Structure-based engineering of designed armadillo repeat proteins.

The specific recognition of macromolecules is key for many applications in biochemical research, medical diagnostics and disease treatment. Currently the development of new recognition molecules depends on the immunization of lab animals or combinatorial biochemistry techniques. Since both approaches are elaborate and require the availability of sufficient amounts of stable target molecules we are developing a modular system that allows a rational design of peptide recognition modules. This system is based on the armadillo repeat scaffold, because natural armadillo repeat proteins bind their targets in extended anti-parallel conformations with very regular binding topologies. The development of designed armadillo repeat proteins (dArmRPs) consisting entirely of identical internal repeats (full-consensus design) has been described [1]. Although dArmRP with 2nd generation capping repeats were predominantly monomeric in solution, crystal structures of dArmRP with different numbers of internal repeats revealed domain-swapped N-caps [2]. Redesign of the N-cap significantly improved thermodynamic stability and abrogated swapping of N-caps. These 3rd generation dArmRP recognize full-consensus peptides with nanomolar affinities. The dissociation constants depend on the lengths of the targeted peptides and the number of internal repeats. Three crystal structures of complexes between dArmRPs with five or six internal repeats and the targeted full-consensus (KR)5 peptide (either free or fused to the N- and C-terminii of globular proteins) confirm that the binding mode fulfills the expected regular topology. Further crystal structures of complexes between dArmRPs and the mismatch (RR)5 peptide revealed that the dArmRPs recognize their target peptides in a side-chain specific manner. Several crystal structures confirm that 3rd generation dArmRPs behave as stable and monomeric molecules that allow the selective recognition of the targeted peptide with the expected topology. Therefore, the rational assembly of binding modules from a pool of dipeptide-specific armadillo repeats to recognize peptides with given sequences should indeed be possible.

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Rigid fusions of designed helical repeat binding proteins efficiently protect a binding surface from crystal contacts

Scientific Reports ◽

10.1038/s41598-019-52121-9 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Patrick Ernst ◽

Annemarie Honegger ◽

Floor van der Valk ◽

Christina Ewald ◽

Peer R. E. Mittl ◽

...

Keyword(s):

Protein Design ◽

Binding Proteins ◽

Peptide Binding ◽

Binding Mode ◽

Equilibrium Structure ◽

Repeat Proteins ◽

Binding Surface ◽

Crystal Contacts ◽

Armadillo Repeat ◽

Ankyrin Repeat Proteins

Abstract Designed armadillo repeat proteins (dArmRPs) bind extended peptides in a modular way. The consensus version recognises alternating arginines and lysines, with one dipeptide per repeat. For generating new binding specificities, the rapid and robust analysis by crystallography is key. Yet, we have previously found that crystal contacts can strongly influence this analysis, by displacing the peptide and potentially distorting the overall geometry of the scaffold. Therefore, we now used protein design to minimise these effects and expand the previously described concept of shared helices to rigidly connect dArmRPs and designed ankyrin repeat proteins (DARPins), which serve as a crystallisation chaperone. To shield the peptide-binding surface from crystal contacts, we rigidly fused two DARPins to the N- and C-terminal repeat of the dArmRP and linked the two DARPins by a disulfide bond. In this ring-like structure, peptide binding, on the inside of the ring, is very regular and undistorted, highlighting the truly modular binding mode. Thus, protein design was utilised to construct a well crystallising scaffold that prevents interference from crystal contacts with peptide binding and maintains the equilibrium structure of the dArmRP. Rigid DARPin-dArmRPs fusions will also be useful when chimeric binding proteins with predefined geometries are required.

