DNA Recognition Helix Research Articles

Val43 residue of NsrR is crucial for the nitric oxide response of Salmonella Typhimurium.

In pathogenic bacteria, the flavohemoglobin Hmp is crucial in metabolizing the cytotoxic levels of nitric oxide (NO) produced in phagocytic cells, contributing to bacterial virulence. Hmp expression is predominantly regulated by the Rrf2 family transcription repressor NsrR in an NO-dependent manner; however, the underlying molecular mechanism in enterobacteria remains poorly understood. In this study, we identified Val43 of Salmonella Typhimurium NsrR (StNsrR) as a critical amino acid residue for regulating Hmp expression. The Val43-to-Ala-substituted mutant NsrR isolated through random and site-directed mutagenesis showed high binding affinity to the target DNA irrespective of NO exposure, resulting in a severe reduction in hmp transcription and slow NO metabolism in Salmonella under NO-producing conditions. Conversely, the Val43-to-Glu-substituted NsrR caused effects similar to nsrR null mutation, which directed hmp transcription and NO metabolism in a constitutive way. Comparative analysis of the primary sequences of NsrR and another NO-sensing Rrf2 family regulator, IscR, from diverse bacteria, revealed that Val43 of enterobacterial NsrR corresponds to Ala in Pseudomonas aeruginosa or Streptomyces coelicolor NsrR and Glu in enterobacterial IscR, all of which are located in the DNA recognition helix α3. The predicted structure of StNsrR in complex with the hmp DNA suggests dissimilar spatial stoichiometry in the interactions of Val43 and its substituted residues with the target DNA, consistent with the observed phenotypic changes in StNsrR Val43 mutants. Our findings highlight the discriminative roles of the NsrR recognition helix in regulating species-specific target gene expression, facilitating effective NO detoxification strategies in bacteria across diverse environments. The precise regulation of flavohemoglobin Hmp expression by NsrR is critical for bacterial fitness, as excessive Hmp expression in the absence of NO can disturb bacterial redox homeostasis. While the molecular structure of Streptomyces coelicolor NsrR has been recently identified, the specific molecular structures of NsrR proteins in enterobacteria remain unknown. Our discovery of the crucial role of Val43 in the DNA recognition helix α3 of Salmonella NsrR offers valuable insights into the Hmp modulation under NO stress. Furthermore, the observed amino acid polymorphisms in the α3 helices of NsrR proteins across different bacterial species suggest the diverse evolution of NsrR structure and gene regulation in response to varying levels of NO pressure within their ecological niches.

Structural basis for activation of DNMT1

DNMT1 is an essential enzyme that maintains genomic DNA methylation, and its function is regulated by mechanisms that are not yet fully understood. Here, we report the cryo-EM structure of human DNMT1 bound to its two natural activators: hemimethylated DNA and ubiquitinated histone H3. We find that a hitherto unstudied linker, between the RFTS and CXXC domains, plays a key role for activation. It contains a conserved α-helix which engages a crucial “Toggle” pocket, displacing a previously described inhibitory linker, and allowing the DNA Recognition Helix to spring into the active conformation. This is accompanied by large-scale reorganization of the inhibitory RFTS and CXXC domains, allowing the enzyme to gain full activity. Our results therefore provide a mechanistic basis for the activation of DNMT1, with consequences for basic research and drug design.

RF15 | PMON291 Crystal Structures of Androgen DNA Binding Domain Interacting with its Response Elements (C3(1) ARE and MMTV-GRE)

