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

Ewald, Christina; Christen, Martin T.; Watson, R.P.; Mihajlovic, Maja; Zhou, Ting; Honegger, Annemarie; Plückthun, Andreas; Caflisch, Amedeo; Zerbe, Oliver

doi:10.1016/j.jmb.2015.02.022

Cited by 6 publications

(6 citation statements)

References 50 publications

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“…The fact that we observe by ITC an approximate 1:1 stoichiometry for the gankyrin-cjoc42 interaction suggests that cjoc42 binds to one site rather than multiple sites; the delocalized nature of the residues found by NMR to be perturbed in the presence would then indicate that the effect of cjoc42 binding at one site is propagated to distant sites throughout the protein’s length. This finding is consistent with long-range effects of ligand binding that have previously been observed both for repeat proteins 17 18 and for numerous other proteins 19 20 21 22 23 .…”

Section: Resultssupporting

confidence: 92%

Discovery of a small-molecule binder of the oncoprotein gankyrin that modulates gankyrin activity in the cell

Chattopadhyay

O'Connor

Zhang

et al. 2016

Sci Rep

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Gankyrin is an ankyrin-repeat oncoprotein whose overexpression has been implicated in the development of many cancer types. Elevated gankyrin levels are linked to aberrant cellular events including enhanced degradation of tumour suppressor protein p53, and inhibition of gankyrin activity has therefore been identified as an attractive anticancer strategy. Gankyrin interacts with several partner proteins, and a number of these protein-protein interactions (PPIs) are of relevance to cancer. Thus, molecules that bind the PPI interface of gankyrin and interrupt these interactions are of considerable interest. Herein, we report the discovery of a small molecule termed cjoc42 that is capable of binding to gankyrin. Cell-based experiments demonstrate that cjoc42 can inhibit gankyrin activity in a dose-dependent manner: cjoc42 prevents the decrease in p53 protein levels normally associated with high amounts of gankyrin, and it restores p53-dependent transcription and sensitivity to DNA damage. The results represent the first evidence that gankyrin is a “druggable” target with small molecules.

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Section: Resultssupporting

confidence: 92%

Discovery of a small-molecule binder of the oncoprotein gankyrin that modulates gankyrin activity in the cell

Chattopadhyay

O'Connor

Zhang

et al. 2016

Sci Rep

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“…From an initial version of a consensus ArmRP library, a binder to neurotensin was selected with a K d of 7 µM [29], but the binding mode was different from the intended canonical binding [30]. More recently, picomolar affinities for the interaction between the designed ArmRP Y III M 6 A II and the (KR) 4 -peptide have been measured [31].…”

Section: Surface Redesign For Peptide Bindingmentioning

confidence: 99%

“…K 29 in CAR2.V1 is located on the binding surface close to binding pocket P2' (see Figure 7C). Thus, the positive charge of K 29 would reduce the charge-charge interaction between the ligand arginine residue and E 30 . This effect is strengthened by the multiple appearance of this pocket in the repetitive binding molecule.…”

Section: Structure Of Car2v1 Complexed With Peptide (Rr)mentioning

confidence: 99%

Computationally Designed Armadillo Repeat Proteins for Modular Peptide Recognition

Reichen

Hansen

Forzani

et al. 2016

Journal of Molecular Biology

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Armadillo repeat proteins (ArmRP) recognize their target peptide in extended conformation and bind, in a first approximation, two residues per repeat. They may thus form the basis for building a modular system, in which each repeat is complementary to a piece of the target peptide. Accordingly, preselected repeats could be assembled into specific binding proteins on demand and thereby avoid the traditional generation of every new binding molecule by an independent selection from a library.Stacked armadillo repeats, each consisting of 42 amino acids arranged in three α-helices, build an elongated superhelical structure. Here, we analyzed curvature variations in natural ArmRPs, and identified a repeat pair from yeast importin-α as having the optimal curvature geometry to be complementary to a peptide over its whole length. We employed a symmetric in silico design to obtain a uniform sequence for a stackable repeat while maintaining the desired curvature geometry.Computationally designed armadillo repeat proteins (dArmRPs) had to be stabilized by mutations to remove regions of higher flexibility, which were identified by molecular dynamics (MD) simulations in explicit solvent. Using an N-capping repeat from the consensus-design approach, two different crystal structures of dArmRP were determined. Although the experimental structures of dArmRP deviated from the designed curvature, the insertion of the most conserved binding pockets of natural ArmRPs onto the surface of dArmRPs resulted in binders against the expected peptide with low nanomolar affinities, similar to the binders from the consensus-design series.

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“…They propagate a right-handed triangular spiral, which exposes a supercoiled binding surface consisting of helix H 3 of each repeat (Michel et al 2018). In view of their potential role as antibody substitutes, they display the favorable feature of binding unstructured peptides, as demonstrated for neurotensin (Ewald et al 2015;Varadamsetty et al 2012) or peptides comprising lysine-arginine (KR) dipeptide repeats (Hansen et al 2017;Reichen et al 2016b). In the latter case, X-ray structures confirmed that the bound peptide was in an extended conformation, and the interactions corresponded to those of the natural ArmRPs, in an extended and idealized way.…”

Section: Introductionmentioning

confidence: 99%

An automated iterative approach for protein structure refinement using pseudocontact shifts

et al. 2021

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NMR structure calculation using NOE-derived distance restraints requires a considerable number of assignments of both backbone and sidechains resonances, often difficult or impossible to get for large or complex proteins. Pseudocontact shifts (PCSs) also play a well-established role in NMR protein structure calculation, usually to augment existing structural, mostly NOE-derived, information. Existing refinement protocols using PCSs usually either require a sizeable number of sidechain assignments or are complemented by other experimental restraints. Here, we present an automated iterative procedure to perform backbone protein structure refinements requiring only a limited amount of backbone amide PCSs. Already known structural features from a starting homology model, in this case modules of repeat proteins, are framed into a scaffold that is subsequently refined by experimental PCSs. The method produces reliable indicators that can be monitored to judge about the performance. We applied it to a system in which sidechain assignments are hardly possible, designed Armadillo repeat proteins (dArmRPs), and we calculated the solution NMR structure of YM4A, a dArmRP containing four sequence-identical internal modules, obtaining high convergence to a single structure. We suggest that this approach is particularly useful when approximate folds are known from other techniques, such as X-ray crystallography, while avoiding inherent artefacts due to, for instance, crystal packing.

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

Cited by 6 publications

References 50 publications

Discovery of a small-molecule binder of the oncoprotein gankyrin that modulates gankyrin activity in the cell

Discovery of a small-molecule binder of the oncoprotein gankyrin that modulates gankyrin activity in the cell

Computationally Designed Armadillo Repeat Proteins for Modular Peptide Recognition

An automated iterative approach for protein structure refinement using pseudocontact shifts

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