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Crystal structures of designed armadillo repeat proteins: Implications of construct design and crystallization conditions on overall structure

Protein Science ◽

10.1002/pro.2535 ◽

2014 ◽

Vol 23 (11) ◽

pp. 1572-1583 ◽

Cited By ~ 12

Author(s):

Christian Reichen ◽

Chaithanya Madhurantakam ◽

Andreas Plückthun ◽

Peer R. E. Mittl

Keyword(s):

Crystal Structures ◽

Repeat Proteins ◽

Crystallization Conditions ◽

Armadillo Repeat

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Fluorescent thermal shift-based method for detection of NF-κB binding to double-stranded DNA

Scientific Reports ◽

10.1038/s41598-021-81743-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Peter D. Leitner ◽

Ilja Vietor ◽

Lukas A. Huber ◽

Taras Valovka

Keyword(s):

Dna Binding ◽

Rational Design ◽

Dissociation Constants ◽

Inflammatory Diseases ◽

Dimethyl Fumarate ◽

Bioinformatic Analysis ◽

Quantitative Detection ◽

Regulatory Sequences ◽

Wide Range ◽

Inhibitory Drug

AbstractThe nuclear factor kappa B (NF-κB) family of dimeric transcription factors regulates a wide range of genes by binding to their specific DNA regulatory sequences. NF-κB is an important therapeutic target linked to a number of cancers as well as autoimmune and inflammatory diseases. Therefore, effective high-throughput methods for the detection of NF-κB DNA binding are essential for studying its transcriptional activity and for inhibitory drug screening. We describe here a novel fluorescence-based assay for quantitative detection of κB consensus double-stranded (ds) DNA binding by measuring the thermal stability of the NF-κB proteins. Specifically, DNA binding proficient NF-κB probes, consisting of the N-terminal p65/RelA (aa 1–306) and p50 (aa 1–367) regions, were designed using bioinformatic analysis of protein hydrophobicity, folding and sequence similarities. By measuring the SYPRO Orange fluorescence during thermal denaturation of the probes, we detected and quantified a shift in the melting temperatures (ΔTm) of p65/RelA and p50 produced by the dsDNA binding. The increase in Tm was proportional to the concentration of dsDNA with apparent dissociation constants (KD) of 2.228 × 10–6 M and 0.794 × 10–6 M, respectively. The use of withaferin A (WFA), dimethyl fumarate (DMF) and p-xyleneselenocyanate (p-XSC) verified the suitability of this assay for measuring dose-dependent antagonistic effects on DNA binding. In addition, the assay can be used to analyse the direct binding of inhibitors and their effects on structural stability of the protein probe. This may facilitate the identification and rational design of new drug candidates interfering with NF-κB functions.

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Crystal Structures of the Human and Fungal Cytosolic Leucyl-tRNA Synthetase Editing Domains: A Structural Basis for the Rational Design of Antifungal Benzoxaboroles

Journal of Molecular Biology ◽

10.1016/j.jmb.2009.04.073 ◽

2009 ◽

Vol 390 (2) ◽

pp. 196-207 ◽

Cited By ~ 65

Author(s):

Elena Seiradake ◽

Weimin Mao ◽

Vincent Hernandez ◽

Stephen J. Baker ◽

Jacob J. Plattner ◽

...

Keyword(s):

Crystal Structures ◽

Rational Design ◽

Trna Synthetase ◽

Structural Basis

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Structures of designed armadillo-repeat proteins show propagation of inter-repeat interface effects

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798315023116 ◽

2016 ◽

Vol 72 (1) ◽

pp. 168-175 ◽

Cited By ~ 7

Author(s):

Christian Reichen ◽

Chaithanya Madhurantakam ◽

Simon Hansen ◽

Markus G. Grütter ◽

Andreas Plückthun ◽

...

