Theory of Site-Specific DNA-Protein Interactions in the Presence of Conformational Fluctuations of DNA Binding Domains

167

Egr-1 is an inducible transcription factor that recognizes 9-bp target DNA sites via three zinc finger domains and activates genes in response to cellular stimuli such as synaptic signals and vascular stresses. Using spectroscopic and computational approaches, we have studied structural, dynamic, and kinetic aspects of the DNAscanning process in which Egr-1 is nonspecifically bound to DNA and perpetually changes its location on DNA. Our NMR data indicate that Egr-1 undergoes highly dynamic domain motions when scanning DNA. In particular, the zinc finger 1 (ZF1) of Egr-1 in the nonspecific complex is mainly dissociated from DNA and undergoes collective motions on a nanosecond timescale, whereas zinc fingers 2 and 3 (ZF2 and ZF3, respectively) are bound to DNA. This was totally unexpected because the previous crystallographic studies of the specific complex indicated that all of Egr-1's three zinc fingers are equally involved in binding to a target DNA site. Mutations that are expected to enhance ZF1's interactions with DNA and with ZF2 were found to reduce ZF1's domain motions in the nonspecific complex suggesting that these interactions dictate the dynamic behavior of ZF1. By experiment and computation, we have also investigated kinetics of Egr-1's translocation between two nonspecific DNA duplexes. Our data on the wild type and mutant proteins suggest that the domain dynamics facilitate Egr-1's intersegment transfer that involves transient bridging of two DNA sites. These results shed light on asymmetrical roles of the zinc finger domains for Egr-1 to scan DNA efficiently in the nucleus.NMR spectroscopy | target search process | interdomain dynamics | protein-DNA interactions | simulation I n cellular responses to various stimuli such as signals and stresses, gene regulation by transcription factors is of fundamental importance. Egr-1 (also known as Zif268) is an inducible transcription factor with crucial roles particularly in the brain and cardiovascular systems in mammals. In the brain, Egr-1 is induced by synaptic signals in an activity-dependent manner and activates genes for long-term memory formation and consolidation (1, 2). In the cardiovascular system, Egr-1 is a stress-inducible transcription factor that activates the genes for initiating defense responses against vascular stress and injury (3, 4). Given the short lifetime of induced Egr-1 (typically ∼2 h) (3), rapid gene activation by Egr-1 is important in these biological processes that require an immediate response to the stimuli.The induced Egr-1 protein has to initiate its role by searching for its target DNA sites among billions of DNA base pairs in the nucleus. In the DNA scanning process, transcription factors need to discriminate their target sites from nonspecific sites based on relatively minor differences in DNA structure and sequence. Crystallographic studies demonstrated that Egr-1 recognizes its 9-bp target sequence, GCGTGGGCG, as a monomer via zinc finger domains 1, 2, and 3 (hereafter referred to as ZF1, ZF2, and ZF3) that contact 3 ...

Section: Discussionsupporting

confidence: 79%

Asymmetrical roles of zinc fingers in dynamic DNA-scanning process by the inducible transcription factor Egr-1

Zandarashvili

Vuzman

Esadze

et al. 2012

167

“…Our previous study suggested the presence of dynamic transitions between conformationally different states of Egr-1 during the DNA-scanning process: one allowing for rapid translocation on DNA (the search mode) and the other allowing for highly specific recognition (the recognition mode) (28). This finding is consistent with the conformational switch model (19,25,(31)(32)(33), which some theoreticians proposed to resolve the speed-stability paradox, originally on the basis of experimental data for the Escherichia coli lac repressor (34,35).…”

Section: Significancesupporting

confidence: 75%

Balancing between affinity and speed in target DNA search by zinc-finger proteins via modulation of dynamic conformational ensemble

