Andreas Kohl - Academia.edu (original) (raw)

Papers by Andreas Kohl

... ELSEVIER Journal of Molecular Catalysis B: Enzymatic 2 (1997)12531255 Letter Preparation of s... more ... ELSEVIER Journal of Molecular Catalysis B: Enzymatic 2 (1997)12531255 Letter Preparation of substituted ( R) 2alkanols by microbial hydroxylation Herbert L. Holland ... rized in Table 1. [7] CJ Sih, JB Heather, R. Sood, P. Price, G. Peruzzotti, LFH Lee and SS Lee, J. Am. Chem. ...

Structure, 2003

Signaling in apoptosis and inflammation is often mediated by proteins of the death domain superfa... more Signaling in apoptosis and inflammation is often mediated by proteins of the death domain superfamily in the Fas/FADD/Caspase-8 or the Apaf-1/Caspase-9 pathways. This superfamily currently comprises the death domain (DD), death effector domain (DED), caspase recruitment domain (CARD), and pyrin domain (PYD) subfamilies. The PYD subfamily is most abundant, but three-dimensional structures are only available for the subfamilies DD, DED, and CARD, which have an antiparallel arrangement of six alpha helices as common fold. This paper presents the NMR structure of PYD of NALP1, a protein that is involved in the innate immune response and is a component of the inflammasome. The structure of NALP1 PYD differs from all other known death domain superfamily structures in that the third alpha helix is replaced by a flexibly disordered loop. This unique feature appears to relate to the molecular basis of familial Mediterranean fever (FMF), a genetic disease caused by single-point mutations.

Science, 2006

CorA family members are ubiquitously distributed transporters of divalent metal cations and are c... more CorA family members are ubiquitously distributed transporters of divalent metal cations and are considered to be the primary Mg2+ transporter of Bacteria and Archaea. We have determined a 2.9 angstrom resolution structure of CorA from Thermotoga maritima that reveals a pentameric cone-shaped protein. Two potential regulatory metal binding sites are found in the N-terminal domain that bind both Mg2+ and Co2+. The structure of CorA supports an efflux system involving dehydration and rehydration of divalent metal ions potentially mediated by a ring of conserved aspartate residues at the cytoplasmic entrance and a carbonyl funnel at the periplasmic side of the pore.

Proteins: Structure, Function, and Bioinformatics, 2006

Consensus-designed ankyrin repeat (AR) proteins are thermodynamically very stable. The structural... more 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.

Proceedings of the National Academy of Sciences, 2003

Ankyrin repeat (AR) proteins mediate innumerable protein-protein interactions in virtually all ph... more Ankyrin repeat (AR) proteins mediate innumerable protein-protein interactions in virtually all phyla. This finding suggested the use of AR proteins as designed binding molecules. Based on sequence and structural analyses, we designed a consensus AR with fixed framework and randomized interacting residues. We generated several combinatorial libraries of AR proteins consisting of defined numbers of this repeat. Randomly chosen library members are expressed in soluble form in the cytoplasm of Escherichia coli constituting up to 30% of total cellular protein and show high thermodynamic stability. We determined the crystal structure of one of those library members to 2.0-Å resolution, providing insight into the consensus AR fold. Besides the highly complementary hydrophobic repeat-repeat interfaces and the absence of structural irregularities in the consensus AR protein, the regular and extended hydrogen bond networks in the ␤-turn and loop regions are noteworthy. Furthermore, all residues found in the turn region of the Ramachandran plot are glycines. Many of these features also occur in natural AR proteins, but not in this rigorous and standardized fashion. We conclude that the AR domain fold is an intrinsically very stable and well-expressed scaffold, able to display randomized interacting residues. This scaffold represents an excellent basis for the design of novel binding molecules.

Nature Biotechnology, 2004

Repeat proteins are ubiquitous binding molecules fundamental to many biological processes 1-3 . T... more Repeat proteins are ubiquitous binding molecules fundamental to many biological processes 1-3 . Their modular architecture is presumably the key to their evolutionary success 4 . Repeat proteins are characterized by consecutive homologous structural units (repeats), which stack to form an elongated protein domain with a continuous hydrophobic core 3 . In principle, this architecture allows their binding specificities to evolve not only by point mutations but also by insertion, deletion or shuffling of repeats 5 . This evolutionary strategy might enable repeat proteins to acquire new functions by adjusting their surface without jeopardizing their overall topology. AR proteins are one prominent repeat protein family illustrating the binding versatility of repeat proteins. They occur throughout all phyla and mediate proteinprotein interactions in the nucleus or cytoplasm, or while anchored to the membrane or when secreted into the extracellular space 6 . AR proteins are built from stacked, 33 amino acid repeats, each forming a β-turn that is followed by two antiparallel α-helices and a loop reaching the β-turn of the next repeat 7 . In most known complexes, the β-turn and the first α-helix mediate the interactions with the target, 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 Å 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.

Nature, 2007

Cysteinyl leukotrienes are key mediators in inflammation and have an important role in acute and ... more Cysteinyl leukotrienes are key mediators in inflammation and have an important role in acute and chronic inflammatory diseases of the cardiovascular and respiratory systems, in particular bronchial asthma. In the biosynthesis of cysteinyl leukotrienes, conversion of arachidonic acid forms the unstable epoxide leukotriene A 4 (LTA 4 ). This intermediate is conjugated with glutathione (GSH) to produce leukotriene C 4 (LTC 4 ) in a reaction catalysed by LTC 4 synthase 1 : this reaction is the key step in cysteinyl leukotriene formation. Here we present the crystal structure of the human LTC 4 synthase in its apo and GSH-complexed forms to 2.00 and 2.15 Å resolution, respectively. The structure reveals a homotrimer, where each monomer is composed of four transmembrane segments. The structure of the enzyme in complex with substrate reveals that the active site enforces a horseshoe-shaped conformation on GSH, and effectively positions the thiol group for activation by a nearby arginine at the membrane-enzyme interface. In addition, the structure provides a model for how the v-end of the lipophilic co-substrate is pinned at one end of a hydrophobic cleft, providing a molecular 'ruler' to align the reactive epoxide at the thiol of glutathione. This provides new structural insights into the mechanism of LTC 4 formation, and also suggests that the observed binding and activation of GSH might be common for a family of homologous proteins important for inflammatory and detoxification responses.

Journal of Molecular Catalysis B: Enzymatic, 1997

The fungus Mortierella isabellina ATCC 42613 catalyses the biotransformation of substituted-aryl ... more The fungus Mortierella isabellina ATCC 42613 catalyses the biotransformation of substituted-aryl methyl sulfides to give sulfoxides of predominantly (R) configuration; of aryl-substituted alkenes to give chiral vicinal diols; and of various phenylalkanes and phenylcycloalkanes ...

Journal of Biological Chemistry, 2005

The specific intracellular inhibition of protein activity at the protein level allows the determi... more 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.

FEBS Letters, 2002

Death e¡ector domains (DEDs) are protein^protein interaction domains found in the death inducing ... more Death e¡ector domains (DEDs) are protein^protein interaction domains found in the death inducing signaling complex (DISC). Performing a structure-based alignment of all DED sequences we identi¢ed a region of high diversity in K K-helix 3 and propose a classi¢cation of DEDs into class I DEDs typically containing a stretch of basic residues in the K K-helix 3 region whereas DEDs of class II do not. Functional assays using mutants of Fas-associated death domain revealed that this basic region in£uences binding and recruitment of caspase-8 and cellular FLICE inhibitor protein to the DISC. ß

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.