Katja Arndt

Improving coiled coil stability while maintaining specificity by a bacterial hitchhiker selection system

by Stefan Gerhardt, Daniel Wohlwend, Katja Arndt, Tim Kükenshöner, and Kristian Müller

Journal of Structural Biology, 2014

The design and selection of peptides targeting cellular proteins is challenging and often yields ... more The design and selection of peptides targeting cellular proteins is challenging and often yields candidates with undesired properties. Therefore we deployed a new selection system based on the twin-arginine translocase (TAT) pathway of Escherichia coli, named hitchhiker translocation (HiT) selection. A pool of α-helix encoding sequences was designed and selected for interference with the coiled coil domain (CC) of a melanoma-associated basic-helix-loop-helix-leucine-zipper (bHLHLZ) protein, the microphthalmia associated transcription factor (MITF). One predominant sequence (iM10) was enriched during selection and showed remarkable protease resistance, high solubility and thermal stability while maintaining its specificity. Furthermore, it exhibited nanomolar range affinity towards the target peptide. A mutation screen indicated that target-binding helices of increased homodimer stability and improved expression rates were preferred in the selection process. The crystal structure of the iM10/MITF-CC heterodimer (2.1Å) provided important structural insights and validated our design predictions. Importantly, iM10 did not only bind to the MITF coiled coil, but also to the markedly more stable HLHLZ domain of MITF. Characterizing the selected variants of the semi-rational library demonstrated the potential of the innovative bacterial selection approach.

Protein Engineering

by Urs Hagemann and Katja Arndt

Molecular Biomethods Handbook, 2008

Protein Engineering

by Urs Hagemann and Katja Arndt

TAT hitchhiker selection expanded to folding helpers, multimeric interactions and combinations with protein fragment complementation

by Tim Kükenshöner and Katja Arndt

Protein Engineering Design and Selection, 2013

The twin-arginine translocation (TAT) pathway of the bacterial cytoplasmic membrane mediates tran... more The twin-arginine translocation (TAT) pathway of the bacterial cytoplasmic membrane mediates translocation only of proteins that accomplished a native-like conformation. We deploy this feature in modular selection systems for directed evolution, in which folding helpers as well as dimeric or oligomeric protein-protein interactions enable TATdependent translocation of the resistance marker TEM b-lactamase (bL). Specifically, we demonstrate and analyze selection of (i) enhancers for folding by direct TAT translocation selection of a target protein interposed between the TorA signal sequence and bL, (ii) dimeric or oligomeric protein-protein interactions by hitchhiker translocation (HiT) selection of proteins fused to the TorA signal sequence and to the bL, respectively and (iii) heterotrimeric protein-protein interactions by combining HiT with protein fragment complementation selection of proteins fused to two split bL fragments and TorA, respectively. The lactamase fragments were additionally engineered for improved activity and stability. Applicability was benchmarked with interaction partners of known affinity and multimerization whereby cellular fitness correlated well with biophysical protein properties. Ultimately, the HiT selection was employed to identify peptides, which specifically bind to leukemia-and melanoma-relevant target proteins (MITF and ETO) by coiled-coil or tetra-helix-bundle formation with high affinity. The various versions of TAT selection led to inhibiting peptides (iPEPs) of diseasepromoting interactions and enabled so far difficult to achieve selections. Downloaded from b Given are only interaction partners relevant for the present work. c NHR2 has been described to form tetramers (Liu et al., 2006), in our hands, size-exclusion chromatography at 208C revealed formation of octamers (not shown), which might derive from the interaction of two tetramers. J.Speck et al. 226

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Analysis of Selected and Designed Chimeric D- and L-α-Helix Assemblies

by Tim Kükenshöner and Katja Arndt

Biomacromolecules, 2014

D-Peptides have been attributed pharmacological advantages over regular L-peptides, yet design ru... more D-Peptides have been attributed pharmacological advantages over regular L-peptides, yet design rules are largely unknown. Based on a designed coiled coil-like D/L heterotetramer, named L-Base/D-Acid, we generated a library offering alternative residues for interaction with the D-peptide. Phage display selection yielded one predominant peptide, named HelixA, that differed at 13 positions from the scaffold helix. In addition to the observed D-/L-heterotetramers, ratiodependent intermediate states were detected by isothermal titration calorimetry. Importantly, the formation of the selected HelixA/D-Acid bundle passes through fewer intermediate states than L-Base/D-Acid. Back mutation of HelixA core residues to L-Base (HelixLL) revealed that the residues at e/g-positions are responsible for the different intermediates. Furthermore, a Val-core variant (PeptideVV) was completely devoid of binding D-Acid, whereas an Ile-core helix (HelixII) interacted with D-Acid in a significantly more specific complex than L-Base.

