Combined inhibition of caspase 3 and caspase 7 by two highly selective DARPins slows down cellular demise.

Caspases play important roles during apoptosis, inflammation and proliferation. The high homology among family members makes selective targeting of individual caspases difficult, which is necessary to precisely define the role of these enzymes. We have selected caspase-7-specific binders from a library of DARPins (designed ankyrin repeat proteins). The DARPins D7.18 and D7.43 bind specifically to procaspase 7 and active caspase 7, but not to other members of the family. Binding of the DARPins does not affect the active enzyme, but interferes with its activation by other caspases. The crystal structure of the caspase 7-D7.18 complex elucidates the high selectivity and the mode of inhibition. Combining these caspase-7-specific DARPins with the previously reported caspase-3-inhibitory DARPin D3.4S76R reduces the activity of caspase 3 and 7 in double-transfected HeLa cells during apoptosis. In addition, these cells showed less susceptibility to TRAIL (tumour-necrosis-factor-related apoptosis-inducing ligand)-induced apoptosis in living cell experiments. D7.18 and D7.43 are therefore novel tools for in vitro studies on procaspase 7 activation as well as for clarifying the role of its activation in different cellular processes. If applied in combination with D3.4S76R, they represent an excellent instrument to increase our understanding of these enzymes during various cellular processes.

The structure of Bradyrhizobium japonicum transcription factor FixK2 unveils sites of DNA binding and oxidation.

FixK2 is a regulatory protein that activates a large number of genes for the anoxic and microoxic, endosymbiotic, and nitrogen-fixing life styles of the α-proteobacterium Bradyrhizobium japonicum. FixK2 belongs to the cAMP receptor protein (CRP) superfamily. Although most CRP family members are coregulated by effector molecules, the activity of FixK2 is negatively controlled by oxidation of its single cysteine (Cys-183) located next to the DNA-binding domain and possibly also by proteolysis. Here, we report the three-dimensional x-ray structure of FixK2, a representative of the FixK subgroup of the CRP superfamily. Crystallization succeeded only when (i) an oxidation- and protease-insensitive protein variant (FixK2(C183S)-His6) was used in which Cys-183 was replaced with serine and the C terminus was fused with a hexahistidine tag and (ii) this protein was allowed to form a complex with a 30-mer double-stranded target DNA. The structure of the FixK2-DNA complex was solved at a resolution of 1.77 Å, at which the protein formed a homodimer. The precise protein-DNA contacts were identified, which led to an affirmation of the canonical target sequence, the so-called FixK2 box. The C terminus is surface-exposed, which might explain its sensitivity to specific cleavage and degradation. The oxidation-sensitive Cys-183 is also surface-exposed and in close proximity to DNA. Therefore, we propose a mechanism whereby the oxo acids generated after oxidation of the cysteine thiol cause an electrostatic repulsion, thus preventing specific DNA binding.

Specific inhibition of caspase-3 by a competitive DARPin: molecular mimicry between native and designed inhibitors.

Dysregulation of apoptosis is associated with several human diseases. The main apoptotic mediators are caspases, which propagate death signals to downstream targets. Executioner caspase-3 is responsible for the majority of cleavage events and its therapeutic potential is of high interest with to date several available active site peptide inhibitors. These molecules inhibit caspase-3, but also homologous caspases. Here, we describe caspase-3 specific inhibitors D3.4 and D3.8, which have been selected from a library of designed ankyrin repeat proteins (DARPins). The crystal structures of D3.4 and mutants thereof show how high specificity and inhibition is achieved. They also show similarities in the binding mode with that of the natural caspase inhibitor XIAP (X-linked inhibitor of apoptosis). The kinetic data reveal a competitive inhibition mechanism. D3.4 is specific for caspase-3 and does not bind the highly homologous caspase-7. D3.4 therefore is an excellent tool to define the precise role of caspase-3 in the various apoptotic pathways.

Crystal structures of BapA complexes with ß-lactam-derived inhibitors illustrate substrate specificity and enantioselectivity of ß-aminopeptidases.

