that hirustasin differs from other serine protease inhibitors in its conformation and its disulfide bond connectivity, making it the prototype for a new class of inhibitor. The disulfide pattern shows that the structure consists of two domains, but only the C-terminal domain interacts with the protease. The disulfide pattern of the N-terminal domain is related to the pattern found in other proteins. Kallikrein recognizes hirustasin by the formation of an antiparallel β sheet between the protease and the inhibitor. The P1 arginine binds in a deep negatively charged pocket of the enzyme. An additional pocket at the periphery of the active site accommodates the sidechain of the P4 valine.

Adenoviruses (Ads) have shown promise as vectors for gene delivery in clinical trials. Efficient viral targeting to a tissue of choice requires both ablation of the virus’ original tropism and engineering of an efficient receptor-mediated uptake by a specific cell population. We have developed a series of adapters binding to the virus with such high affinity that they remain fully bound for >10 d, block its natural receptor binding site and mediate interaction with a surface receptor of choice. The adapter contains two fused modules, both consisting of designed ankyrin repeat proteins (DARPins), one binding to the fiber knob of adenovirus serotype 5 and the other binding to various tumor markers. By solving the crystal structure of the complex of the trimeric knob with three bound DARPins at 1.95-Å resolution, we could use computer modeling to design a link to a trimeric protein of extraordinary kinetic stability, the capsid protein SHP from the lambdoid phage 21. We arrived at a module which binds the knob like a trimeric clamp. When this clamp was fused with DARPins of varying specificities, it enabled adenovirus serotype 5-mediated delivery of a transgene in a human epidermal growth factor receptor 2-, epidermal growth factor receptor-, or epithelial cell adhesion molecule-dependent manner with transduction efficiencies comparable to or even exceeding those of Ad itself. With these adapters, efficiently produced in Escherichia coli , Ad can be converted rapidly to new receptor specificities using any ligand as the receptor-binding moiety. Prefabricated Ads with different payloads thus can be retargeted readily to many cell types of choice.