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

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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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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Advances in the design and engineering of peptide-binding repeat proteins

Biological Chemistry ◽

10.1515/hsz-2016-0233 ◽

2017 ◽

Vol 398 (1) ◽

pp. 23-29 ◽

Cited By ~ 7

Author(s):

Patrick Ernst ◽

Andreas Plückthun

Keyword(s):

Binding Proteins ◽

De Novo ◽

Peptide Binding ◽

Peptide Sequence ◽

Cellular Interactions ◽

Specific Recognition ◽

Repeat Proteins ◽

Biochemical Assays ◽

Selection Of ◽

Unstructured Regions

Abstract The specific recognition of peptides, which we define to include unstructured regions or denatured forms of proteins, is an intrinsic part of a multitude of biochemical assays and procedures. Many cellular interactions are also based on this principle as well. While it would be highly desirable to have a stockpile of sequence-specific binders for essentially any sequence, a de novo selection of individual binders against every possible target peptide sequence would be rather difficult to reduce to practice. Modular peptide binders could overcome this problem, as preselected and/or predesigned modules could be reused for the generation of new binders and thereby revolutionize the generation of binding proteins. This minireview summarizes advances in the development of peptide binders and possible scaffolds for their design.

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Structure-based engineering of designed armadillo repeat proteins.

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314088469 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1153-C1153

Author(s):

Simon Hansen ◽

Christian Reichen ◽

Chaithanya Madhurantakam ◽

Markus Grütter ◽

Andreas Plückthun ◽

...

Keyword(s):

Crystal Structures ◽

Rational Design ◽

Dissociation Constants ◽

Binding Mode ◽

Medical Diagnostics ◽

Modular System ◽

Repeat Proteins ◽

Armadillo Repeat ◽

Biochemical Research ◽

Full Consensus

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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Modular peptide binding: From a comparison of natural binders to designed armadillo repeat proteins

Journal of Structural Biology ◽

10.1016/j.jsb.2013.07.012 ◽

2014 ◽

Vol 185 (2) ◽

pp. 147-162 ◽

Cited By ~ 35

Author(s):

Christian Reichen ◽

Simon Hansen ◽

Andreas Plückthun

Keyword(s):

Peptide Binding ◽

Repeat Proteins ◽

Armadillo Repeat

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Designed Armadillo Repeat Proteins as General Peptide-Binding Scaffolds: Consensus Design and Computational Optimization of the Hydrophobic Core

Journal of Molecular Biology ◽

10.1016/j.jmb.2007.12.014 ◽

2008 ◽

Vol 376 (5) ◽

pp. 1282-1304 ◽

Cited By ~ 92

Author(s):

Fabio Parmeggiani ◽

Riccardo Pellarin ◽

Anders Peter Larsen ◽

Gautham Varadamsetty ◽

Michael T. Stumpp ◽

...

Keyword(s):

Peptide Binding ◽

Hydrophobic Core ◽

Computational Optimization ◽

Repeat Proteins ◽

Armadillo Repeat

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Abstract 413: Designed Ankyrin Repeat Proteins Against Activation Specific Binding Sites of The Leukocyte Integrin αmβ2: Novel Strategies in Vascular Inflammation

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.35.suppl_1.413 ◽

2015 ◽

Vol 35 (suppl_1) ◽

Author(s):

Philipp Diehl ◽

Patrick Siegel ◽

Jessica Holien ◽

Nicole Bassler ◽

Ulrike Flierl ◽

...

Keyword(s):

Binding Proteins ◽

Specific Binding ◽

Vascular Inflammation ◽

Docking Studies ◽

Ankyrin Repeat ◽

Interaction Sites ◽

Binding Behavior ◽

Repeat Proteins ◽

Anti Inflammatory ◽

Ankyrin Repeat Proteins

Introduction: Vascular inflammation is the underlying condition of several cardiovascular diseases and is mainly mediated by activated leukocytes. The leukocyte integrin αMβ2 with its activation specific epitope (I domain) is strongly involved in leukocyte adhesion to endothelial cells and thus represents an interesting therapeutic target. Designed Ankyrin Repeat Proteins (DARPins) are a novel class of linear, thermostable, highly specific recombinant binding proteins that overcome several limitations of immunoglobulins. Hypothesis: DARPins selected against the mouse I domain (mId) of αMβ2 bind specifically to activated leukocytes and can be used as a novel diagnostic tool as well as a therapeutic, anti-inflammatory agent. Methods: Using phage display, binding proteins were selected against recombinant I domain. Specific binding behavior to only activated leukocytes was assessed in FACS. Docking studies were used to define specific interaction sites of selected DARPins with the I domain. Therapeutic, anti-inflammatory effects of anti-mId DARPins was assessed in a sepsis mouse model. Results: DARPins selected against the I domain bind in FACS specifically to activated monocytes (activated vs. non-activated 61±4 % vs. 19±6 %, p<0.05). Docking studies revealed amino acid positions responsible for the specific binding behavior. Mutagenesis of these residues showed significantly reduced binding of the mutated DARPin using FACS analysis (anti-mId DARPin vs. mutated anti-mId DARPin 61±4 vs. 29±7, p<0.05) proving that binding of the wild type DARPin to its target is specific. Furthermore, anti- I domain DARPins showed anti-inflammatory effects in a mouse sepsis model (peritoneal cells: anti-mId DARPin vs control: 2,049±189 103/ml vs. 3,382±213 103/ml, p<0.01). Conclusions: DARPins selected against the I domain of αMβ2 bind specifically to activated leukocytes and inhibit leukocyte function as a new class of anti-inflammatory agents under in vivo conditions.

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Insight into microtubule nucleation from tubulin-capping proteins

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1813559116 ◽

2019 ◽

Vol 116 (20) ◽

pp. 9859-9864 ◽

Cited By ~ 2

Author(s):

Valérie Campanacci ◽

Agathe Urvoas ◽

Soraya Cantos-Fernandes ◽

Magali Aumont-Nicaise ◽

Ana-Andreea Arteni ◽

...

Keyword(s):

Binding Mode ◽

Artificial Proteins ◽

Capping Proteins ◽

Repeat Proteins ◽

Ring Complex ◽

Ankyrin Repeat Proteins ◽

Inhibition Potency ◽

Insight Into ◽

Β Tubulin

Nucleation is one of the least understood steps of microtubule dynamics. It is a kinetically unfavorable process that is templated in the cell by the γ-tubulin ring complex or by preexisting microtubules; it also occurs in vitro from pure tubulin. Here we study the nucleation inhibition potency of natural or artificial proteins in connection with their binding mode to the longitudinal surface of α- or β-tubulin. The structure of tubulin-bound CopN, a Chlamydia protein that delays nucleation, suggests that this protein may interfere with two protofilaments at the (+) end of a nucleus. Designed ankyrin repeat proteins that share a binding mode similar to that of CopN also impede nucleation, whereas those that target only one protofilament do not. In addition, an αRep protein predicted to target two protofilaments at the (−) end does not delay nucleation, pointing to different behaviors at both ends of the nucleus. Our results link the interference with protofilaments at the (+) end and the inhibition of nucleation.

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