ß-Aminopeptidases have exclusive biocatalytic potential because they react with peptides composed of ß-amino acids, which serve as building blocks for the design of non-natural peptidomimetics. We have identified the ß-lactam antibiotic ampicillin and the ampicillin-derived penicilloic acid as novel inhibitors of the ß-aminopeptidase BapA from Sphingosinicella xenopeptidilytica (K(i) values of 0.69 and 0.74 mM, respectively). We report high-resolution crystal structures of BapA in noncovalent complexes with these inhibitors and with the serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride. All three inhibitors showed similar binding characteristics; the aromatic moiety extended into a hydrophobic binding pocket of the active site, and the free amino group formed a salt bridge with Glu133 of BapA. The exact position of the inhibitors and structural details of the ligand binding pocket illustrate the specificity and the enantioselectivity of BapA-catalyzed reactions with ß-peptide substrates.

Autoproteolytic and catalytic mechanisms for the ß-aminopeptidase BapA--a member of the Ntn hydrolase family.

The ß-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 ß-amino acid residues from peptides. We determined the crystal structures of the native (αß)4 heterooctamer and of the 153 kDa precursor homotetramer at a resolution of 1.45 and 1.8 Å, 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.

Crystal structure of a heterodimeric ABC transporter in its inward-facing conformation.

ATP-binding cassette (ABC) transporters shuttle a wide variety of molecules across cell membranes by alternating between inward- and outward-facing conformations, harnessing the energy of ATP binding and hydrolysis at their nucleotide binding domains (NBDs). Here we present the 2.9-Å crystal structure of the heterodimeric ABC transporter TM287-TM288 (TM287/288) from Thermotoga maritima in its inward-facing state. In contrast to previous studies, we found that the NBDs only partially separate, remaining in contact through an interface involving conserved motifs that connect the two ATP hydrolysis sites. We observed AMP-PNP binding to the degenerate catalytic site, which deviates from the consensus sequence in the same positions as the eukaryotic homologs CFTR and TAP1-TAP2 (TAP1/2). The TM287/288 structure provides unprecedented insights into the mechanism of heterodimeric ABC exporters and will enable future studies on this large transporter superfamily.

Analysis of AcrB and AcrB/DARPin ligand complexes by LILBID MS.

The AcrA/AcrB/TolC complex is responsible for intrinsic multidrug resistance (MDR) in Escherichia coli. Together with the periplasmic adaptor protein AcrA and the outer membrane channel TolC, the inner membrane component AcrB forms an efflux complex that spans both the inner and outer membrane and bridges the periplasm of the Gram-negative cell. Within the entire tripartite complex, homotrimeric AcrB plays a central role in energy transduction and substrate selection. In vitro selected designed ankyrin repeat proteins (DARPin) that specifically bind to the periplasmic domain of AcrB were shown to ameliorate diffraction resolution of AcrB/DARPin protein co-crystals (G. Sennhauser, P. Amstutz, C. Briand, O. Storchenegger, M.G. Grutter, Drug export pathway of multidrug exporter AcrB revealed by DARPin inhibitors, PLoS Biol 5 (2007) e7). Structural analysis by X-ray crystallography revealed that 2 DARPin molecules were bound to the trimeric AcrB wildtype protein in the crystal, whereas the V612F and G616N AcrB variant crystal structures show 3 DARPin molecules bound to the trimer. These specific stoichiometric differences were analyzed in solution via densitometry after microchannel electrophoresis, analytical ultracentrifugation and via laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS). Using the latter technology, we investigated the gradual disassembly of the AcrB trimer and bound DARPin ligands in dependence on laser intensity in solution. At low laser intensity, the release of the detergent molecule micelle from the AcrB/DARPin complex was observed. By increasing laser intensity, dimeric and monomeric AcrB species with bound DARPin molecules were detected showing the high affinity binding of DARPin to monomeric AcrB species. High laser intensity LILBID MS experiments indicated a spectral shift of the monomeric AcrB peak of 3.1kDa, representing a low molecular weight ligand in all detergent-solubilized AcrB samples and in the AcrB crystal. The identity of this ligand was further investigated using phospholipid analysis of purified AcrB and AcrB variant samples, and indicated the presence of phosphatidylethanolamine and possibly cardiolipin, both constituents of the Escherichia coli membrane.

Designed ankyrin repeat protein binders for the crystallization of AcrB: plasticity of the dominant interface.