Abstract Prostate cancer (PCa) is the second leading cancer in the world in men. The major therapeutic target in advanced PCa is the ligand-binding pocket (LBP) of the androgen receptor (AR). The anti-androgens used to treat cancer are binding to this domain and compete with the natural agonist. Over time the cancer grows resistant towards this therapy. Nevertheless, the cancer still typically depends on the AR, indicating other sites of the AR may still serve as drug targets. The DNA binding domain (DBD) is a likely good therapeutic target as it is involved in essential processes such AR dimerization, translocation to the nucleus and binding to the androgen response elements (AREs). However the structure and the DNA binding interactions with AREs are not fully explored yet. Only one crystal structure (pdb: 1R4I) is available. In this structure, the DBD is bound to an artificial direct repeat DNA sequences (ADR3), resulting in an unexpected dimeric head-to-head arrangement. A better understanding on how the AR recognizes and binds to different native DNA sequences which are mainly organized as semi inverted repeats is crucial for rational drug design. Here, we performed protein crystallography of the DBD in complex with various natural ARE sequences, including the mouse mammary tumour virus (MMTV-GRE) motif. Surprisingly, this latter element is still able to be activated by a dimerization deficient AR. Our first crystal structures were hallmarked by a high degree of disorder and appeared to bind both in a head-to-head as a head-to-tail fashion. Rational engineering of the DNA duplexes used for crystallography yielded well diffracting crystals for two different complexes. These two structures were diffracted to a range of 2 to 2.7 Å. Both structures with natural sequences appeared to bind in the head-to-head arrangement including the MMTV-GRE which consist out of semi inverted repeat DNA, whereas the C3(1)ARE contains the canonical fully inverted repeats. The structure reveals a slightly bend arrangement from the previously reported structure. The displacement is most visible at the DNA recognition helix bound to the half site and the so-called lever arm, which positions this helix in this region with a RMSD difference of 1.3 A. This indicates that the structural conformation changes of the protein is driven towards improved DNA-base pair recognition. Indeed the protein is able to make an additional set of non-covalent interactions with the base pairs of MMTV-GRE, leading to higher affinity. Both these extra interactions and conformational change lead to a higher affinity binding of protein-DNA, which seems to overcome the barrier introduced by the dimerization breaking mutations. This interaction surface could be a druggable site for structure-based drug design in the future, targeting AR-DBD dimerization. Presentation: Sunday, June 12, 2022 12:30 p.m. - 12:35 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Conformational Plasticity and DNA-Binding Specificity of the Eukaryotic Transcription Factor Pax5.

The eukaryotic transcription factor Pax5 has a DNA-binding Paired domain composed of two independent helical bundle subdomains joined by a flexible linker. Previously, we showed distinct biophysical properties of the N-terminal (NTD) and C-terminal (CTD) subdomains, with implications for how these two regions cooperate to distinguish nonspecific and cognate DNA sites [Perez-Borrajero, C., et al. (2016) J. Mol. Biol. 428, 2372-2391]. In this study, we combined experimental methods and molecular dynamics (MD) simulations to dissect the mechanisms underlying the functional differences between the Pax5 subdomains. Both subdomains showed a similar dependence of DNA-binding affinity on ionic strength. However, due to a greater contribution of non-ionic interactions, the NTD bound its cognate DNA half-site with an affinity approximately 10-fold higher than that of the CTD with its half-site. These interactions involve base-mediated contacts as evidenced by nuclear magnetic resonance spectroscopy-monitored chemical shift perturbations. Isothermal titration calorimetry revealed that favorable enthalpic and compensating unfavorable entropic changes were substantially larger for DNA binding by the NTD than by the CTD. Complementary MD simulations indicated that the DNA recognition helix H3 of the NTD is particularly flexible in the absence of DNA and undergoes the largest changes in conformational dynamics upon binding. Overall, these data suggest that the differences observed for the subdomains of Pax5 are due to the coupling of DNA binding with dampening of motions in the NTD required for specific base contacts. Thus, the conformational plasticity of the Pax5 Paired domain underpins the differing roles of its subdomains in association with nonspecific versus cognate DNA sites.

Allosteric Response of DNA Recognition Helices of Catabolite Activator Protein to cAMP and DNA Binding.

