Markus Grütter - Academia.edu (original) (raw)

Papers by Markus Grütter

Scientific Reports, 2015

Many tripartite motif-containing (TRIM) proteins, comprising RING-finger, B-Box, and coiled-coil ... more 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.

Current opinion in virology, 2012

TRIM5 is a restriction factor that blocks retrovirus infection soon after the virion core enters ... more TRIM5 is a restriction factor that blocks retrovirus infection soon after the virion core enters the cell cytoplasm. Restriction activity is targeted to the virion core via recognition of the capsid protein lattice that encases the viral genomic RNA. In common with all of the many TRIM family members, TRIM5 has RING, B-box, and coiled-coil domains. As an E3 ubiquitin ligase TRIM5 cooperates with the heterodimeric E2, UBC13/UEV1A, to activate the TAK1 (MAP3K7) kinase, NF-κB and AP-1 signaling, and the transcription of inflammatory cytokines and chemokines. TAK1, UBC13, and UEV1A all contribute to TRIM5-mediated retrovirus restriction activity. Interaction of the carboxy-terminal PRYSPRY or cyclophilin domains of TRIM5 with the retroviral capsid lattice stimulates the formation of a complementary lattice by TRIM5, with greatly increased TRIM5 E3 activity, and host cell signal transduction. Structural and biochemical studies on TRIM5 have opened a much needed window on how the innate i...

Biochemical Journal, 2014

Caspases play important roles during apoptosis, inflammation and proliferation. The high homology... more 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.

Nature Structural Biology, 1996

Within the scope of the ALTZOMONI campaign in March 2006 it was possible to sample various times ... more Within the scope of the ALTZOMONI campaign in March 2006 it was possible to sample various times the urban plume of Mexico City at a site located at about 4000 m a.s.l. and approximately 60 km to the southeast of the city. Continuous GC-ECD measurements of peroxiacetic nitric anhydride (PAN) and peroxipropionic nitric anhydride (PPN) were used to determine the

European Journal of Biochemistry, 1996

beta-1,4-Galactosyltransferase (Gal-T, EC 2.4.1.38) transfers galactose (Gal) from UDP-Gal to N-a... more beta-1,4-Galactosyltransferase (Gal-T, EC 2.4.1.38) transfers galactose (Gal) from UDP-Gal to N-acetyl-D-glucosamine or a derivative GlcNAc-R. Soluble Gal-T, purified from human breast milk, was shown to be very heterogeneous by isoelectric focusing (IEF). In order to produce sufficient homogeneous enzyme for three-dimensional analysis, the human enzyme (hGal-T) has been expressed in Saccharomyces cerevisiae, production scaled up to 187 U recombinant Gal-T (rGal-T) and purified. The purification protocol was based on chromatography on concanavalin-A-Sepharose followed by affinity chromatographies on GlcNAc-Sepharose and alpha-lactalbumin-Sepharose. Analysis by SDS/PAGE revealed hyperglycosylation at the single N-glycosylation site, preventing recognition by antibodies. Analysis by IEF revealed considerable heterogeneity of rGal-T. The N-glycan could be removed by treatment with endoglycosidase H (endo H). The N-deglycosylated form of rGal-T retained full activity and showed only three isoforms by IEF analysis. Then we abolished the single N-glycosylation consensus sequence by site-directed mutagenesis changing Asn69-->Asp. The soluble mutated enzyme (N-deglycosylated rGal-T) was expressed in S. cerevisiae and its production scaled up to 60 U.N-deglycosylated rGal-T was purified to electrophoretic homogeneity. When analyzed by IEF, N-deglycosylated rGal-T was resolved in two bands. The O-glycans could be removed by jack bean alpha-mannosidase treatment and the completely deglycosylated Gal-T appeared homogeneous by IEF. The kinetic parameters of N-deglycosylated rGal-T were shown not to differ to any significant extent from those of the hGal-T. No significant changes in CD spectra were observed between hGal-T and N-deglycosylated rGal-T. Light-scattering analysis revealed dimerization of both enzymes. These data indicate that N-deglycosylated rGal-T was correctly folded, homogeneous and thus suitable for crystallization experiments.

The EMBO Journal, 2004

DsbD from Escherichia coli catalyzes the transport of electrons from cytoplasmic thioredoxin to t... more DsbD from Escherichia coli catalyzes the transport of electrons from cytoplasmic thioredoxin to the periplasmic disulfide isomerase DsbC. DsbD contains two periplasmically oriented domains at the N-and C-terminus (nDsbD and cDsbD) that are connected by a central transmembrane (TM) domain. Each domain contains a pair of cysteines that are essential for catalysis. Here, we show that Cys109 and Cys461 form a transient interdomain disulfide bond between nDsbD and cDsbD in the reaction cycle of DsbD. We solved the crystal structure of this catalytic intermediate at 2.85 Å resolution, which revealed large relative domain movements in DsbD as a consequence of a strong overlap between the surface areas of nDsbD that interact with DsbC and cDsbD. In addition, we have measured the kinetics of all functional and nonfunctional disulfide exchange reactions between redox-active, periplasmic proteins and protein domains from the oxidative DsbA/B and the reductive DsbC/D pathway. We show that both pathways are separated by large kinetic barriers for nonfunctional disulfide exchange between components from different pathways.