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Speck et al TAT HiT selection supplement

by Tim Kükenshöner and Katja Arndt

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Selectional and Mutational Scope of Peptides Sequestering the Jun–Fos Coiled-Coil Domain

by Urs Hagemann and Katja Arndt

Journal of Molecular Biology, 2008

The activator protein-1 (AP-1) complex plays a crucial role in numerous pathways, and its ability... more The activator protein-1 (AP-1) complex plays a crucial role in numerous pathways, and its ability to induce tumorigenesis is well documented. Thus, AP-1 represents an interesting therapeutic target. We selected peptides from phage display and compared their ability to disrupt the cFos/cJun interaction to a previously described in vivo protein-fragment complementation assay (PCA). A cJun-based library was screened to enrich for peptides that disrupt the AP-1 complex by binding to the cFos coiled-coil domain. Interestingly, phage display identified one helix, JunW Ph1 [phage-selected winning peptide (clone 1) targeting cFos], which differs in only 2 out of 10 randomized positions to JunW (PCA-selected winning peptide targeting cFos). Phage-selected peptides revealed higher affinity to cFos than wildtype cJun, harboring a T m of 53°C compared to 16°C for cFos/cJun or 44°C for cFos/JunW. In PCA growth assays in the presence of cJun as competitor, phage-selected JunW Ph1 conferred shorter generation times than JunW. Bacterial growth was barely detectable, using JunW Ph1 as a competitor for the wild-type cJun/cFos interaction, indicating efficient cFos removal from the dimeric wild-type complex. Importantly, all inhibitory peptides were able to interfere with DNA binding as demonstrated in gel shift assays. The selected sequences have consequently improved our 'bZIP coiled-coil interaction prediction algorithm' in distinguishing interacting from noninteracting coiled-coil sequences. Predicting and manipulating protein interaction will accelerate the systems biology field, and generated peptides will be valuable tools for analytical and biomedical applications.

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Improved Stability of the Jun-Fos Activator Protein-1 Coiled Coil Motif: A STOPPED-FLOW CIRCULAR DICHROISM KINETIC ANALYSIS

by Urs Hagemann and Katja Arndt

Journal of Biological Chemistry, 2007

Two c-Jun leucine zipper variants that bind with high affinity to c-Fos have been selected using ... more Two c-Jun leucine zipper variants that bind with high affinity to c-Fos have been selected using semirational design combined with protein-fragment complementation assays (JunW) or phage display selection (JunW Ph1 ). Enriched winners differ from each other in only two of ten semi-randomized positions, with ⌬T m values of 28°C and 37°C over wild-type. cFos-JunW, cFos-JunW Ph1 , and two intermediate mutants (cFos-JunW Q21R and cFos-JunW E23K ) display biphasic kinetics in the folding direction, indicating the existence of a folding intermediate.

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Analysis of Selected and Designed Chimeric D- and L-α-Helix Assemblies

by Urs Hagemann, Daniel Wohlwend, and Katja Arndt

Biomacromolecules, 2014

D-Peptides have been attributed pharmacological advantages over regular L-peptides, yet design ru... more D-Peptides have been attributed pharmacological advantages over regular L-peptides, yet design rules are largely unknown. Based on a designed coiled coil-like D/L heterotetramer, named L-Base/D-Acid, we generated a library offering alternative residues for interaction with the D-peptide. Phage display selection yielded one predominant peptide, named HelixA, that differed at 13 positions from the scaffold helix. In addition to the observed D-/L-heterotetramers, ratiodependent intermediate states were detected by isothermal titration calorimetry. Importantly, the formation of the selected HelixA/D-Acid bundle passes through fewer intermediate states than L-Base/D-Acid. Back mutation of HelixA core residues to L-Base (HelixLL) revealed that the residues at e/g-positions are responsible for the different intermediates. Furthermore, a Val-core variant (PeptideVV) was completely devoid of binding D-Acid, whereas an Ile-core helix (HelixII) interacted with D-Acid in a significantly more specific complex than L-Base.