The formation of well-diffracting crystals is a major bottleneck in structural analysis of membrane proteins by X-ray crystallography. One approach to improve crystal quality is the use of DARPins as crystallization chaperones. Here, we present a detailed analysis of the interaction between DARPins and the integral membrane protein AcrB. We find that binders selected in vitro by ribosome display share a common epitope. The comparative analysis of three crystal structures of AcrB-DARPin complexes allowed us to study the plasticity of the interaction with this dominant binding site. Seemingly redundant AcrB-DARPin crystals show substantially different diffraction quality as a result of subtle differences in the binding geometry. This work exemplifies the importance to screen a number of crystallization chaperones to obtain optimal diffraction data. Crystallographic analysis is complemented by biophysical characterization of nine AcrB binders. We observe that small variations in the interface can lead to differing behavior of the DARPins with regards to affinity, stoichiometry of the complexes and specificity for their target.

The crystal structure of caspase-6, a selective effector of axonal degeneration.

Neurodegenerative diseases pose one of the most pressing unmet medical needs today. It has long been recognized that caspase-6 may play a role in several neurodegenerative diseases for which there are currently no disease-modifying therapies. Thus it is a potential target for neurodegenerative drug development. In the present study we report on the biochemistry and structure of caspase-6. As an effector caspase, caspase-6 is a constitutive dimer independent of the maturation state of the enzyme. The ligand-free structure shows caspase-6 in a partially mature but latent conformation. The cleaved inter-domain linker remains partially inserted in the central groove of the dimer, as observed in other caspases. However, in contrast with the structures of other caspases, not only is the catalytic machinery misaligned, but several structural elements required for substrate recognition are missing. Most importantly, residues forming a short anti-parallel beta-sheet abutting the substrate in other caspase structures are part of an elongation of the central alpha-helix. Despite the dramatic structural changes that are required to adopt a canonical catalytically competent conformation, the pre-steady-state kinetics exhibit no lag phase in substrate turnover. This suggests that the observed conformation does not play a regulatory role in caspase-6 activity. However, targeting the latent conformation in search for specific and bio-available caspase-6 inhibitors might offer an alternative to active-site-directed approaches.

Crystal structure of the multidrug exporter MexB from Pseudomonas aeruginosa.

We report here the crystal structure of the Pseudomonas aeruginosa multidrug exporter MexB, an intensively studied member of the resistance-nodulation-cell division family of secondary active transporters, at 3.0 A. MexB forms an asymmetric homotrimer where each subunit adopts a different conformation representing three snapshots of the transport cycle similar to the recently determined structures of its close homologue AcrB from Escherichia coli, so far the sole structurally characterized member of the superfamily. As for AcrB, the conformations of two subunits can be clearly assigned to either the binding step or the extrusion step in the transport process. Unexpectedly, a remarkable conformational shift in the third subunit is observed in MexB, which has potential implications for the assembly of the tripartite MexAB-OprM drug efflux system. Furthermore, an n-dodecyl-d-maltoside molecule was found bound to the internal multidrug-binding cavity, which might indicate that MexB binds and transports detergent molecules as substrates. As the only missing piece of the puzzle in the MexAB-OprM system, the X-ray structure of MexB completes the molecular picture of the major pump mediating intrinsic and acquired multidrug resistance in P. aeruginosa.

Inhibition of caspase-2 by a designed ankyrin repeat protein: specificity, structure, and inhibition mechanism.

Specific and potent caspase inhibitors are indispensable for the dissection of the intricate pathways leading to apoptosis. We selected a designed ankyrin repeat protein (DARPin) from a combinatorial library that inhibits caspase-2 in vitro with a subnanomolar inhibition constant and, in contrast to the peptidic caspase inhibitors, with very high specificity for this particular caspase. The crystal structure of this inhibitor (AR_F8) in complex with caspase-2 reveals the molecular basis for the specificity and, together with kinetic analyses, the allosteric mechanism of inhibition. The structure also shows a conformation of the active site that can be exploited for the design of inhibitory compounds. AR_F8 is a specific inhibitor of an initiator caspase and has the potential to help identify the function of caspase-2 in the complex biological apoptotic signaling network.

Drug export pathway of multidrug exporter AcrB revealed by DARPin inhibitors.