The EMBO Journal, 2005

Adhesive type 1 pili from uropathogenic Escherichia coli are filamentous protein complexes that a... more Adhesive type 1 pili from uropathogenic Escherichia coli are filamentous protein complexes that are attached to the assembly platform FimD in the outer membrane. During pilus assembly, FimD binds complexes between the chaperone FimC and type 1 pilus subunits in the periplasm and mediates subunit translocation to the cell surface.

The EMBO Journal, 2006

Escherichia coli and other enterobacteria exploit the H þconsuming reaction catalysed by glutamat... more Escherichia coli and other enterobacteria exploit the H þconsuming reaction catalysed by glutamate decarboxylase to survive the stomach acidity before reaching the intestine. Here we show that chloride, extremely abundant in gastric secretions, is an allosteric activator producing a 10-fold increase in the decarboxylase activity at pH 5.6. Cooperativity and sensitivity to chloride were lost when the N-terminal 14 residues, involved in the formation of two triple-helix bundles, were deleted by mutagenesis. X-ray structures, obtained in the presence of the substrate analogue acetate, identified halide-binding sites at the base of each N-terminal helix, showed how halide binding is responsible for bundle stability and demonstrated that the interconversion between active and inactive forms of the enzyme is a stepwise process. We also discovered an entirely novel structure of the cofactor pyridoxal 5 0 -phosphate (aldamine) to be responsible for the reversibly inactivated enzyme. Our results link the entry of chloride ions, via the H þ /Cl À exchange activities of ClC-ec1, to the trigger of the acid stress response in the cell when the intracellular proton concentration has not yet reached fatal values.

Structure, 2006

Dimethylarginine dimethylaminohydrolase (DDAH) is involved in the regulation of nitric oxide synt... more 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, 2008

Adhesive type 1 pili from uropathogenic Escherichia coli strains are heat and denaturant resistan... more Adhesive type 1 pili from uropathogenic Escherichia coli strains are heat and denaturant resistant, filamentous protein complexes. Individual pilus subunits associate through ''donor strand complementation,'' whereby the incomplete immunoglobulin-like fold of each subunit is completed by the N-terminal extension of a neighboring subunit. We show that antiparallel donor strand insertion generally causes nonequilibrium behavior in protein folding and extreme activation energy barriers for dissociation of subunit-subunit complexes. We identify the most kinetically stable, noncovalent protein complex known to date. The complex between the pilus subunit FimG and the donor strand peptide of the subunit FimF shows an extrapolated dissociation half-life of 3 3 10 9 years. The 15 residue peptide forms ideal intermolecular b sheet H-bonds with FimG over 10 residues, and its hydrophobic side chains strongly interact with the hydrophobic core of FimG. The results show that kinetic stability and nonequilibrium behavior in protein folding confers infinite stability against dissociation in extracellular protein complexes.

Structure, 2007

Specific and potent caspase inhibitors are indispensable for the dissection of the intricate path... more 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.

Structure, 2005

DsbD from Escherichia coli transports two electrons from cytoplasmic thioredoxin to the periplasm... more DsbD from Escherichia coli transports two electrons from cytoplasmic thioredoxin to the periplasmic substrate proteins DsbC, DsbG and CcmG. DsbD consists of an N-terminal periplasmic domain (nDsbD), a C-terminal periplasmic domain, and a central transmembrane domain. Each domain possesses two cysteines required for electron transport. Herein, we demonstrate fast (3.9 x 10(5) M(-1)s(-1)) and direct disulfide exchange between nDsbD and CcmG, a highly specific disulfide reductase essential for cytochrome c maturation. We determined the crystal structure of the disulfide-linked complex between nDsbD and the soluble part of CcmG at 1.94 A resolution. In contrast to the other two known complexes of nDsbD with target proteins, the N-terminal segment of nDsbD contributes to specific recognition of CcmG. This and other features, like the possibility of using an additional interaction surface, constitute the structural basis for the adaptability of nDsbD to different protein substrates.

Structure, 1997

Hirustasin belongs to a class of serine protease inhibitors characterized by a well conserved pat... more 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. The crystal structure of the complex between tissue kallikrein and hirustasin was analyzed at 2.4 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. 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 beta 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.