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Selection of peptides interfering with protein-protein interaction

by Urs Hagemann and Katja Arndt

Methods in molecular biology (Clifton, N.J.), 2009

Cell physiology depends on a fine-tuned network of protein-protein interactions, and misguided in... more Cell physiology depends on a fine-tuned network of protein-protein interactions, and misguided interactions are often associated with various diseases. Consequently, peptides, which are able to specifically interfere with such adventitious interactions, are of high interest for analytical as well as medical purposes. One of the most abundant protein interaction domains is the coiled-coil motif, and thus provides a premier target. Coiled coils, which consist of two or more alpha-helices wrapped around each other, have one of the simplest interaction interfaces, yet they are able to confer highly specific homo- and heterotypic interactions involved in virtually any cellular process. While there are several ways to generate interfering peptides, the combination of library design with a powerful selection system seems to be one of the most effective and promising approaches. This chapter guides through all steps of such a process, starting with library options and cloning, detailing sui...

Role of Hydrophobic and Electrostatic Interactions in Coiled Coil Stability and Specificity

by Urs Hagemann and Katja Arndt

Biochemistry, 2009

We have screened two coiled coil-forming libraries in which core a and electrostatic e/g position... more We have screened two coiled coil-forming libraries in which core a and electrostatic e/g positions have been partially randomized. We observed the relative ability of these residues to confer coiled coil stability using a protein-fragment complementation assay. Our studies continue with the Jun/Fos activator protein-1 (AP-1) leucine zipper complex, as it provides a valid therapeutic target, while representing one of the more simplistic examples of quaternary structure. In mammalian cells, 28 possible dimeric interactions result from combinations of cJun, JunB, JunD, cFos, FosB, Fra1, and Fra2. Consequently, peptides designed to target particular oncogenic members must bind with high affinity and also be specific if they are to function as desired. We have therefore tested the ability of core and electrostatic interactions to confer stable and specific peptides. A previously selected peptide (FosW) formed the template for the core and electrostatic libraries. The winner from the core randomization (FosW Core ) bound specifically to cJun relative to cFos, FosB, Fra1, Fra2, and the FosW Core homodimer, as verified by thermal melting analyses and growth competitions in the presence of either a negative control "mock" peptide or a competitor fusion peptide (cFos-FosB-Fra1-Fra2). In contrast, the winner from the electrostatic e/g randomization (FosW e/g ) bound to all respective complexes with high stability, suggesting that the more significant energetic contributions made by core residues may be enough to generate specificity as a consequence of positive design. 1 Abbreviations: DHFR, dihydrofolate reductase; PCA, protein-fragment complementation assay; AP-1, activator protein-1.

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Targeting and Inhibiting Oncogenic Transcription Factors with D- and L-Peptides

by Urs Hagemann and Katja Arndt

GBM Fall meeting Hamburg 2007, 2007

Role of Hydrophobic and Electrostatic Interactions in Coiled Coil Stability and Specificity

by Urs Hagemann and Katja Arndt

Biochemistry Usa, Oct 1, 2009

We have screened two coiled coil-forming libraries in which core a and electrostatic e/g position... more We have screened two coiled coil-forming libraries in which core a and electrostatic e/g positions have been partially randomized. We observed the relative ability of these residues to confer coiled coil stability using a protein-fragment complementation assay. Our studies continue with the Jun/Fos activator protein-1 (AP-1) leucine zipper complex, as it provides a valid therapeutic target, while representing one of the more simplistic examples of quaternary structure. In mammalian cells, 28 possible dimeric interactions result from combinations of cJun, JunB, JunD, cFos, FosB, Fra1, and Fra2. Consequently, peptides designed to target particular oncogenic members must bind with high affinity and also be specific if they are to function as desired. We have therefore tested the ability of core and electrostatic interactions to confer stable and specific peptides. A previously selected peptide (FosW) formed the template for the core and electrostatic libraries. The winner from the core randomization (FosW Core ) bound specifically to cJun relative to cFos, FosB, Fra1, Fra2, and the FosW Core homodimer, as verified by thermal melting analyses and growth competitions in the presence of either a negative control "mock" peptide or a competitor fusion peptide (cFos-FosB-Fra1-Fra2). In contrast, the winner from the electrostatic e/g randomization (FosW e/g ) bound to all respective complexes with high stability, suggesting that the more significant energetic contributions made by core residues may be enough to generate specificity as a consequence of positive design. 1 Abbreviations: DHFR, dihydrofolate reductase; PCA, protein-fragment complementation assay; AP-1, activator protein-1.