The homodimeric catabolite activator protein (CAP) regulates the transcription of several bacterial genes based on the cellular concentration of cyclic adenosine monophosphate (cAMP). The binding of cAMP to CAP triggers allosteric communication between the cAMP binding domains (CBD) and DNA binding domains (DBD) of CAP, which entails repositioning of DNA recognition helices (F-helices) in the DBD to dock favorably to the target DNA. Despite considerable progress, much remains to be understood about the mechanistic details of DNA recognition by CAP and about the map of allosteric pathways involved in CAP-mediated gene transcription. The present study uses molecular dynamics and umbrella sampling simulations to investigate the mechanism of cAMP- and DNA-induced changes in the conformation and energetics of F-helices observed during the allosteric regulation of CAP by cAMP and the subsequent binding to the DNA promoter region. Using novel collective variables, the free energy profiles associated with the orientation and dynamics of F-helices in the unliganded, cAMP-bound, and cAMP-DNA-bound states of CAP are calculated and compared. The binding-induced alterations in the resultant free energy profiles reveal important flexibility constraints imposed on DBD upon cAMP and DNA binding. A comprehensive analysis of residue-wise interaction maps reveals potential allosteric pathways between CBD and DBD that facilitate the allosteric transduction of regulatory signals in CAP. The revelation that the predicted allosteric pathways crisscross the intersubunit interface offers important clues on the microscopic origin of the intersubunit cooperativity and dimer stability of CAP.

Protein Diffusion on Charged Biopolymers: DNA versus Microtubule

Protein diffusion in lower-dimensional spaces is used for various cellular functions. For example, sliding on DNA is essential for proteins searching for their target sites, and protein diffusion on microtubules is important for proper cell division and neuronal development. On the one hand, these linear diffusion processes are mediated by long-range electrostatic interactions between positively charged proteins and negatively charged biopolymers and have similar characteristic diffusion coefficients. On the other hand, DNA and microtubules have different structural properties. Here, using computational approaches, we studied the mechanism of protein diffusion along DNA and microtubules by exploring the diffusion of both protein types on both biopolymers. We found that DNA-binding and microtubule-binding proteins can diffuse on each other’s substrates; however, the adopted diffusion mechanism depends on the molecular properties of the diffusing proteins and the biopolymers. On the protein side, only DNA-binding proteins can perform rotation-coupled diffusion along DNA, with this being due to their higher net charge and its spatial organization at the DNA recognition helix. By contrast, the lower net charge on microtubule-binding proteins enables them to diffuse more quickly than DNA-binding proteins on both biopolymers. On the biopolymer side, microtubules possess intrinsically disordered, negatively charged C-terminal tails that interact with microtubule-binding proteins, thus supporting their diffusion. Thus, although both DNA-binding and microtubule-binding proteins can diffuse on the negatively charged biopolymers, the unique molecular features of the biopolymers and of their natural substrates are essential for function.

Structural basis of G-quadruplex DNA recognition by the yeast telomeric protein Rap1.

G-quadruplexes are four-stranded nucleic acid structures involved in multiple cellular pathways including DNA replication and telomere maintenance. Such structures are formed by G-rich DNA sequences typified by telomeric DNA repeats. Whilst there is evidence for proteins that bind and regulate G-quadruplex formation, the molecular basis for this remains poorly understood. The budding yeast telomeric protein Rap1, originally identified as a transcriptional regulator functioning by recognizing double-stranded DNA binding sites, was one of the first proteins to be discovered to also bind and promote G-quadruplex formation in vitro. Here, we present the 2.4 Å resolution crystal structure of the Rap1 DNA-binding domain in complex with a G-quadruplex. Our structure not only provides a detailed insight into the structural basis for G-quadruplex recognition by a protein, but also gives a mechanistic understanding of how the same DNA-binding domain adapts to specifically recognize different DNA structures. The key observation is the DNA-recognition helix functions in a bimodal manner: In double-stranded DNA recognition one helix face makes electrostatic interactions with the major groove of DNA, whereas in G-quadruplex recognition a different helix face is used to make primarily hydrophobic interactions with the planar face of a G-tetrad.