Structure, 1995

The human immunodeficiency virus (HIV) is the causative agent of acquired immunodeficiency syndro... more The human immunodeficiency virus (HIV) is the causative agent of acquired immunodeficiency syndrome (AIDS). Two subtypes of the virus, HIV-1 and HIV-2, have been characterized. The protease enzymes from these two subtypes, which are aspartic acid proteases and have been found to be essential for maturation of the infectious particle, share about 50% sequence identity. Differences in substrate and inhibitor binding between these enzymes have been previously reported. We report the X-ray crystal structures of both HIV-1 and HIV-2 proteases each in complex with the pseudosymmetric inhibitor, CGP 53820, to 2.2 A and 2.3 A, respectively. In both structures, the entire enzyme and inhibitor could be located. The structures confirmed earlier modeling studies. Differences between the CGP 53820 inhibitory binding constants for the two enzymes could be correlated with structural differences. Minor sequence changes in subsites at the active site can explain some of the observed differences in substrate and inhibitor binding between the two enzymes. The information gained from this investigation may help in the design of equipotent HIV-1/HIV-2 protease inhibitors.

Structure, 1999

In the initial stages of Fas-mediated apoptosis the cysteine protease caspase-8 is recruited to t... more In the initial stages of Fas-mediated apoptosis the cysteine protease caspase-8 is recruited to the cell receptor as a zymogen (procaspase-8) and is incorporated into the death-signalling complex. Procaspase-8 is subsequently activated leading to a cascade of proteolytic events, one of them being the activation of caspase-3, and ultimately resulting in cell destruction. Variations in the substrate specificity of different caspases have been reported.

Structure, 1999

Background: Leech-derived inhibitors have a prominent role in the development of new antithrombot... more Background: Leech-derived inhibitors have a prominent role in the development of new antithrombotic drugs, because some of them are able to block the blood coagulation cascade. Hirustasin, a serine protease inhibitor from the leech Hirudo medicinalis, binds specifically to tissue kallikrein and possesses structural similarity with antistasin, a potent factor Xa inhibitor from Haementeria officinalis. Although the 2.4 Å structure

Structure, 2005

example, the mitogen-activated protein kinase p38 1 Department of Biochemistry [Wang et al., 1997... more example, the mitogen-activated protein kinase p38 1 Department of Biochemistry [Wang et al., 1997], the insulin receptor kinase [Hub-University of Zürich bard et al., 1994], or the Abelson tyrosin kinase [ABL] Winterthurerstrasse 190 [Schindler et al., 2000]). It was concluded that APH and CH-8057 Zürich EPKs share a common ancestor, which suggests that Switzerland APH is an ideal model system for all kinases sharing this fold (Hon et al., 1997). EPKs are of great biological and medical importance Summary because of their fundamental role in signal transduction and regulatory pathways in eukaryotic cells. Diseases, Aminoglycoside phosphotransferase (3)-IIIa (APH) is including cancer, inflammation and diabetes, are often a bacterial kinase that confers antibiotic resistance to directly linked to the malfunctioning of EPKs (Noble et many pathogenic bacteria and shares structural hoal., 2004). The human genome encodes a total of 518 mology with eukaryotic protein kinases. We report kinases (Manning et al., 2002). The highly conserved here the crystal structure of APH, trapped in an incatalytic domain of kinases consists of an N-terminal, active conformation by a tailor-made inhibitory anmostly β sheet-containing lobe and a C-terminal, kyrin repeat (AR) protein, at 2.15 Å resolution. The α-helical lobe. The ATP binding pocket is located in the inhibitor was selected from a combinatorial library of groove between the two lobes. Most of the known EPK designed AR proteins. The AR protein binds the inhibitors bind in the conserved ATP binding site, and C-terminal lobe of APH and thereby stabilizes three ␣ a degree of specificity is only achieved by making use helices, which are necessary for substrate binding, in of small individual differences in the ATP binding pocka significantly displaced conformation. BIAcore analets. This often leads to cross-reactivity and unwanted ysis and kinetic enzyme inhibition experiments are side effects of EPK inhibitors (Noble et al., 2004). Alterconsistent with the proposed allosteric inhibition native inhibition mechanisms, not directly or exclusively mechanism. In contrast to most small-molecule kitargeting the active site, have proven to be highly effinase inhibitors, the AR proteins are not restricted to cient, albeit very hard to achieve. A prime example is active site binding, allowing for higher specificity. Inthe drug Gleevec, which only partially binds in the ATP active conformations of pharmaceutically relevant enpocket and inhibits ABL by stabilizing an inactive conzymes, as can be elucidated with the approach preformation of the kinase (Schindler et al., 2000). sented here, represent powerful starting points for We have developed proteinaceous APH inhibitors rational drug design. based on designed ankyrin repeat (AR) proteins (Amstutz et al., 2005). Designed AR proteins (Binz et al., Introduction 2003; Forrer et al., 2003, 2004) are built from single 33 amino acid repeat modules, which stack together to Bacteria have developed a variety of pathways and form elongated protein domains (Sedgwick and Smermechanisms by which to inactivate antibiotics. Aminodon, 1999). Molecules selected from designed AR proglycosides, including kanamycin, amikacin, and streptein libraries have been shown to bind different target tomycin, are widely used in hospital care today, and, proteins with high affinity and specificity (Binz et al., therefore, bacterial resistance to these antibiotics is a 2004). Furthermore, AR proteins are highly stable, wellmajor concern in the health care field (Boehr et al., expressed, and do not contain disulfide bonds, allow-2003). In general, aminoglycosides interact with the ing for intracellular applications. The selected APH inbacterial ribosome, thereby disrupting protein synthehibitors (Amstutz et al., 2005) inhibit the enzyme both sis of the target cell. One way by which resistance to in vitro and in vivo and confer kanamycin and amikacin aminoglycosides can be achieved is by phosphorylasensitivity to a level comparable to the gene knockout. tion of hydroxy groups of the antibiotic. The aminogly-Here, we describe the crystal structure of APH in coside phosphotransferase (3#)-IIIa (APH) is a protocomplex with one of the most potent AR protein inhibitype enzyme for this mechanism (Boehr et al., 2001). tors, AR_3a, to 2.15 Å resolution. It shows that the AR It was found to mediate aminoglycoside resistance in protein binds to the C-terminal lobe of APH outside the Enterococci and Staphylococci. The crystal structure of substrate binding pocket and stabilizes a significantly APH has been determined (Hon et al., 1997), followed altered APH conformation with a distorted active site. by a very detailed functional and structural analysis of Based on the structural data combined with the kinetic the enzymatic mechanism (Thompson et al., 1999, measurements, we suggest an allosteric inhibition mechanism. A comparison to other known allosteric protein *Correspondence: gruetter@bioc.unizh.ch (M.G.G.); plueckthun@ kinase inhibitors reveals similarities and differences in bioc.unizh.ch (A.P.) 2 These authors contributed equally to this work.