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Comparison of In Vivo Selection and Rational Design of Heterodimeric Coiled Coils

by Joelle Pelletier and Katja Arndt

Structure, 2002

One of the oligomerization domains used commonly in protein design is the ␣-helical coiled coil [... more One of the oligomerization domains used commonly in protein design is the ␣-helical coiled coil [3-6]. Coiled coils consist of two or more ␣ helices that wrap around one another in a left-handed supercoil. Coiled-coil sequences are characterized by a repeat of seven amino acids, denoted a-g ( ). Most of the residues at positions a and d are hydrophobic, forming a character-2 Biochemisches Institut Universitä t Zü rich istic 3,4 repeat, and residues at positions e and g are often charged . Charged residues at the g position Winterthurerstr. 190 CH-8057 Zü rich often form interhelical ion pairs with residues at the eЈ position of the next heptad of the neighboring helix. The Switzerland simplicity and small size of this motif has made it a popular choice for designing protein fusions with defined oligomerization states [9].

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An in vivo library-versus-library selection of optimized protein-protein interactions

by Joelle Pelletier and Katja Arndt

Nature biotechnology, 1999

We describe a rapid and efficient in vivo library-versus-library screening strategy for identifyi... more We describe a rapid and efficient in vivo library-versus-library screening strategy for identifying optimally interacting pairs of heterodimerizing polypeptides. Two leucine zipper libraries, semi-randomized at the positions adjacent to the hydrophobic core, were genetically fused to either one of two designed fragments of the enzyme murine dihydrofolate reductase (mDHFR), and cotransformed into Escherichia coli. Interaction between the library polypeptides reconstituted enzymatic activity of mDHFR, allowing bacterial growth. Analysis of the resulting colonies revealed important biases in the zipper sequences relative to the original libraries, which are consistent with selection for stable, heterodimerizing pairs. Using more weakly associating mDHFR fragments, we increased the stringency of selection. We enriched the best-performing leucine zipper pairs by multiple passaging of the pooled, selected colonies in liquid culture, as the best pairs allowed for better bacterial propagati...

A Heterodimeric Coiled-coil Peptide Pair Selected from a Designed Library-Library Ensemble

by Joelle Pelletier, Kristian Müller, and Katja Arndt

of approximately 24 nM. As a result of having been selected in vivo it possesses all characterist... more of approximately 24 nM. As a result of having been selected in vivo it possesses all characteristics required for a general in vivo heterodimerization module. The combination of rational library design and in vivo selection presented here is a very powerful strategy for protein design, and it can reveal new structural relationships. # 2000 Academic Press Keywords: coiled coil; leucine zipper; PCA; protein design; protein engineering *Corresponding author Introduction #-Helical coiled coils are involved in the oligomerization of a wide variety of proteins. Because of their small size and structural regularity, they have also been used as articial domains to mediate oligomerization of various proteins (Pack & Plu ckthun, 1992; Pack et al., 1995; Weissenhorn et al., 1997). Coiled coils consist of two or more amphipathic helices wrapping around each other with a slight supercoil. They contain a characteristic heptad repeat (#############) n with a distinct pattern of hydrophobic and hy

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A heterodimeric coiled-coil peptide pair selected in vivo from a designed library-versus-library ensemble