The multidrug exporter AcrB is the inner membrane component of the AcrAB-TolC drug efflux system in Escherichia coli and is responsible for the resistance of this organism to a wide range of drugs. Here we describe the crystal structure of the trimeric AcrB in complex with a designed ankyrin-repeat protein (DARPin) inhibitor at 2.5-A resolution. The three subunits of AcrB are locked in different conformations revealing distinct channels in each subunit. There seems to be remote conformational coupling between the channel access, exit, and the putative proton-translocation site, explaining how the proton motive force is used for drug export. Thus our structure suggests a transport pathway not through the central pore but through the identified channels in the individual subunits, which greatly advances our understanding of the multidrug export mechanism.

Structure of the mammalian NOS regulator dimethylarginine dimethylaminohydrolase: A basis for the design of specific inhibitors.

Dimethylarginine dimethylaminohydrolase (DDAH) is involved in the regulation of nitric oxide synthase (NOS) by metabolizing the free endogenous arginine derivatives N(omega)-methyl-L-arginine (MMA) and N(omega),N(omega)-dimethyl-L-arginine (ADMA), which are competitive inhibitors of NOS. Here, we present high-resolution crystal structures of DDAH isoform 1 (DDAH-1) isolated from bovine brain in complex with different inhibitors, including S-nitroso-L-homocysteine and Zn2+, a regulator of this mammalian enzyme. The structure of DDAH-1 consists of a propeller-like fold similar to other arginine-modifying enzymes and a flexible loop, which adopts different conformations and acts as a lid at the entrance of the active site. The orientation and interaction mode of inhibitors in the active site give insight into the regulation and the molecular mechanism of the enzyme. The presented structures provide a basis for the structure-based development of specific DDAH-1 inhibitors that might be useful in the therapeutic treatment of NOS dysfunction-related diseases.

Structure of the PRYSPRY-domain: implications for autoinflammatory diseases.

We determined the first structure of PRYSPRY, a domain found in over 500 different proteins, involved in innate immune signaling, cytokine signaling suppression, development, cell growth and retroviral restriction. The fold encompasses a 7-stranded and a 6-stranded antiparallel beta-sheet, arranged in a beta-sandwich. In the crystal, PRYSPRY forms a dimer where the C-terminus of an acceptor molecule binds to the concave surface of a donor molecule, which represents a putative interaction site. Mutations in the PRYSPRY domains of Pyrin, which are responsible for familial Mediterranean fever, map on the putative PRYSPRY interaction site.

Allosteric inhibition of aminoglycoside phosphotransferase by a designed ankyrin repeat protein.

Aminoglycoside phosphotransferase (3')-IIIa (APH) is a bacterial kinase that confers antibiotic resistance to many pathogenic bacteria and shares structural homology with eukaryotic protein kinases. We report here the crystal structure of APH, trapped in an inactive conformation by a tailor-made inhibitory ankyrin repeat (AR) protein, at 2.15 A resolution. The inhibitor was selected from a combinatorial library of designed AR proteins. The AR protein binds the C-terminal lobe of APH and thereby stabilizes three alpha helices, which are necessary for substrate binding, in a significantly displaced conformation. BIAcore analysis and kinetic enzyme inhibition experiments are consistent with the proposed allosteric inhibition mechanism. In contrast to most small-molecule kinase inhibitors, the AR proteins are not restricted to active site binding, allowing for higher specificity. Inactive conformations of pharmaceutically relevant enzymes, as can be elucidated with the approach presented here, represent powerful starting points for rational drug design.

High-affinity binders selected from designed ankyrin repeat protein libraries.

We report here the evolution of ankyrin repeat (AR) proteins in vitro for specific, high-affinity target binding. Using a consensus design strategy, we generated combinatorial libraries of AR proteins of varying repeat numbers with diversified binding surfaces. Libraries of two and three repeats, flanked by 'capping repeats,' were used in ribosome-display selections against maltose binding protein (MBP) and two eukaryotic kinases. We rapidly enriched target-specific binders with affinities in the low nanomolar range and determined the crystal structure of one of the selected AR proteins in complex with MBP at 2.3 A resolution. The interaction relies on the randomized positions of the designed AR protein and is comparable to natural, heterodimeric protein-protein interactions. Thus, our AR protein libraries are valuable sources for binding molecules and, because of the very favorable biophysical properties of the designed AR proteins, an attractive alternative to antibody libraries.

Crystal structure of caspase-2, apical initiator of the intrinsic apoptotic pathway.