Structure, 2009

Caspases are proteases with an active-site cysteine and aspartate specificity in their substrates... more Caspases are proteases with an active-site cysteine and aspartate specificity in their substrates. They are involved in apoptotic cell death and inflammation, and dysfunction of these enzymes is directly linked to a variety of diseases. Caspase-8 initiates an apoptotic pathway triggered by external stimuli. It was previously characterized in its active inhibitor bound state by crystallography. Here we present the solution structure of the monomeric unprocessed catalytic domain of the caspase-8 zymogen, procaspase-8, showing for the first time the position of the linker and flexibility of the active site forming loops. Biophysical studies of carefully designed mutants allowed disentangling dimerization and processing, and we could demonstrate lack of activity of monomeric uncleaved procaspase-8 and of a processed but dimerization-incompetent mutant. The data provide experimental support in so-far unprecedented detail, and reveal why caspase-8 (and most likely other initiator caspases) needs the dimerization platform during activation.

Proteins: Structure, Function, and Bioinformatics, 2005

The 65 kDa human isoform of glutamate decarboxylase, GAD65, plays a central role in neurotransmis... more The 65 kDa human isoform of glutamate decarboxylase, GAD65, plays a central role in neurotransmission in higher vertebrates and is a typical autoantigen in several human autoimmune diseases, such as insulin-dependent diabetes mellitus (IDDM), Stiff-man syndrome and autoimmune polyendocrine syndrome type I. In autoimmune diabetes, an attack of inflammatory cells to endocrine pancreatic beta-cells leads to their complete destruction, eventually resulting in the inability to produce sufficient insulin for the body's requirements. Even though the etiology of beta-cell destruction is still a matter of debate, the role and antigenic potency of GAD65 are widely recognized. Herein a model of GAD65 is presented, which is based on the recently solved crystal structures of mammalian DOPA decarboxylase and of bacterial glutamate decarboxylase. The model provides for the first time a detailed and accurate structure of the GAD65 subunit (all three domains) and of its dimeric quaternary assembly. It reveals the structural basis for specific antibody recognition to GAD65 as opposed to GAD67, the other human isoform, which shares 81% sequence similarity with GAD65 and is much less antigenic. Literature data on monoclonal antibody binding are perfectly consistent with the detailed features of the model, which allows explanation of several findings on GAD65 immunogenicity. Importantly, by analyzing the active site, we identified the residues most likely involved in catalysis and substrate recognition, paving the way for rational mutagenesis studies of the GAD65 reaction mechanism, specificity and inhibition.