by Joelle Pelletier and Katja Arndt

Journal of Molecular Biology, 2000

Novel heterodimeric coiled-coil pairs were selected simultaneously from two DNA libraries using a... more Novel heterodimeric coiled-coil pairs were selected simultaneously from two DNA libraries using an in vivo protein-fragment complementation assay with dihydrofolate reductase, and the best pair was biophysically characterized. We randomized the interface-¯anking e and g positions to Gln, Glu, Arg or Lys, and the core a position to Asn or Val in both helices simultaneously, using trinucleotide codons in DNA synthesis. Selection cycles with three different stringencies yielded sets of coiled-coil pairs, of which 80 clones were statistically analyzed. Thereby, properties most crucial for successful heterodimerization could be distinguished from those mediating more subtle optimization. A strong bias towards an Asn pair in the core a position indicated selection for structural uniqueness, and a reduction of charge repulsions at the e/g positions indicated selection for stability. Increased stringency led to additional selection for heterospeci®city by destabilizing the respective homodimers. Interestingly, the best heterodimers did not contain exclusively complementary charges. The dominant pair, WinZip-A1B1, proved to be at least as stable in vitro as naturally occurring coiled coils, and was shown to be dimeric and highly heterospeci®c with a K D of approximately 24 nM. As a result of having been selected in vivo it possesses all characteristics required for a general in vivo heterodimerization module. The combination of rational library design and in vivo selection presented here is a very powerful strategy for protein design, and it can reveal new structural relationships.

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An in vivo library-versus-library selection of optimized protein-protein interactions

Nature biotechnology, 1999

We describe a rapid and efficient in vivo library-versus-library screening strategy for identifyi... more We describe a rapid and efficient in vivo library-versus-library screening strategy for identifying optimally interacting pairs of heterodimerizing polypeptides. Two leucine zipper libraries, semi-randomized at the positions adjacent to the hydrophobic core, were genetically fused to either one of two designed fragments of the enzyme murine dihydrofolate reductase (mDHFR), and cotransformed into Escherichia coli. Interaction between the library polypeptides reconstituted enzymatic activity of mDHFR, allowing bacterial growth. Analysis of the resulting colonies revealed important biases in the zipper sequences relative to the original libraries, which are consistent with selection for stable, heterodimerizing pairs. Using more weakly associating mDHFR fragments, we increased the stringency of selection. We enriched the best-performing leucine zipper pairs by multiple passaging of the pooled, selected colonies in liquid culture, as the best pairs allowed for better bacterial propagati...

Helix-stabilized fv (hsfv) antibody fragments: substituting the constant domains of a fab fragment for a heterodimeric coiled-coil domain

Journal of Molecular Biology, 2001

Antibody Fv fragments would in principle be useful for a variety of biotechnological applications... more Antibody Fv fragments would in principle be useful for a variety of biotechnological applications because of their small size and the possibility to produce them in relatively large amounts in recombinant form; however, their limited stability is a drawback. To solve this problem, both domains are usually fused via a peptide linker to form a single-chain Fv (scFv) fragment, but in some cases this leads to a dimerization. We present an alternative format for stabilizing antibody Fv fragments. The C H 1 and C L domain of the Fab fragment were replaced with a heterodimeric coiled coil (WinZip-A2B1), which had previously been selected using a protein-fragment complementation assay in Escherichia coli. This new antibody format was termed helix-stabilized Fv fragment (hsFv), and was compared to the corresponding Fv, Fab and single-chain Fv format. Bacterial growth and expression of the hsFv was signi®cantly improved compared to the Fab fragment. The hsFv fragment formed a heterodimer of heavy and light chain with the expected molecular mass, also under conditions where the scFv fragment was predominantly dimeric. The hsFv fragment was signi®cantly more stable than the Fv fragment, and nearly as stable as the scFv fragment under the conditions used (80 nM protein concentration). Thus, the format of a helix-stabilized Fv (hsFv) fragment can be a useful alternative to existing recombinant antibody formats, especially in cases where poor expression of Fab fragments or multimerization of scFv fragments is a problem.

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A dimeric bispecific miniantibody combines two specificities with avidity

FEBS Letters, 1998

Bispecific antibodies extend the capabilities of nature and might be applied in immunotherapy and... more Bispecific antibodies extend the capabilities of nature and might be applied in immunotherapy and biotechnology. By fusing the gene of a single-chain Fv (scFv) fragment to a helical dimerization domain, followed by a second scFv fragment of different specificity, we were able to express a functional protein in E. coli, which is bispecific and has two valencies for each specificity. The dimeric bispecific (DiBi) miniantibody preserves the natural avidity of antibodies in a very small-sized molecule of only 120 kDa. The generality of the principle was shown with a scFv fragment binding the EGF-receptor (named scFv 425) in three combinations with scFv fragments either directed against CD2 (ACID2.M1), phosphorylcholine (McPC603) or fluorescein (FITC-E2). Binding was analyzed by sandwich surface plasmon resonance biosensor (BIAcore) measurements.

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