The cell death protease caspase-2 has recently been recognized as the most apical caspase in the apoptotic cascade ignited during cell stress signaling. Cytotoxic stress, such as that caused by cancer therapies, leads to activation of caspase-2, which acts as a direct effector of the mitochondrion-dependent apoptotic pathway resulting in programmed cell death. Here we report the x-ray structure of caspase-2 in complex with the inhibitor acetyl-Leu-Asp-Glu-Ser-Asp-aldehyde at 1.65-A resolution. Compared with other caspases, significant structural differences prevail in the active site region and the dimer interface. The structure reveals the hydrophobic properties of the S5 specificity pocket, which is unique to caspase-2, and provides the details of the inhibitor-protein interactions in subsites S1-S4. These features form the basis of caspase-2 specificity and allow the design of caspase-2-directed ligands for medical and analytical use. Another unique feature of caspase-2 is a disulfide bridge at the dimer interface, which covalently links the two monomers. Consistent with this finding, caspase-2 exists as a (p19/p12)2 dimer in solution, even in the absence of substrates or inhibitors. The intersubunit disulfide bridge stabilizes the dimeric form of caspase-2, whereas all other long prodomain caspases exist as monomers in solution, and dimer formation is driven by ligand binding. Therefore, the central disulfide bridge appears to represent a novel way of dimer stabilization in caspases.

Structure and oligomeric state of the mammalian tumour-associated antigen UK114.

The tumour-associated antigen UK114, isolated from goat liver, belongs to the YER057c/YIL051c/YjgF protein family, which has members in both the prokaryotes and eukaryotes. The crystal structure of a mammalian representative, goat UK114, was determined, revealing a trimeric arrangement in the crystal. It was confirmed by ultracentrifugation that UK114 is a trimer in solution. These results are in agreement with the published structures of homologues from unicellular organisms, but contrast with those reported for the rat homologue of UK114, for which a dimeric quaternary structure was proposed.

Insights into the regulatory mechanism for caspase-8 activation.

In the death receptor induced apoptotic pathway, caspase-8 autocatalytically cleaves itself at specific cleavage sites. To better understand the regulatory mechanisms behind caspase-8 activation, we compared active wild-type caspase-8 (wtC8) and an uncleavable form of procaspase-8 (uncleavable C8). We demonstrate that wtC8 predominantly exists as a monomer and dimerizes in a concentration and inhibitor binding-dependent fashion. The K(D) for dimeric wtC8 is approximately 50 micro M and decreases when inhibitor bound. Uncleavable C8 is mainly monomeric, but a small amount that dimerizes is as active as wtC8. Inhibitor binding does not favor dimerization but induces active site rearrangements in uncleavable C8. Our findings suggest that dimerization is the crucial factor for caspase-8 activation.

The long form of FLIP is an activator of caspase-8 at the Fas death-inducing signaling complex.

Death receptors, such as Fas and tumor necrosis factor-related apoptosis-inducing ligand receptors, recruit Fas-associated death domain and pro-caspase-8 homodimers, which are then autoproteolytically activated. Active caspase-8 is released into the cytoplasm, where it cleaves various proteins including pro-caspase-3, resulting in apoptosis. The cellular Fas-associated death domain-like interleukin-1-beta-converting enzyme-inhibitory protein long form (FLIP(L)), a structural homologue of caspase-8 lacking caspase activity because of several mutations in the active site, is a potent inhibitor of death receptor-induced apoptosis. FLIP(L) is proposed to block caspase-8 activity by forming a proteolytically inactive heterodimer with caspase-8. In contrast, we propose that FLIP(L)-bound caspase-8 is an active protease. Upon heterocomplex formation, a limited caspase-8 autoprocessing occurs resulting in the generation of the p43/41 and the p12 subunits. This partially processed form but also the non-cleaved FLIP(L)-caspase-8 heterocomplex are proteolytically active because they both bind synthetic substrates efficiently. Moreover, FLIP(L) expression favors receptor-interacting kinase (RIP) processing within the Fas-signaling complex. We propose that FLIP(L) inhibits caspase-8 release-dependent pro-apoptotic signals, whereas the single, membrane-restricted active site of the FLIP(L)-caspase-8 heterocomplex is proteolytically active and acts on local substrates such as RIP.