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

Abstract

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 ...

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

“…1A). Our previous studies suggested that the interdomain dynamics could harness the rapid conformational switch between the search and recognition modes, thereby allowing Egr-1 to overcome the speedstability paradox (24,28). Our current work is based on a hypothesis that the balance between the search and recognition modes is a major determinant of the kinetic efficiency in target DNA search by ZF proteins.…”

“…Our previous NMR and computational investigations on nonspecific and specific DNA complexes of Egr-1 suggested that, while scanning DNA, Egr-1 undergoes the dynamic conformational equilibrium between the search and recognition modes (28). We consider the conformational ensemble involving three major states in the DNA-scanning process as shown in Fig.…”

“…relatively short lifetime (∼0.5-1 h) (29). 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).…”

“…This situation is often called the speed-stability paradox (19,24,25). In our previous work (26)(27)(28), we studied how the inducible transcription factor Egr-1 (also known as Zif268) can overcome this paradox. The Egr-1 protein recognizes its 9-bp target sequence via three ZFs and plays important roles in the brain and cardiovascular system (29,30).…”

Asymmetric perturbations of signalling oligomers

“…Functional asymmetry can be observed when identical domains perform distinct tasks: i) the zinc fingers of the Egr-1 transcription factor perform rapid scanning and stable recognition (Zandarashvili et al, 2012) and ii) histidine kinase receptor dimers possess either kinase or phosphatase activities (Moore and Hendrickson, 2012). Operational asymmetry arises when the symmetry of enzyme reaction cycles is broken, which can lead to evolutionary consequences (García-Bellido, 1996).…”