Scientific Reports, 2015

Many tripartite motif-containing (TRIM) proteins, comprising RING-finger, B-Box, and coiled-coil ... more 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.

Current opinion in virology, 2012

TRIM5 is a restriction factor that blocks retrovirus infection soon after the virion core enters ... more TRIM5 is a restriction factor that blocks retrovirus infection soon after the virion core enters the cell cytoplasm. Restriction activity is targeted to the virion core via recognition of the capsid protein lattice that encases the viral genomic RNA. In common with all of the many TRIM family members, TRIM5 has RING, B-box, and coiled-coil domains. As an E3 ubiquitin ligase TRIM5 cooperates with the heterodimeric E2, UBC13/UEV1A, to activate the TAK1 (MAP3K7) kinase, NF-κB and AP-1 signaling, and the transcription of inflammatory cytokines and chemokines. TAK1, UBC13, and UEV1A all contribute to TRIM5-mediated retrovirus restriction activity. Interaction of the carboxy-terminal PRYSPRY or cyclophilin domains of TRIM5 with the retroviral capsid lattice stimulates the formation of a complementary lattice by TRIM5, with greatly increased TRIM5 E3 activity, and host cell signal transduction. Structural and biochemical studies on TRIM5 have opened a much needed window on how the innate i...

Biochemical Journal, 2014

Caspases play important roles during apoptosis, inflammation and proliferation. The high homology... more 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.

Nature Structural Biology, 1996

Within the scope of the ALTZOMONI campaign in March 2006 it was possible to sample various times ... more Within the scope of the ALTZOMONI campaign in March 2006 it was possible to sample various times the urban plume of Mexico City at a site located at about 4000 m a.s.l. and approximately 60 km to the southeast of the city. Continuous GC-ECD measurements of peroxiacetic nitric anhydride (PAN) and peroxipropionic nitric anhydride (PPN) were used to determine the

European Journal of Biochemistry, 1996

beta-1,4-Galactosyltransferase (Gal-T, EC 2.4.1.38) transfers galactose (Gal) from UDP-Gal to N-a... more beta-1,4-Galactosyltransferase (Gal-T, EC 2.4.1.38) transfers galactose (Gal) from UDP-Gal to N-acetyl-D-glucosamine or a derivative GlcNAc-R. Soluble Gal-T, purified from human breast milk, was shown to be very heterogeneous by isoelectric focusing (IEF). In order to produce sufficient homogeneous enzyme for three-dimensional analysis, the human enzyme (hGal-T) has been expressed in Saccharomyces cerevisiae, production scaled up to 187 U recombinant Gal-T (rGal-T) and purified. The purification protocol was based on chromatography on concanavalin-A-Sepharose followed by affinity chromatographies on GlcNAc-Sepharose and alpha-lactalbumin-Sepharose. Analysis by SDS/PAGE revealed hyperglycosylation at the single N-glycosylation site, preventing recognition by antibodies. Analysis by IEF revealed considerable heterogeneity of rGal-T. The N-glycan could be removed by treatment with endoglycosidase H (endo H). The N-deglycosylated form of rGal-T retained full activity and showed only three isoforms by IEF analysis. Then we abolished the single N-glycosylation consensus sequence by site-directed mutagenesis changing Asn69-->Asp. The soluble mutated enzyme (N-deglycosylated rGal-T) was expressed in S. cerevisiae and its production scaled up to 60 U.N-deglycosylated rGal-T was purified to electrophoretic homogeneity. When analyzed by IEF, N-deglycosylated rGal-T was resolved in two bands. The O-glycans could be removed by jack bean alpha-mannosidase treatment and the completely deglycosylated Gal-T appeared homogeneous by IEF. The kinetic parameters of N-deglycosylated rGal-T were shown not to differ to any significant extent from those of the hGal-T. No significant changes in CD spectra were observed between hGal-T and N-deglycosylated rGal-T. Light-scattering analysis revealed dimerization of both enzymes. These data indicate that N-deglycosylated rGal-T was correctly folded, homogeneous and thus suitable for crystallization experiments.

The EMBO Journal, 2004

DsbD from Escherichia coli catalyzes the transport of electrons from cytoplasmic thioredoxin to t... more DsbD from Escherichia coli catalyzes the transport of electrons from cytoplasmic thioredoxin to the periplasmic disulfide isomerase DsbC. DsbD contains two periplasmically oriented domains at the N-and C-terminus (nDsbD and cDsbD) that are connected by a central transmembrane (TM) domain. Each domain contains a pair of cysteines that are essential for catalysis. Here, we show that Cys109 and Cys461 form a transient interdomain disulfide bond between nDsbD and cDsbD in the reaction cycle of DsbD. We solved the crystal structure of this catalytic intermediate at 2.85 Å resolution, which revealed large relative domain movements in DsbD as a consequence of a strong overlap between the surface areas of nDsbD that interact with DsbC and cDsbD. In addition, we have measured the kinetics of all functional and nonfunctional disulfide exchange reactions between redox-active, periplasmic proteins and protein domains from the oxidative DsbA/B and the reductive DsbC/D pathway. We show that both pathways are separated by large kinetic barriers for nonfunctional disulfide exchange between components from different pathways.

