Facilitated dissociation of transcription factors from single DNA binding sites

Kamar, Ramsey I.; Banigan, Edward J.; Erbaş, Aykut; Giuntoli, Rebecca D.; Cruz, Mónica Olvera de la; Johnson, Reid C.; Marko, John F.

doi:10.1073/pnas.1701884114

Cited by 74 publications

(105 citation statements)

References 55 publications

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“…For the sake of simplicity and to minimize the number of free fit parameters, here we assume a two-step fashion and that only the first step has a salt dependence. This assumption is consistent with a previously reported analyses of the experimental data [27]. Thus, the off-rate can be expressed as the sum of two reaction times as…”

Section: A Spontaneous Dissociationsupporting

confidence: 92%

“…2b to a simplified version of a theoretical model. This model has been previously suggested for the unbinding rates of transcription factors (i.e., nucleic-acid binding proteins) from single binding sites along double stranded (ds)DNA [23,27]. The model assumes that unbinding of a multimeric ligand occurs in a multi-step process.…”

Section: A Spontaneous Dissociationmentioning

confidence: 99%

“…[27]). The exponent m usually takes a value near unity for the familiar Langmuir binding isotherm in the absence of any cooperativity (i.e., binding events are not altered by already bound ligands) [23,27]. The dashed curves in Fig.…”

Section: Short-ranged Non-electrostatic Interactions Modulate the Ratmentioning

confidence: 99%

“…The free ligands can be identical to the initially bound ligand [21,22,[27][28][29][30]. However, different ligand molecules [27,30,31] or even nucleic acid fragments [20] can lead to the facilitated dissociation (FD).…”

Section: Introductionmentioning

confidence: 99%

“…However, different ligand molecules [27,30,31] or even nucleic acid fragments [20] can lead to the facilitated dissociation (FD). According to the proposed molecular mechanism for FD, a free ligand binds to an already occupied binding site and destabilizes the complex by forming a ternary complex (i.e., two ligands on the same site) [19,23,27]. This destabilized complex promotes shorter binding lifetimes for the ligands, while imposing an upper limit on the unbinding rates.…”

Section: Introductionmentioning

confidence: 99%

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Effects of electrostatic interactions on ligand dissociation kinetics

2018

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We study unbinding of multivalent cationic ligands from oppositely charged polymeric binding sites sparsely grafted on a flat neutral substrate. Our molecular dynamics (MD) simulations are suggested by single-molecule studies of protein-DNA interactions. We consider univalent salt concentrations spanning roughly a thousandfold range, together with various concentrations of excess ligands in solution. To reveal the ionic effects on unbinding kinetics of spontaneous and facilitated dissociation mechanisms, we treat electrostatic interactions both at a Debye-Hückel (DH, or 'implicit' ions, i.e., use of an electrostatic potential with a prescribed decay length) level, as well as by the more precise approach of considering all ionic species explicitly in the simulations. We find that the DH approach systematically overestimates unbinding rates, relative to the calculations where all ion pairs are present explicitly in solution, although many aspects of the two types of calculation are qualitatively similar. For facilitated dissociation (FD, acceleration of unbinding by free ligands in solution) explicit ion simulations lead to unbinding at lower free ligand concentrations. Our simulations predict a variety of FD regimes as a function of free ligand and ion concentrations; a particularly interesting regime is at intermediate concentrations of ligands where non-electrostatic binding strength controls FD. We conclude that explicit-ion electrostatic modeling is an essential component to quantitatively tackle problems in molecular ligand dissociation, including nucleicacid-binding proteins.

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Section: A Spontaneous Dissociationsupporting

confidence: 92%

Section: A Spontaneous Dissociationmentioning

confidence: 99%

Section: Short-ranged Non-electrostatic Interactions Modulate the Ratmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Effects of electrostatic interactions on ligand dissociation kinetics

2018

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From Immunogenic Peptides to Intrinsically Disordered Proteins

Dyson

Wright

2023

Israel Journal of Chemistry

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It is hard to evaluate the role of individual mentors in the genesis of important ideas. In the case of our realization that proteins do not have to be stably folded to be functional, the influence of Richard Lerner and our collaborative work in the 1980s on the conformations of immunogenic peptides provided a base level of thinking about the nature of polypeptides in water solutions that led us to formulate and develop our ideas on the importance of intrinsic disorder in proteins. This review describes how the insights gained into the behavior of peptides led directly to the realization that proteins were not only capable of being functional while disordered, but also that disorder provided a distinct functional advantage in many important cellular processes.

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Ter‐Seq: A high‐throughput method to stabilize transient ternary complexes and measure associated kinetics

et al. 2022

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Regulation of biological processes by proteins often involves the formation of transient, multimeric complexes whose characterization is mechanistically important but challenging. The bacterial toxin CcdB binds and poisons DNA Gyrase. The corresponding antitoxin CcdA extracts CcdB from its complex with Gyrase through the formation of a transient ternary complex, thus rejuvenating Gyrase. We describe a high throughput methodology called Ter-Seq to stabilize probable ternary complexes and measure associated kinetics using the CcdA-CcdB-GyrA14 ternary complex as a model system. The method involves screening a yeast surface display (YSD) saturation mutagenesis library of one partner (CcdB) for mutants that show enhanced ternary complex formation. We also isolated CcdB mutants that were either resistant or sensitive to rejuvenation, and used surface plasmon resonance (SPR) with purified proteins to validate the kinetics measured using the surface display. Positions, where CcdB mutations lead to slower rejuvenation rates, are largely involved in CcdA-binding, though there were several notable exceptions suggesting allostery. Mutations at these positions reduce the affinity towards CcdA, thereby slowing down the rejuvenation process. Mutations at GyrA14-interacting positions significantly enhanced rejuvenation rates, either due to reduced affinity or complete loss of CcdB binding to GyrA14. We examined the effect of different parameters (CcdA affinity, GyrA14 affinity, surface accessibilities, evolutionary conservation) on the rate of rejuvenation. Finally, we further validated the Ter-Seq results by monitoring the kinetics of ternary complex formation for individual CcdB mutants in solution by fluorescence resonance energy transfer (FRET) studies.

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Facilitated dissociation of transcription factors from single DNA binding sites

Cited by 74 publications

References 55 publications

Effects of electrostatic interactions on ligand dissociation kinetics

Effects of electrostatic interactions on ligand dissociation kinetics

From Immunogenic Peptides to Intrinsically Disordered Proteins

Ter‐Seq: A high‐throughput method to stabilize transient ternary complexes and measure associated kinetics

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