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 ...

The inducible transcription factor Egr-1, which recognizes a 9-bp target DNA sequence via three zinc-finger domains, rapidly activates particular genes upon cellular stimuli such as neuronal signals and vascular stresses. Here, using the stopped-flow fluorescence method, we measured the target search kinetics of the Egr-1 zinc-finger protein at various ionic strengths between 40 and 400 mM KCl and found the most efficient search at 150 mM KCl. We further investigated the kinetics of intersegment transfer, dissociation, and sliding of this protein on DNA at distinct concentrations of KCl. Our data suggest that Egr-1's kinetic properties are well suited for efficient scanning of chromosomal DNA in vivo. Based on a newly developed theory, we analyzed the origin of the optimal search efficiency at physiological ionic strength. Target association is accelerated by nonspecific binding to nearby sites and subsequent sliding to the target as well as by intersegment transfer. Although these effects are stronger at lower ionic strengths, such conditions also favor trapping of the protein at distant nonspecific sites, decelerating the target association. Our data demonstrate that Egr-1 achieves the optimal search at physiological ionic strength through a compromise between the positive and negative impacts of nonspecific interactions with DNA.

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...

Despite their importance in macromolecular interactions and functions, the dynamics of lysine side-chain amino groups in proteins are not well understood. In this study, we have developed the methodology for the investigations of the dynamics of lysine NH3(+) groups by NMR spectroscopy and computation. By using 1H−15N heteronuclear correlation experiments optimized for 15NH3(+) moieties, we have analyzed the dynamic behavior of individual lysine NH3(+) groups in human ubiquitin at 2 °C and pH 5. We modified the theoretical framework developed previously for CH3 groups and used it to analyze 15N relaxation data for the NH3(+) groups. For six lysine NH3(+) groups out of seven in ubiquitin, we have determined model-free order parameters, correlation times for bond rotation, and reorientation of the symmetry axis occurring on a pico- to nanosecond time scale. From CPMG relaxation dispersion experiment for lysine NH3(+) groups, slower dynamics occurring on a millisecond time scale have also been detected for Lys27. The NH3(+) groups of Lys48, which plays a key role as the linkage site in ubiquitination for proteasomal degradation, was found to be highly mobile with the lowest order parameter among the six NH3(+) groups analyzed by NMR. We compared the experimental order parameters for the lysine NH3(+) groups with those from a 1 μs molecular dynamics simulation in explicit solvent and found good agreement between the two. Furthermore, both the computer simulation and the experimental correlation times for the bond rotations of NH3(+) groups suggest that their hydrogen bonding is highly dynamic with a subnanosecond lifetime. This study demonstrates the utility of combining NMR experiment and simulation for an in-depth characterization of the dynamics of these functionally most important side-chains of ubiquitin.

Ion pairing is one of the most fundamental chemical interactions and is essential for molecular recognition by biological macromolecules. From an experimental standpoint, very little is known to date about ion-pair dynamics in biological macromolecular systems. Absorption, infrared, and Raman spectroscopic methods were previously used to characterize dynamic properties of ion pairs, but these methods can be applied only to small compounds. Here, using NMR 15N relaxation and hydrogen-bond scalar 15N-31P J-couplings (h3JNP), we have investigated the dynamics of the ion pairs between lysine side-chain NH3+ amino groups and DNA phosphate groups at the molecular interface of the HoxD9 homeodomain-DNA complex. We have determined the order parameters and the correlation times for C-N bond rotation and reorientation of the lysine NH3+ groups. Our data indicate that the NH3+ groups in the intermolecular ion pairs are highly dynamic at the protein-DNA interface, which should lower the entropic costs for protein-DNA association. Judging from the C-N bond-rotation correlation times along with experimental and quantum-chemically derived h3JNP hydrogen-bond scalar couplings, it seems that breakage of hydrogen bonds in the ion pairs occurs on a sub-nanosecond timescale. Interestingly, the oxygen-to-sulfur substitution in a DNA phosphate group was found to enhance the mobility of the NH3+ group in the intermolecular ion pair. This can partially account for the affinity enhancement of the protein-DNA association by the oxygen-to-sulfur substitution, which is a previously observed but poorly understood phenomenon.

Kinetic characterizations of protein translocation on DNA are nontrivial because the simultaneous presence of multiple different mechanisms makes it difficult to extract the information specific to a particular translocation mechanism. In this study, we have developed new approaches for the kinetic investigations of proteins' sliding and intersegment transfer (also known as "direct transfer") in the target DNA search process. Based on the analytical expression of the mean search time for the discrete-state stochastic model, we derived analytical forms of the apparent rate constant kapp for protein-target association in systems involving competitor DNA and the intersegment transfer mechanism. Our analytical forms of kapp facilitate the experimental determination of the kinetic rate constants for intersegment transfer and sliding in the target association process. Using stopped-flow fluorescence data for the target association kinetics along with the analytical forms of kapp, we have studied the translocation of the Egr-1 zinc-finger protein in the target DNA association process. Sliding was analyzed using the DNA-length-dependent kapp data. Using the dependence of kapp on the concentration of competitor DNA, we determined the second-order rate constant for intersegment transfer. Our results indicate that a major pathway in the target association process for the Egr-1 zinc-finger protein is the one involving intersegment transfer to a nonspecific site and the subsequent sliding to the target.

Intermolecular ion pairs (salt bridges) are crucial for protein–DNA association. For two protein–DNA complexes, we demonstrate that the ion pairs of protein side-chain NH3+ and DNA phosphate groups undergo dynamic transitions between distinct states in which the charged moieties are either in direct contact or separated by water. While the crystal structures of the complexes show only the solvent-separated ion pair (SIP) state for some interfacial lysine side chains, our NMR hydrogen-bond scalar coupling data clearly indicate the presence of the contact ion pair (CIP) state for the same residues. The 0.6-μs molecular dynamics (MD) simulations confirm dynamic transitions between the CIP and SIP states. This behavior is consistent with our NMR order parameters and scalar coupling data for the lysine side chains. Using the MD trajectories, we also analyze the free energies of the CIP–SIP equilibria. This work illustrates the dynamic nature of short-range electrostatic interactions in DNA recognition by proteins.

Basic side chains play major roles in recognition of nucleic acids by proteins. However, dynamic properties of these positively charged side chains are not well understood. In this work, we studied changes in conformational dynamics of basic side chains upon protein–DNA association for the zinc-finger protein Egr-1. By nuclear magnetic resonance (NMR) spectroscopy, we characterized the dynamics of all side-chain cationic groups in the free protein and in the complex with target DNA. Our NMR order parameters indicate that the arginine guanidino groups interacting with DNA bases are strongly immobilized, forming rigid interfaces. Despite the strong short-range electrostatic interactions, the majority of the basic side chains interacting with the DNA phosphates exhibited high mobility, forming dynamic interfaces. In particular, the lysine side-chain amino groups exhibited only small changes in the order parameters upon DNA-binding. We found a similar trend in the molecular dynamics (MD) simulations for the free Egr-1 and the Egr-1–DNA complex. Using the MD trajectories, we also analyzed side-chain conformational entropy. The interfacial arginine side chains exhibited substantial entropic loss upon binding to DNA, whereas the interfacial lysine side chains showed relatively small changes in conformational entropy. These data illustrate different dynamic characteristics of the interfacial arginine and lysine side chains.