The EMBO Journal, 2005

Adhesive type 1 pili from uropathogenic Escherichia coli are filamentous protein complexes that a... more Adhesive type 1 pili from uropathogenic Escherichia coli are filamentous protein complexes that are attached to the assembly platform FimD in the outer membrane. During pilus assembly, FimD binds complexes between the chaperone FimC and type 1 pilus subunits in the periplasm and mediates subunit translocation to the cell surface.

The EMBO Journal, 2006

Escherichia coli and other enterobacteria exploit the H þconsuming reaction catalysed by glutamat... more Escherichia coli and other enterobacteria exploit the H þconsuming reaction catalysed by glutamate decarboxylase to survive the stomach acidity before reaching the intestine. Here we show that chloride, extremely abundant in gastric secretions, is an allosteric activator producing a 10-fold increase in the decarboxylase activity at pH 5.6. Cooperativity and sensitivity to chloride were lost when the N-terminal 14 residues, involved in the formation of two triple-helix bundles, were deleted by mutagenesis. X-ray structures, obtained in the presence of the substrate analogue acetate, identified halide-binding sites at the base of each N-terminal helix, showed how halide binding is responsible for bundle stability and demonstrated that the interconversion between active and inactive forms of the enzyme is a stepwise process. We also discovered an entirely novel structure of the cofactor pyridoxal 5 0 -phosphate (aldamine) to be responsible for the reversibly inactivated enzyme. Our results link the entry of chloride ions, via the H þ /Cl À exchange activities of ClC-ec1, to the trigger of the acid stress response in the cell when the intracellular proton concentration has not yet reached fatal values.

Structure, 2006

Dimethylarginine dimethylaminohydrolase (DDAH) is involved in the regulation of nitric oxide synt... more 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, 2008

Adhesive type 1 pili from uropathogenic Escherichia coli strains are heat and denaturant resistan... more Adhesive type 1 pili from uropathogenic Escherichia coli strains are heat and denaturant resistant, filamentous protein complexes. Individual pilus subunits associate through ''donor strand complementation,'' whereby the incomplete immunoglobulin-like fold of each subunit is completed by the N-terminal extension of a neighboring subunit. We show that antiparallel donor strand insertion generally causes nonequilibrium behavior in protein folding and extreme activation energy barriers for dissociation of subunit-subunit complexes. We identify the most kinetically stable, noncovalent protein complex known to date. The complex between the pilus subunit FimG and the donor strand peptide of the subunit FimF shows an extrapolated dissociation half-life of 3 3 10 9 years. The 15 residue peptide forms ideal intermolecular b sheet H-bonds with FimG over 10 residues, and its hydrophobic side chains strongly interact with the hydrophobic core of FimG. The results show that kinetic stability and nonequilibrium behavior in protein folding confers infinite stability against dissociation in extracellular protein complexes.

Structure, 2007

Specific and potent caspase inhibitors are indispensable for the dissection of the intricate path... more 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.

Structure, 2005

DsbD from Escherichia coli transports two electrons from cytoplasmic thioredoxin to the periplasm... more DsbD from Escherichia coli transports two electrons from cytoplasmic thioredoxin to the periplasmic substrate proteins DsbC, DsbG and CcmG. DsbD consists of an N-terminal periplasmic domain (nDsbD), a C-terminal periplasmic domain, and a central transmembrane domain. Each domain possesses two cysteines required for electron transport. Herein, we demonstrate fast (3.9 x 10(5) M(-1)s(-1)) and direct disulfide exchange between nDsbD and CcmG, a highly specific disulfide reductase essential for cytochrome c maturation. We determined the crystal structure of the disulfide-linked complex between nDsbD and the soluble part of CcmG at 1.94 A resolution. In contrast to the other two known complexes of nDsbD with target proteins, the N-terminal segment of nDsbD contributes to specific recognition of CcmG. This and other features, like the possibility of using an additional interaction surface, constitute the structural basis for the adaptability of nDsbD to different protein substrates.

Structure, 1997

Hirustasin belongs to a class of serine protease inhibitors characterized by a well conserved pat... more 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. The crystal structure of the complex between tissue kallikrein and hirustasin was analyzed at 2.4 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. 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 beta 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.

