Modulating the DNA Affinity of Elk-1 with Computationally Selected Mutations

Park, Sheldon; Boder, Eric T.; Saven, Jeffery G.

doi:10.1016/j.jmb.2004.12.062

Cited by 7 publications

(8 citation statements)

References 38 publications

(40 reference statements)

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“…However, Saven et al . [23] performed molecular dynamics (MD) simulations of a single Elk-1 ETS domain taken from the dimeric structure. They discerned regions within the simulated monomeric structure which showed large structural deviation with respect to the structure of the domain in the dimeric conformation.…”

Section: Introductionmentioning

confidence: 99%

“…Thus far, work on characterising the mechanism for protein-DNA recognition in TCFs has been abundant [18,23-27]. However, there has been little on understanding the basis of Elk-1 dimerisation for transcriptional activity.…”

Section: Introductionmentioning

confidence: 99%

“…Using a docking-based approach, we screened entire libraries of all possible di- and tri-peptides against the Elk-1 ETS domain, targeting the stability region of the domain identified by Shaw et al [21]. Given the findings of Saven et al ., [23] it was essential to consider possible structural deviations or fluctuations in the α 1 β 1 loop region that may affect binding of such inhibitors. Therefore, we performed MD simulations for an Elk-1 ETS monomer, to generate an ensemble of monomeric ETS conformations to use as docking targets.…”

Section: Introductionmentioning

confidence: 99%

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Molecular dynamics simulations and in silico peptide ligand screening of the Elk-1 ETS domain

2011

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BackgroundThe Elk-1 transcription factor is a member of a group of proteins called ternary complex factors, which serve as a paradigm for gene regulation in response to extracellular signals. Its deregulation has been linked to multiple human diseases including the development of tumours. The work herein aims to inform the design of potential peptidomimetic compounds that can inhibit the formation of the Elk-1 dimer, which is key to Elk-1 stability. We have conducted molecular dynamics simulations of the Elk-1 ETS domain followed by virtual screening.ResultsWe show the ETS dimerisation site undergoes conformational reorganisation at the α1β1 loop. Through exhaustive screening of di- and tri-peptide libraries against a collection of ETS domain conformations representing the dynamics of the loop, we identified a series of potential binders for the Elk-1 dimer interface. The di-peptides showed no particular preference toward the binding site; however, the tri-peptides made specific interactions with residues: Glu17, Gln18 and Arg49 that are pivotal to the dimer interface.ConclusionsWe have shown molecular dynamics simulations can be combined with virtual peptide screening to obtain an exhaustive docking protocol that incorporates dynamic fluctuations in a receptor. Based on our findings, we suggest experimental binding studies to be performed on the 12 SILE ranked tri-peptides as possible compounds for the design of inhibitors of Elk-1 dimerisation. It would also be reasonable to consider the score-ranked tri-peptides as a comparative test to establish whether peptide size is a determinant factor of binding to the ETS domain.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Molecular dynamics simulations and in silico peptide ligand screening of the Elk-1 ETS domain

2011

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“…Computational redesign of existing proteins has previously yielded structures with novel properties. ,− Examples include the creation of maquettes that bind porphyrins or metals 47 and proteins with catalytic activity . Several different algorithms allow sequence searches for energy minima, including pruning and dead-end elimination, Monte Carlo, and simulated annealing. , Complementary to these approaches are statistical and probabilistic approaches to protein design, , which yield the site-specific likelihoods of the amino acids among sequences likely to fold to a given three-dimensional structure. ,− Recent experiments validating such a probabilisitic approach include the design of a water-soluble membrane potassium channel protein, four-helix bundles that bind metals 47 or nonbiological cofactors, and redox-active rubredoxin mimics which facilitated the present study.…”

Section: Introductionmentioning

confidence: 99%

“…57,64 Complementary to these approaches are statistical and probabilistic approaches to protein design, 48,65 which yield the site-specific likelihoods of the amino acids among sequences likely to fold to a given three-dimensional structure. 64,[66][67][68][69] Recent experiments validating such a probabilisitic approach include the design of a water-soluble membrane potassium channel protein, 70 four-helix bundles that bind metals 47 or nonbiological cofactors, 60 and redox-active rubredoxin mimics. 71 This method was extended recently to include symmetric quaternary structures, 69 which facilitated the present study.…”

Section: Introductionmentioning

confidence: 99%

Design of Functional Ferritin-Like Proteins with Hydrophobic Cavities

et al. 2006

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Ferritin four-helix bundle subunits self-assemble to create a stable multimer with a large central hydrophilic cavity where metal ions bind. To explore the versatility of this reaction vessel, computational design was used to generate cavities with increasingly apolar surface areas inside a dodecameric ferritin-like protein, Dps. Cavity mutants, in which as many as 120 surface accessible hydrophilic residues were replaced with hydrophobic amino acids, were shown to still assemble properly using size-exclusion chromatography and dynamic light scattering measurements. Wild-type Dps exhibited highly cooperative subunit folding and assembly, which was monitored by changes in Trp fluorescence and UV circular dichroism. The hydrophobic cavity mutants showed distinctly less cooperative unfolding behavior, with one mutant forming a partially assembled intermediate upon guanidine denaturation. Although the stability of Dps to such denaturation decreased with increasing apolar surface area, all proteins exhibited high melting temperatures, T(m) = 74-90 degrees C. Despite the large number of mutations, near-native ability to mineralize iron was maintained. This work illustrates the versatility of the ferritin scaffold for engineering large protein cavities with novel properties.

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Engineered Streptavidin Monomer and Dimer with Improved Stability and Function

et al. 2011

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Although streptavidin's high affinity for biotin has made it a widely used and studied binding protein and labeling tool, its tetrameric structure may interfere with some assays. A streptavidin mutant with a simpler quaternary structure would demonstrate a molecular-level understanding of its structural organization and lead to the development of a novel molecular reagent. However, modulating the tetrameric structure without disrupting biotin binding has been extremely difficult. In this study, we describe the design of a stable monomer that binds biotin both in vitro and in vivo. To this end, we constructed and characterized monomers containing rationally designed mutations. The mutations improved the stability of the monomer (increase in T(m) from 31 to 47 °C) as well as its affinity (increase in K(d) from 123 to 38 nM). We also used the stability-improved monomer to construct a dimer consisting of two streptavidin subunits that interact across the dimer-dimer interface, which we call the A/D dimer. The biotin binding pocket is conserved between the tetramer and the A/D dimer, and therefore, the dimer is expected to have a significantly higher affinity than the monomer. The affinity of the dimer (K(d) = 17 nM) is higher than that of the monomer but is still many orders of magnitude lower than that of the wild-type tetramer, which suggests there are other factors important for high-affinity biotin binding. We show that the engineered streptavidin monomer and dimer can selectively bind biotinylated targets in vivo by labeling the cells displaying biotinylated receptors. Therefore, the designed mutants may be useful in novel applications as well as in future studies in elucidating the role of oligomerization in streptavidin function.

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Modulating the DNA Affinity of Elk-1 with Computationally Selected Mutations

Cited by 7 publications

References 38 publications

Molecular dynamics simulations and in silico peptide ligand screening of the Elk-1 ETS domain

Molecular dynamics simulations and in silico peptide ligand screening of the Elk-1 ETS domain

Design of Functional Ferritin-Like Proteins with Hydrophobic Cavities

Engineered Streptavidin Monomer and Dimer with Improved Stability and Function

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