Residue-Resolved Stability of Full-Consensus Ankyrin Repeat Proteins Probed by NMR

Wetzel, Svava K.; Ewald, Christina; Settanni, Giovanni; Jurt, Simon; Plückthun, Andreas; Zerbe, Oliver

doi:10.1016/j.jmb.2010.07.031

Cited by 29 publications

(33 citation statements)

References 36 publications

(75 reference statements)

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“…To test this reasoning, we chose to design a highly stable trimeric module to link three copies of the knob-binding DARPin 1D3 in an optimal arrangement to clamp the knob. First, the consensus C-capping repeat (C-cap) of the knob-binding DARPin was replaced by a C-cap of higher thermal stability and fewer structural fluctuations, as previously reported (41,42). This approach seemed reasonable, because the C-cap was not involved in directly binding to the knob, and, indeed, with the new C-cap no differences in affinity were observed (Fig.…”

Section: Resultsmentioning

confidence: 96%

“…The knob-binding DARPins 1D3 and 2E6 were equipped with a stabilized C-cap (41,42) and cloned into the expression vector pDST72 (44), a derivative of pQE30, by a PCR-based strategy. To construct adapters, the C terminus of the knob-binding DARPins (omitting the last three residues of the DARPin, which were disordered in the crystal structure) were fused to the N terminus of the phage capsid protein SHP, used as trimerization domain (43) (SHP, V13-P115), and was spaced by different linkers (SHP1, G; SHP2, GA; SHP3, GLKAGADVNA), introduced by a PCR-based strategy and cloned into pDST72.…”

Section: Methodsmentioning

confidence: 99%

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Development of a generic adenovirus delivery system based on structure-guided design of bispecific trimeric DARPin adapters

Dreier

Honegger

Hess

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Adenoviruses (Ads) have shown promise as vectors for gene delivery in clinical trials. Efficient viral targeting to a tissue of choice requires both ablation of the virus' original tropism and engineering of an efficient receptor-mediated uptake by a specific cell population. We have developed a series of adapters binding to the virus with such high affinity that they remain fully bound for >10 d, block its natural receptor binding site and mediate interaction with a surface receptor of choice. The adapter contains two fused modules, both consisting of designed ankyrin repeat proteins (DARPins), one binding to the fiber knob of adenovirus serotype 5 and the other binding to various tumor markers. By solving the crystal structure of the complex of the trimeric knob with three bound DARPins at 1.95-Å resolution, we could use computer modeling to design a link to a trimeric protein of extraordinary kinetic stability, the capsid protein SHP from the lambdoid phage 21. We arrived at a module which binds the knob like a trimeric clamp. When this clamp was fused with DARPins of varying specificities, it enabled adenovirus serotype 5-mediated delivery of a transgene in a human epidermal growth factor receptor 2-, epidermal growth factor receptor-, or epithelial cell adhesion molecule-dependent manner with transduction efficiencies comparable to or even exceeding those of Ad itself. With these adapters, efficiently produced in Escherichia coli, Ad can be converted rapidly to new receptor specificities using any ligand as the receptor-binding moiety. Prefabricated Ads with different payloads thus can be retargeted readily to many cell types of choice.protein design | tumor targeting | viral retargeting | X-ray crystallography | protein engineering

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Section: Resultsmentioning

confidence: 96%

Section: Methodsmentioning

confidence: 99%

Development of a generic adenovirus delivery system based on structure-guided design of bispecific trimeric DARPin adapters

Dreier

Honegger

Hess

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…This observation is supported by results from our previous study of proteins with Ankyrin repeats, in which we observed a similar influence of the stability of capping repeats on the overall protein stability. 22,25,26 In fact, the Ising model predicts that the stability of these proteins arises from mutual stabilization of neighboring repeats, [25][26][27][28] and these effects are therefore expected to propagate throughout the entire protein.…”

Section: Discussionmentioning

confidence: 99%

Optimization of designed armadillo repeat proteins by molecular dynamics simulations and NMR spectroscopy

et al. 2012

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A multidisciplinary approach based on molecular dynamics (MD) simulations using homology models, NMR spectroscopy, and a variety of biophysical techniques was used to efficiently improve the thermodynamic stability of armadillo repeat proteins (ArmRPs). ArmRPs can form the basis of modular peptide recognition and the ArmRP version on which synthetic libraries are based must be as stable as possible. The 42-residue internal Arm repeats had been designed previously using a sequence-consensus method. Heteronuclear NMR revealed unfavorable interactions present at neutral but absent at high pH. Two lysines per repeat were involved in repulsive interactions, and stability was increased by mutating both to glutamine. Five point mutations in the capping repeats were suggested by the analysis of positional fluctuations and configurational entropy along multiple MD simulations. The most stabilizing single C-cap mutation Q240L was inferred from explicit solvent MD simulations, in which water penetrated the ArmRP. All mutants were characterized by temperature-and denaturant-unfolding studies and the improved mutants were established as monomeric species with cooperative folding and increased stability against heat and denaturant. Importantly, the mutations tested resulted in a cumulative decrease of flexibility of the folded state in silico and a cumulative increase of thermodynamic stability in vitro. The final construct has a melting temperature of about 85°C, 14.5°higher than the starting sequence. This work indicates that in silico studies in combination with heteronuclear NMR and other biophysical tools may provide a basis for successfully selecting mutations that rapidly improve biophysical properties of the target proteins.

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“…Unfortunately, the repetitive nature of the armadillo sequence results in a number of technical challenges, some of which were already encountered in our previous studies of Ankyrin repeat proteins [28]. To facilitate this process we employed a wide range of techniques, from isotopic labeling and fragmentation of ArmRPs, to the selective deletion of repeats.…”

Section: Accepted M Manuscriptmentioning

confidence: 99%

A Combined NMR and Computational Approach to Investigate Peptide Binding to a Designed Armadillo Repeat Protein

Ewald

Christen

Watson

et al. 2015

Journal of Molecular Biology

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Residue-Resolved Stability of Full-Consensus Ankyrin Repeat Proteins Probed by NMR

Cited by 29 publications

References 36 publications

Development of a generic adenovirus delivery system based on structure-guided design of bispecific trimeric DARPin adapters

Development of a generic adenovirus delivery system based on structure-guided design of bispecific trimeric DARPin adapters

Optimization of designed armadillo repeat proteins by molecular dynamics simulations and NMR spectroscopy

A Combined NMR and Computational Approach to Investigate Peptide Binding to a Designed Armadillo Repeat Protein

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