Structure, 1995

The human immunodeficiency virus (HIV) is the causative agent of acquired immunodeficiency syndro... more The human immunodeficiency virus (HIV) is the causative agent of acquired immunodeficiency syndrome (AIDS). Two subtypes of the virus, HIV-1 and HIV-2, have been characterized. The protease enzymes from these two subtypes, which are aspartic acid proteases and have been found to be essential for maturation of the infectious particle, share about 50% sequence identity. Differences in substrate and inhibitor binding between these enzymes have been previously reported. We report the X-ray crystal structures of both HIV-1 and HIV-2 proteases each in complex with the pseudosymmetric inhibitor, CGP 53820, to 2.2 A and 2.3 A, respectively. In both structures, the entire enzyme and inhibitor could be located. The structures confirmed earlier modeling studies. Differences between the CGP 53820 inhibitory binding constants for the two enzymes could be correlated with structural differences. Minor sequence changes in subsites at the active site can explain some of the observed differences in substrate and inhibitor binding between the two enzymes. The information gained from this investigation may help in the design of equipotent HIV-1/HIV-2 protease inhibitors.

Structure, 1999

In the initial stages of Fas-mediated apoptosis the cysteine protease caspase-8 is recruited to t... more In the initial stages of Fas-mediated apoptosis the cysteine protease caspase-8 is recruited to the cell receptor as a zymogen (procaspase-8) and is incorporated into the death-signalling complex. Procaspase-8 is subsequently activated leading to a cascade of proteolytic events, one of them being the activation of caspase-3, and ultimately resulting in cell destruction. Variations in the substrate specificity of different caspases have been reported.

Structure, 1999

Background: Leech-derived inhibitors have a prominent role in the development of new antithrombot... more Background: Leech-derived inhibitors have a prominent role in the development of new antithrombotic drugs, because some of them are able to block the blood coagulation cascade. Hirustasin, a serine protease inhibitor from the leech Hirudo medicinalis, binds specifically to tissue kallikrein and possesses structural similarity with antistasin, a potent factor Xa inhibitor from Haementeria officinalis. Although the 2.4 Å structure

Structure, 2005

example, the mitogen-activated protein kinase p38 1 Department of Biochemistry [Wang et al., 1997... more example, the mitogen-activated protein kinase p38 1 Department of Biochemistry [Wang et al., 1997], the insulin receptor kinase [Hub-University of Zürich bard et al., 1994], or the Abelson tyrosin kinase [ABL] Winterthurerstrasse 190 [Schindler et al., 2000]). It was concluded that APH and CH-8057 Zürich EPKs share a common ancestor, which suggests that Switzerland APH is an ideal model system for all kinases sharing this fold (Hon et al., 1997). EPKs are of great biological and medical importance Summary because of their fundamental role in signal transduction and regulatory pathways in eukaryotic cells. Diseases, Aminoglycoside phosphotransferase (3)-IIIa (APH) is including cancer, inflammation and diabetes, are often a bacterial kinase that confers antibiotic resistance to directly linked to the malfunctioning of EPKs (Noble et many pathogenic bacteria and shares structural hoal., 2004). The human genome encodes a total of 518 mology with eukaryotic protein kinases. We report kinases (Manning et al., 2002). The highly conserved here the crystal structure of APH, trapped in an incatalytic domain of kinases consists of an N-terminal, active conformation by a tailor-made inhibitory anmostly β sheet-containing lobe and a C-terminal, kyrin repeat (AR) protein, at 2.15 Å resolution. The α-helical lobe. The ATP binding pocket is located in the inhibitor was selected from a combinatorial library of groove between the two lobes. Most of the known EPK designed AR proteins. The AR protein binds the inhibitors bind in the conserved ATP binding site, and C-terminal lobe of APH and thereby stabilizes three ␣ a degree of specificity is only achieved by making use helices, which are necessary for substrate binding, in of small individual differences in the ATP binding pocka significantly displaced conformation. BIAcore analets. This often leads to cross-reactivity and unwanted ysis and kinetic enzyme inhibition experiments are side effects of EPK inhibitors (Noble et al., 2004). Alterconsistent with the proposed allosteric inhibition native inhibition mechanisms, not directly or exclusively mechanism. In contrast to most small-molecule kitargeting the active site, have proven to be highly effinase inhibitors, the AR proteins are not restricted to cient, albeit very hard to achieve. A prime example is active site binding, allowing for higher specificity. Inthe drug Gleevec, which only partially binds in the ATP active conformations of pharmaceutically relevant enpocket and inhibits ABL by stabilizing an inactive conzymes, as can be elucidated with the approach preformation of the kinase (Schindler et al., 2000). sented here, represent powerful starting points for We have developed proteinaceous APH inhibitors rational drug design. based on designed ankyrin repeat (AR) proteins (Amstutz et al., 2005). Designed AR proteins (Binz et al., Introduction 2003; Forrer et al., 2003, 2004) are built from single 33 amino acid repeat modules, which stack together to Bacteria have developed a variety of pathways and form elongated protein domains (Sedgwick and Smermechanisms by which to inactivate antibiotics. Aminodon, 1999). Molecules selected from designed AR proglycosides, including kanamycin, amikacin, and streptein libraries have been shown to bind different target tomycin, are widely used in hospital care today, and, proteins with high affinity and specificity (Binz et al., therefore, bacterial resistance to these antibiotics is a 2004). Furthermore, AR proteins are highly stable, wellmajor concern in the health care field (Boehr et al., expressed, and do not contain disulfide bonds, allow-2003). In general, aminoglycosides interact with the ing for intracellular applications. The selected APH inbacterial ribosome, thereby disrupting protein synthehibitors (Amstutz et al., 2005) inhibit the enzyme both sis of the target cell. One way by which resistance to in vitro and in vivo and confer kanamycin and amikacin aminoglycosides can be achieved is by phosphorylasensitivity to a level comparable to the gene knockout. tion of hydroxy groups of the antibiotic. The aminogly-Here, we describe the crystal structure of APH in coside phosphotransferase (3#)-IIIa (APH) is a protocomplex with one of the most potent AR protein inhibitype enzyme for this mechanism (Boehr et al., 2001). tors, AR_3a, to 2.15 Å resolution. It shows that the AR It was found to mediate aminoglycoside resistance in protein binds to the C-terminal lobe of APH outside the Enterococci and Staphylococci. The crystal structure of substrate binding pocket and stabilizes a significantly APH has been determined (Hon et al., 1997), followed altered APH conformation with a distorted active site. by a very detailed functional and structural analysis of Based on the structural data combined with the kinetic the enzymatic mechanism (Thompson et al., 1999, measurements, we suggest an allosteric inhibition mechanism. A comparison to other known allosteric protein *Correspondence: gruetter@bioc.unizh.ch (M.G.G.); plueckthun@ kinase inhibitors reveals similarities and differences in bioc.unizh.ch (A.P.) 2 These authors contributed equally to this work.

