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Markus G Grütter

Publications and source records attributed to Markus G Grütter.

At least 19 recordsLinked to original sources
PubMed2007

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.

Ankyrin Repeat↗
PubMed2006

Evidence for a novel domain of bacterial outer membrane ushers.

Many pathogenic bacteria possess adhesive surface organelles (called pili), anchored to their outer membrane, which mediate the first step of infection by binding to host tissue. Pilus biogenesis occurs via the "chaperone-usher" pathway: the usher, a large outer membrane protein, binds complexes of a periplasmic chaperone with pilus subunits, unloads the subunits from the chaperone, and assembles them into the pilus, which is extruded into the extracellular space. Ushers comprise an N-terminal periplasmic domain, a large transmembrane beta-barrel central domain, and a C-terminal periplasmic domain. Since structural data are available only for the N-terminal domain, we performed an in-depth bioinformatic analysis of bacterial ushers. Our analysis led us to the conclusion that the transmembrane beta-barrel region of ushers contains a so far unrecognized soluble domain, the "middle domain", which possesses a beta-sandwich fold. Two other bacterial beta-sandwich domains, the TT0351 protein from Thermus thermophilus and the carbohydrate binding module CBM36 from Paenibacillus polymyxa, are possible distant relatives of the usher "middle domain". Several mutations reported to abolish in vivo pilus formation cluster in this region, underlining its functional importance.

Bacterial Outer Membrane Proteins↗
PubMed2006

Opportunities for structure-based design of protease-directed drugs.

As a result of the recent enormous technological progress, experimental structure determination has become an integral part of the development of drugs against disease-related target proteins. The post-translational modification of proteins is an important regulatory process in living organisms; one such example is lytic processing by peptidases. Many different peptidases represent disease targets and are being used in structure-based drug design approaches. The development of drugs such as aliskiren and tipranavir, which inhibit renin and HIV protease, respectively, testifies to the success of this approach.

Aspartic Acid Endopeptidases↗
PubMed2006

Crystal structure of a consensus-designed ankyrin repeat protein: implications for stability.

Consensus-designed ankyrin repeat (AR) proteins are thermodynamically very stable. The structural analysis of the designed AR protein E3_5 revealed that this stability is due to a regular fold with highly conserved structural motifs and H-bonding networks. However, the designed AR protein E3_19 exhibits a significantly lower stability than E3_5 (9.6 vs. 14.8 kcal/mol), despite 88% sequence identity. To investigate the structural correlations of this stability difference between E3_5 and E3_19, we determined the crystal structure of E3_19 at 1.9 A resolution. E3_19 as well has a regular AR domain fold with the characteristic H-bonding patterns. All structural features of the E3_5 and E3_19 molecules appear to be virtually identical (RMSD(Calpha) approximately 0.7 A). However, clear differences are observed in the surface charge distribution of the two AR proteins. E3_19 features clusters of charged residues and more exposed hydrophobic residues than E3_5. The atomic coordinates of E3_19 have been deposited in the Protein Data Bank. PDB ID: 2BKG.

Amino Acid Sequence↗
PubMed2006

Molecular dynamics study of the stabilities of consensus designed ankyrin repeat proteins.

Two designed ankyrin repeat (AR) proteins (E3_5 and E3_19) are high homologous (with about 87% sequence identity) and their crystal structures have a Calpha atom-positional root-mean-square difference of about 0.14 nm. However, it was found that E3_5 is considerably more stable than E3_19 in guanidinium hydrochloride and thermal denaturation experiments. With the goal of providing insights into the various factors contributing to the stabilities of the designed AR proteins and suggesting possible mutations to enhance their stabilities, homology modeling and molecular dynamics (MD) simulations with explicit solvent have been performed. Because the crystal structure of E3_19 was solved later than that of E3_5, a homology model of E3_19 based on the crystal structure of E3_5 was also used in the simulations. E3_5 shows a very stable trajectory in both crystal and solution simulations. In contrast, the C-terminal repeat of E3_19 unfolds in the simulations starting from either the modeled structure or the crystal structure, although it has a sequence identical to that of E3_5. A continuum electrostatic model was used to estimate the effect of single mutations on protein stability and to study the interaction between the internal ARs and the C-terminal capping AR. Mutations involving charged residues were found to have large effects on stability. Due to the difference in charge distribution in the internal ARs of E3_19 and E3_5, their interaction with the C-terminal capping AR is less favorable in E3_19. The simulation trajectories suggest that the stability of the designed AR proteins can be increased by optimizing the electrostatic interactions within and between the different repeats.

Ankyrin Repeat↗
PubMed2006

Design, synthesis, and evaluation of aza-peptide Michael acceptors as selective and potent inhibitors of caspases-2, -3, -6, -7, -8, -9, and -10.