The wing of the ToxR winged helix-turn-helix domain is required for DNA binding and activation of toxT and ompU.

ToxR and TcpP, two winged helix-turn-helix (w-HTH) family transcription factors, co-activate expression of the toxT promoter in Vibrio cholerae. ToxT then directly regulates a number of genes required for virulence. In addition to co-activation of toxT, ToxR can directly activate the ompU promoter and repress the ompT promoter. Based on a previous study suggesting that certain wing residues of ToxR are preferentially involved in toxT co-activation compared to direct ompU activation, we employed alanine-scanning mutagenesis to determine which residues in the wing of ToxR are required for activation of each promoter. All of the ToxR wing residues tested that were critical for transcriptional activation of toxT and/or ompU were also critical for DNA binding. While some ToxR wing mutants had reduced interaction with TcpP, that reduced interaction did not correlate with a specific defect in toxT activation. Rather, such mutants also affected ompU activation and DNA binding. Based on these findings we conclude that the primary role of the wing of ToxR is to bind DNA, along with the DNA recognition helix of ToxR, and this function is required both for direct activation of ompU and co-activation of toxT.

Two amino acids missing of MtrA resulted in increased erythromycin level and altered phenotypes in Saccharopolyspora erythraea.

The MtrA-MtrB two-component regulatory system is highly conserved in Actinobacteria and plays crucial roles in cell cycle progression, cell morphology, antibiotic resistance, and osmoprotection. Previously, we revealed that the MtrA protein of Saccharopolyspora erythraea E3 strain (a high erythromycin-producing strain) had a two amino acid (H197 and V198) deletion in the DNA recognition helices of the C-terminal domain compared to the wild type S. erythraea strain NRRL2338. Here, we identified mepA (encoding a membrane protein related to metalloendopeptidases) as an MtrA target gene, and found that deleting the two amino acids in MtrA (MtrAdel) resulted in the loss of its DNA-binding activity for the mepA gene. The mutant MtrAdel lost its regulatory activity and affected various physiological functions consistent with mtrA deletion, including increased erythromycin biosynthesis, enhanced antibiotic resistance, deregulated osmoprotection, and improved transport of substances. The introduction of the wild type mtrA gene into the S. erythraea E3 strain with the mtrAdel gene decreased the erythromycin yield by approximately 50%, confirming that MtrA repressed erythromycin production. These findings demonstrate that MtrA is an important pleiotropic regulator of erythromycin biosynthesis, antibiotic resistance, osmoprotection, and substance transport in S. erythraea and provide new insights for improving erythromycin production. Future studies linking the molecular effects of MtrA to these phenotypes will improve our understanding of the MtrA-MtrB two-component regulatory system in Actinobacteria.

Deciphering the Molecular Mechanism Underpinning Phage Arbitrium Communication Systems

SummaryBacillus phages use a communication system, termed “arbitrium,” to coordinate lysis-lysogeny decisions. Arbitrium communication is mediated by the production and secretion of a hexapeptide (AimP) during lytic cycle. Once internalized, AimP reduces the expression of the negative regulator of lysogeny, AimX, by binding to the transcription factor, AimR, promoting lysogeny. We have elucidated the crystal structures of AimR from the Bacillus subtilis SPbeta phage in its apo form, bound to its DNA operator and in complex with AimP. AimR presents intrinsic plasticity, sharing structural features with the RRNPP quorum-sensing family. Remarkably, AimR binds to an unusual operator with a long spacer that interacts nonspecifically with the receptor TPR domain, while the HTH domain canonically recognizes two inverted repeats. AimP stabilizes a compact conformation of AimR that approximates the DNA-recognition helices, preventing AimR binding to the aimX promoter region. Our results establish the molecular basis of the arbitrium communication system.