Structure, 2009

Caspases are proteases with an active-site cysteine and aspartate specificity in their substrates... more Caspases are proteases with an active-site cysteine and aspartate specificity in their substrates. They are involved in apoptotic cell death and inflammation, and dysfunction of these enzymes is directly linked to a variety of diseases. Caspase-8 initiates an apoptotic pathway triggered by external stimuli. It was previously characterized in its active inhibitor bound state by crystallography. Here we present the solution structure of the monomeric unprocessed catalytic domain of the caspase-8 zymogen, procaspase-8, showing for the first time the position of the linker and flexibility of the active site forming loops. Biophysical studies of carefully designed mutants allowed disentangling dimerization and processing, and we could demonstrate lack of activity of monomeric uncleaved procaspase-8 and of a processed but dimerization-incompetent mutant. The data provide experimental support in so-far unprecedented detail, and reveal why caspase-8 (and most likely other initiator caspases) needs the dimerization platform during activation.

Proteins: Structure, Function, and Bioinformatics, 2005

The 65 kDa human isoform of glutamate decarboxylase, GAD65, plays a central role in neurotransmis... more The 65 kDa human isoform of glutamate decarboxylase, GAD65, plays a central role in neurotransmission in higher vertebrates and is a typical autoantigen in several human autoimmune diseases, such as insulin-dependent diabetes mellitus (IDDM), Stiff-man syndrome and autoimmune polyendocrine syndrome type I. In autoimmune diabetes, an attack of inflammatory cells to endocrine pancreatic beta-cells leads to their complete destruction, eventually resulting in the inability to produce sufficient insulin for the body's requirements. Even though the etiology of beta-cell destruction is still a matter of debate, the role and antigenic potency of GAD65 are widely recognized. Herein a model of GAD65 is presented, which is based on the recently solved crystal structures of mammalian DOPA decarboxylase and of bacterial glutamate decarboxylase. The model provides for the first time a detailed and accurate structure of the GAD65 subunit (all three domains) and of its dimeric quaternary assembly. It reveals the structural basis for specific antibody recognition to GAD65 as opposed to GAD67, the other human isoform, which shares 81% sequence similarity with GAD65 and is much less antigenic. Literature data on monoclonal antibody binding are perfectly consistent with the detailed features of the model, which allows explanation of several findings on GAD65 immunogenicity. Importantly, by analyzing the active site, we identified the residues most likely involved in catalysis and substrate recognition, paving the way for rational mutagenesis studies of the GAD65 reaction mechanism, specificity and inhibition.