Aza-peptide Michael acceptors are a novel class of inhibitors that are potent and specific for caspases-2, -3, -6, -7, -8, -9, and -10. The second-order rate constants are in the order of 10(6) M(-1) s(-1). The aza-peptide Michael acceptor inhibitor 18t (Cbz-Asp-Glu-Val-AAsp-trans-CH=CH-CON(CH(2)-1-Naphth)(2) is the most potent compound and it inhibits caspase-3 with a k(2) value of 5620000 M(-1) s(-1). The inhibitor 18t is 13700, 190, 6.4, 594, 37500, and 173-fold more selective for caspase-3 over caspases-2, -6, -7, -8, -9, and -10, respectively. Aza-peptide Michael acceptors designed with caspase specific sequences are selective and do not show any cross reactivity with clan CA cysteine proteases such as papain, cathepsin B, and calpains. High-resolution crystal structures of caspase-3 and caspase-8 in complex with aza-peptide Michael acceptor inhibitors demonstrate the nucleophilic attack on C2 and provide insight into the selectivity and potency of the inhibitors with respect to the P1' moiety.

Aza Compounds↗
PubMed2006

Exploring the S4 and S1 prime subsite specificities in caspase-3 with aza-peptide epoxide inhibitors.

Caspase-3 is a prototypic executioner caspase that plays a central role in apoptosis. Aza-peptide epoxides are a novel class of irreversible inhibitors that are highly specific for clan CD cysteine proteases. The five crystal structures of caspase-3-aza-peptide epoxide inhibitor complexes reported here reveal the structural basis for the mechanism of inhibition and the specificities at the S1' and the S4 subsites. Unlike the clan CA cysteine proteases, the catalytic histidine in caspase-3 plays a critical role during protonation and subsequent ring opening of the epoxide moiety and facilitates the nucleophilic attack by the active site cysteine. The nucleophilic attack takes place on the C3 carbon atom of the epoxide and results in an irreversible alkylation of the active site cysteine residue. A favorable network of hydrogen bonds involving the oxyanion hole, catalytic histidine, and the atoms in the prime site of the inhibitor enhance the binding affinity and specificity of the aza-peptide epoxide inhibitors toward caspase-3. The studies also reveal that subtle movements of the N-terminal loop of the beta-subunit occur when the P4 Asp is replaced by a P4 Ile, whereas the N-terminal loop and the safety catch Asp179 are completely disordered when the P4 Asp is replaced by P4 Cbz group.

Aza Compounds↗
PubMed2006

Extended substrate recognition in caspase-3 revealed by high resolution X-ray structure analysis.

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.

Binding Sites↗
PubMed2006

Escherichia coli acid resistance: pH-sensing, activation by chloride and autoinhibition in GadB.

Escherichia coli and other enterobacteria exploit the H+ -consuming 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'-phosphate (aldamine) to be responsible for the reversibly inactivated enzyme. Our results link the entry of chloride ions, via the H+/Cl- 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.

Acids↗
PubMed2006

High-resolution structures of Escherichia coli cDsbD in different redox states: A combined crystallographic, biochemical and computational study.

Escherichia coli DsbD transports electrons from cytoplasmic thioredoxin to periplasmic target proteins. DsbD is composed of an N-terminal (nDsbD) and a C-terminal (cDsbD) periplasmic domain, connected by a central transmembrane domain. Each domain possesses two cysteine residues essential for electron transport. The transport proceeds via disulfide exchange reactions from cytoplasmic thioredoxin to the central transmembrane domain and via cDsbD to nDsbD, which then reduces the periplasmic target proteins. We determined four high-resolution structures of cDsbD: oxidized (1.65 A resolution), chemically reduced (1.3 A), photo-reduced (1.1 A) and chemically reduced at pH increased from 4.6 to 7. The latter structure was refined at 0.99 A resolution, the highest achieved so far for a thioredoxin superfamily member. The data reveal unprecedented structural details of cDsbD, demonstrating that the domain is very rigid and undergoes hardly any conformational change upon disulfide reduction or interaction with nDsbD. In full agreement with the crystallographic results, guanidinium chloride-induced unfolding and refolding experiments indicate that oxidized and reduced cDsbD are equally stable. We confirmed the structural rigidity of cDsbD by molecular dynamics simulations. A remarkable feature of cDsbD is the pKa of 9.3 for the active site Cys461: this value, determined using two different experimental methods, surprisingly was around 2.5 units higher than expected on the basis of the redox potential. Additionally, taking advantage of the very high quality of the cDsbD structures, we carried out pKa calculations, which gave results in agreement with the experimental findings. In conclusion, our wide-scope analysis of cDsbD, encompassing atomic-resolution crystallography, computational chemistry and biophysical measurements, highlighted two so far unrecognized key aspects of this domain: its unusual redox properties and extreme rigidity. Both are likely to be correlated to the role of cDsbD as a covalently linked electron shuttle between the membrane domain and the N-terminal periplasmic domain of DsbD.

Amino Acid Sequence↗
PubMed2006

Crystallization of the NADH-oxidizing domain of the Na+-translocating NADH:ubiquinone oxidoreductase from Vibrio cholerae.

The Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) from pathogenic and marine bacteria is a respiratory complex that couples the exergonic oxidation of NADH by quinone to the transport of Na+ across the membrane. The NqrF subunit oxidizes NADH and transfers the electrons to other redox cofactors in the enzyme. The FAD-containing domain of NqrF has been expressed, purified and crystallized. The purified NqrF FAD domain exhibited high rates of NADH oxidation and contained stoichiometric amounts of the FAD cofactor. Initial crystallization of the flavin domain was achieved by the sitting-drop technique using a Cartesian MicroSys4000 robot. Optimization of the crystallization conditions yielded yellow hexagonal crystals with dimensions of 30 x 30 x 70 microm. The protein mainly crystallizes in long hexagonal needles with a diameter of up to 30 microm. Crystals diffract to 2.8 A and belong to space group P622, with unit-cell parameters a = b = 145.3, c = 90.2 A, alpha = beta = 90, gamma = 120 degrees.

Bacterial Proteins↗
PubMed2006

Structure-activity studies in a family of beta-hairpin protein epitope mimetic inhibitors of the p53-HDM2 protein-protein interaction.

Inhibitors of the interaction between the p53 tumor-suppressor protein and its natural human inhibitor HDM2 are attractive as potential anticancer agents. In earlier work we explored designing beta-hairpin peptidomimetics of the alpha-helical epitope on p53 that would bind tightly to the p53-binding site on HDM2. The beta-hairpin is used as a scaffold to display energetically hot residues in an optimal array for interaction with HDM2. The initial lead beta-hairpin mimetic, with a weak inhibitory activity (IC(50)=125 microM), was optimized to afford cyclo-(L-Pro-Phe-Glu-6ClTrp-Leu-Asp-Trp-Glu-Phe-D-Pro) (where 6ClTrp=L-6-chlorotryptophan), which has an affinity almost 1,000 times higher (IC(50)=140 nM). In this work, insights into the origins of this affinity maturation based on structure-activity studies and an X-ray crystal structure of the inhibitor/HDM2(residues 17-125) complex at 1.4 A resolution are described. The crystal structure confirms the beta-hairpin conformation of the bound ligand, and also reveals that a significant component of the affinity increase arises through new aromatic/aromatic stacking interactions between side chains around the hairpin and groups on the surface of HDM2.

Amino Acid Sequence↗
PubMed2006

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.

Amidohydrolases↗
PubMed2005

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.

Amino Acid Motifs↗
PubMed2005

Structural basis of chaperone-subunit complex recognition by the type 1 pilus assembly platform FimD.

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. Here we report nuclear magnetic resonance and X-ray protein structures of the N-terminal substrate recognition domain of FimD (FimD(N)) before and after binding of a chaperone-subunit complex. FimD(N) consists of a flexible N-terminal segment of 24 residues, a structured core with a novel fold, and a C-terminal hinge segment. In the ternary complex, residues 1-24 of FimD(N) specifically interact with both FimC and the subunit, acting as a sensor for loaded FimC molecules. Together with in vivo complementation studies, we show how this mechanism enables recognition and discrimination of different chaperone-subunit complexes by bacterial pilus assembly platforms.

Adhesins, Escherichia coli↗
PubMed2005

Structure of ACC synthase inactivated by the mechanism-based inhibitor L-vinylglycine.

L-Vinylglycine (L-VG) is both a substrate for and a mechanism-based inhibitor of 1-aminocyclopropane-1-carboxylate (ACC) synthase. The ratio of the rate constants for catalytic conversion to alpha-ketobutyrate and ammonia to inactivation is 500/1. The crystal structure of the covalent adduct of the inactivated enzyme was determined at 2.25 Angstroms resolution. The active site contains an external aldimine of the adduct of L-VG with the pyridoxal 5'-phosphate cofactor. The side chain gamma-carbon of L-VG is covalently bound to the epsilon-amino group of Lys273. This species corresponds to one of the two alternatives proposed by Feng and Kirsch [Feng, L. and Kirsch, J.F. (2000) L-Vinylglycine is an alternative substrate as well as a mechanism-based inhibitor of 1-aminocyclopropane-1-carboxylate synthase. Biochemistry 39, 2436-2444] and presumably results from Michael addition to a vinylglycine ketimine intermediate.

Crystallography, X-Ray↗
PubMed2005

Intracellular kinase inhibitors selected from combinatorial libraries of designed ankyrin repeat proteins.

The specific intracellular inhibition of protein activity at the protein level allows the determination of protein function in the cellular context. We demonstrate here the use of designed ankyrin repeat proteins as tailor-made intracellular kinase inhibitors. The target was aminoglycoside phosphotransferase (3')-IIIa (APH), which mediates resistance to aminoglycoside antibiotics in pathogenic bacteria and shares structural homology with eukaryotic protein kinases. Combining a selection and screening approach, we isolated 198 potential APH inhibitors from highly diverse combinatorial libraries of designed ankyrin repeat proteins. A detailed analysis of several inhibitors revealed that they bind APH with high specificity and with affinities down to the subnanomolar range. In vitro, the most potent inhibitors showed complete enzyme inhibition, and in vivo, a phenotype comparable with the gene knockout was observed, fully restoring antibiotic sensitivity in resistant bacteria. These results underline the great potential of designed ankyrin repeat proteins for modulation of intracellular protein function.

Amikacin↗