Zandarashvili

Esadze

Vuzman

et al. 2015

145

Although engineering of transcription factors and DNA-modifying enzymes has drawn substantial attention for artificial gene regulation and genome editing, most efforts focus on affinity and specificity of the DNA-binding proteins, typically overlooking the kinetic properties of these proteins. However, a simplistic pursuit of high affinity can lead to kinetically deficient proteins that spend too much time at nonspecific sites before reaching their targets on DNA. We demonstrate that structural dynamic knowledge of the DNA-scanning process allows for kinetically and thermodynamically balanced engineering of DNA-binding proteins. Our current study of the zinc-finger protein Egr-1 (also known as Zif268) and its nuclease derivatives reveals kinetic and thermodynamic roles of the dynamic conformational equilibrium between two modes during the DNAscanning process: one mode suitable for search and the other for recognition. By mutagenesis, we were able to shift this equilibrium, as confirmed by NMR spectroscopy. Using fluorescence and biochemical assays as well as computational simulations, we analyzed how the shifts of the conformational equilibrium influence binding affinity, target search kinetics, and efficiency in displacing other proteins from the target sites. A shift toward the recognition mode caused an increase in affinity for DNA and a decrease in search efficiency. In contrast, a shift toward the search mode caused a decrease in affinity and an increase in search efficiency. This accelerated site-specific DNA cleavage by the zinc-finger nuclease, without enhancing off-target cleavage. Our study shows that appropriate modulation of the dynamic conformational ensemble can greatly improve zinc-finger technology, which has used Egr-1 (Zif268) as a major scaffold for engineering.protein-DNA interactions | DNA scanning | target search | kinetics | dynamics A rtificial transcription factors and DNA-modifying enzymes have gained popularity as powerful means for artificial gene regulation and genome editing (1-7). Successful applications were reported on artificial zinc-finger (ZF) proteins engineered to exhibit a desired sequence specificity in binding to DNA (1-5). Artificial ZF transcription factors, comprising engineered ZFs and transactivation or repression domains, are used to regulate particular genes (1-3). ZF nucleases (ZFNs), comprising engineered ZFs and a FokI nuclease domain (ND), can site-specifically cleave DNA at particular sequences and allow for genome editing in vivo (4, 5). ZFN-based gene therapy for HIV infection is currently under phase 2 clinical trials, yielding some successful cases (8). Other studies suggested that ZF-based gene control could also be therapeutically effective for hemophilia (9) and Parkinson's disease (10). For the success of these technologies, however, two issues, toxicity and low efficiency, should be resolved (4, 5, 11). Regarding the latter issue, some studies (11)(12)(13)(14) suggest that, despite high affinities for target DNA, the artificial proteins do not necessa...

“…For protein-DNA binding, Slutsky and Mirny (12) explicitly considered the conformational switch of the protein while nonspecifically bound to the DNA. This and other follow-up studies (16,19) were based on the two-state model, but with an unjustified specification ofn. Using our approach based on the position-dependent capture probability, we show that a DNA-binding protein can stay mostly in a fast diffusing "inactive" conformation until it encounters the specific site, whereupon interactions with the specific site induce it to quickly switch to the active conformation for recognition.…”

mentioning

confidence: 99%

“…conformational change | nonspecific binding | binding kinetics E ver since the first demonstration that proteins can bind to specific DNA sequences (1,2), numerous studies have been carried out to address the question of how a protein can readily find a short specific site on a long DNA (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). It is widely accepted that the search is accomplished by coupled 3D diffusion in the bulk solution and 1D diffusion, while specifically bound, along the DNA surface.…”

mentioning

confidence: 99%

Rapid search for specific sites on DNA through conformational switch of nonspecifically bound proteins

Zhou

2011

We develop a theory for the rapid search of specific sites on DNA, via a mechanism in which a nonspecifically-bound protein can switch between two conformations. In the "inactive" conformation, the bound protein has favorable, nonspecific interactions with the DNA, but cannot be recognized by the target site. In the "active" conformation, the protein is recognized by the target site but has a very rugged energy surface elsewhere on the DNA. The rate constant for protein binding to the specific site is calculated by an approach in which the protein, after reaching the DNA surface via 3D diffusion, searches for the target site via 1D diffusion while being allowed to escape to the bulk solution. Mindful of the pitfalls of many previous approximate treatments, we validate our approach against a rigorous solution of the problem when the protein has a fixed conformation. In the 1D diffusion toward the specific site, a conformationally switchable protein predominantly adopts the inactive conformation due to the favorable nonspecific interactions with the DNA, thus maximizing the 1D diffusion constant and minimizing the chance of escape to the bulk solution. Once at the target site, a transition to the active conformation allows the protein to be captured. This induced-switch mechanism provides robust speedup of protein-DNA binding rates, and appears to be adopted by many transcription factors and DNA-modifying enzymes.conformational change | nonspecific binding | binding kinetics E ver since the first demonstration that proteins can bind to specific DNA sequences (1, 2), numerous studies have been carried out to address the question of how a protein can readily find a short specific site on a long . It is widely accepted that the search is accomplished by coupled 3D diffusion in the bulk solution and 1D diffusion, while specifically bound, along the DNA surface. Many theoretical models have focused on the 1D diffusion. Appealing terms such as hopping and jumping have been used to treat excursions into the bulk solution. However, the lack of rigor in previous treatments has led to conflicting results. In particular, whether 1D diffusion can play a significant rate-enhancement role under physiological conditions has been questioned (15). Here we present an approach for calculating the protein-DNA binding rate constant k a that allows for proper coupling between 3D and 1D diffusion. We use this approach to treat the conformational switch of a nonspecifically bound protein, and demonstrate that it provides a robust mechanism for speeding up the search of specific sites.The bimolecular rate constant k a can be rigorously determined from the equation governing the relative translational diffusion, rotational diffusion, and internal motions of the two binding molecules (21). This governing equation involves the potential of mean force in these degrees of freedom and parameters characterizing the external and internal dynamics, such as a position-dependent diffusion constant for the translational diffusion and transition rates...