Keyword(s):

Second Generation ◽

Body Movement ◽

Recognition System ◽

Third Generation ◽

Peptide Recognition ◽

Repeat Proteins ◽

Peptide Binding Site ◽

Armadillo Repeat ◽

The Stability ◽

Interface Effects

The armadillo repeat serves as a scaffold for the development of modular peptide-recognition modules. In order to develop such a system, three crystal structures of designed armadillo-repeat proteins with third-generation N-caps (YIII-type), four or five internal repeats (M-type) and second-generation C-caps (AII-type) were determined at 1.8 Å (His-YIIIM4AII), 2.0 Å (His-YIIIM5AII) and 1.95 Å (YIIIM5AII) resolution and compared with those of variants with third-generation C-caps. All constructs are full consensus designs in which the internal repeats have exactly the same sequence, and hence identical conformations of the internal repeats are expected. The N-cap and internal repeats M1to M3are indeed extremely similar, but the comparison reveals structural differences in internal repeats M4and M5and the C-cap. These differences are caused by long-range effects of the C-cap, contacting molecules in the crystal, and the intrinsic design of the repeat. Unfortunately, the rigid-body movement of the C-terminal part impairs the regular arrangement of internal repeats that forms the putative peptide-binding site. The second-generation C-cap improves the packing of buried residues and thereby the stability of the protein. These considerations are useful for future improvements of an armadillo-repeat-based peptide-recognition system.

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Curvature of designed armadillo repeat proteins allows modular peptide binding

Journal of Structural Biology ◽

10.1016/j.jsb.2017.08.009 ◽

2018 ◽

Vol 201 (2) ◽

pp. 108-117 ◽

Cited By ~ 5

Author(s):

Simon Hansen ◽

Patrick Ernst ◽

Sebastian L.B. König ◽

Christian Reichen ◽

Christina Ewald ◽

...

Keyword(s):

Peptide Binding ◽

Repeat Proteins ◽

Armadillo Repeat

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Crystal Structures of Inhibitor Complexes Reveal an Alternate Binding Mode in Orotidine-5′-monophosphate Decarboxylase

Journal of Biological Chemistry ◽

10.1074/jbc.m202362200 ◽

2002 ◽

Vol 277 (31) ◽

pp. 28080-28087 ◽

Cited By ~ 25

Author(s):

Ning Wu ◽

Emil F. Pai

Keyword(s):

Crystal Structures ◽

Binding Mode ◽

Monophosphate Decarboxylase

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Rational design of cyanide-bridged heterometallic M(I)–Mn(II) (M = Ag, Au) one-dimensional chain complexes: synthesis, crystal structures and magnetic properties

CrystEngComm ◽

10.1039/b903174j ◽

2009 ◽

Vol 11 (11) ◽

pp. 2447 ◽

Cited By ~ 22

Author(s):

Daopeng Zhang ◽

Hailong Wang ◽

Laijin Tian ◽

Jianzhuang Jiang ◽

Zhong-Hai Ni

Keyword(s):

Magnetic Properties ◽

Crystal Structures ◽

Rational Design ◽

Dimensional Chain ◽

One Dimensional ◽

Chain Complexes

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Expert-Knowledge Gating Mechanism in the Hierarchical Modular System

Journal of Advanced Computational Intelligence and Intelligent Informatics ◽

10.20965/jaciii.2004.p0410 ◽

2004 ◽

Vol 8 (4) ◽

pp. 410-414

Author(s):

Jeong-Yon Shim ◽

Keyword(s):

Knowledge Acquisition ◽

Associative Memory ◽

Expert Knowledge ◽

Medical Diagnostics ◽

Knowledge System ◽

Knowledge Systems ◽

Modular System ◽

Gating Mechanism ◽

Knowledge Learning

To maximize the efficiency of knowledge learning, it is essential that the knowledge system itself be well structured. Well designed knowledge systems make easy to access for knowledge acquisition and extraction. Expert knowledge plays a role controlling. We propose a Hierarchical modular system with an expert-knowledge gating mechanism that consists of mechanisms for acquiring knowledge, constructing associative memory and enabling knowledge inference and extraction based on expert-knowledge gating. We applied this to medical diagnostics for classifying Viruses (coxackie, echovirus and cold virus), Rhinitis (Nonallergic and allergic) and tested using symptom data.

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