A combined computational and experimental approach reveals the structure of a C/EBPβ–Spi1 interaction required for IL1B gene transcription

We previously reported that transcription of the human IL1B gene, encoding the proinflammatory cytokine interleukin 1β, depends on long-distance chromatin looping that is stabilized by a mutual interaction between the DNA-binding domains (DBDs) of two transcription factors: Spi1 proto-oncogene at the promoter and CCAAT enhancer-binding protein (C/EBPβ) at a far-upstream enhancer. We have also reported that the C-terminal tail sequence beyond the C/EBPβ leucine zipper is critical for its association with Spi1 via an exposed residue (Arg-232) located within a pocket at one end of the Spi1 DNA-recognition helix. Here, combining in vitro interaction studies with computational docking and molecular dynamics of existing X-ray structures for the Spi1 and C/EBPβ DBDs, along with the C/EBPβ C-terminal tail sequence, we found that the tail sequence is intimately associated with Arg-232 of Spi1. The Arg-232 pocket was computationally screened for small-molecule binding aimed at IL1B transcription inhibition, yielding l-arginine, a known anti-inflammatory amino acid, revealing a potential for disrupting the C/EBPβ-Spi1 interaction. As evaluated by ChIP, cultured lipopolysaccharide (LPS)-activated THP-1 cells incubated with l-arginine had significantly decreased IL1B transcription and reduced C/EBPβ's association with Spi1 on the IL1B promoter. No significant change was observed in direct binding of either Spi1 or C/EBPβ to cognate DNA and in transcription of the C/EBPβ-dependent IL6 gene in the same cells. These results support the notion that disordered sequences extending from a leucine zipper can mediate protein-protein interactions and can serve as druggable targets for regulating gene promoter activity.

Loss-of-function mutations in Zn-finger DNA-binding domain of HNF4A cause aberrant transcriptional regulation in liver cancer.

Hepatocyte nuclear factors (HNF) are transcription factors that crucially regulate cell-specific gene expression in many tissues, including the liver. Of these factors, HNF4A acts both as a master regulator of liver organogenesis and a tumor suppressor in the liver. In our whole genome sequencing analysis, we found seven somatic mutations (three Zn-finger mutations, three deletion mutants, and one intron mutation) of HNF4A in liver cancers. Interestingly, three out of seven mutations were clustered in its Zn-finger DNA-binding domain; G79 and F83 are positioned in the DNA recognition helix and the sidechain of M125 is sticking into the core of domain. These mutations are likely to affect DNA interaction from a structural point of view. We then generated these mutants and performed in-vitro promoter assays as well as DNA binding assays. These three mutations reduced HNF4 transcriptional activity at promoter sites of HNF4A-target genes. Expectedly, this decrease in transcriptional activity was associated with a change in DNA binding. RNA-Seq analysis observed a strong correlation between HNF4A expression and expression of its target genes, ApoB and HNF1A, in liver cancers. Since knockdown of HNF4A caused a reduction in ApoB and HNF1A expression, possibly loss of HNF4 reduces the expression of these genes and subsequently tumor growth is triggered. Therefore, we propose that HNF4A mutations G79C, F83C, and M125I are functional mutations found in liver cancers and that loss of HNF4A function, through its mutation, leads to a reduction in HNF1A and ApoB gene expression with a concomitant increased risk of liver tumorigenesis.

Proposed 3-D Structure of a Critical Protein-Protein Interaction Required for Transcription of the IL1B Gene Coding for Human Interleukin 1β

Abstract We previously reported that expression of the human IL1B gene coding for Interleukin 1β in LPS-activated macrophages requires long-range chromatin looping mediated by a mutual DNA binding domain (DBD) interaction between two transcription factors, Spi-1/PU.1 (Spi1) at the promoter and C/EBPβ at a far-upstream enhancer. We have now combined preexisting X-ray structures and advanced computational techniques with additional biochemical and cell-based studies in order to further investigate the structure of this critical interaction. In vitro binding was used to generate a surface interaction map for the two proteins that suggested an association similar to that of a C/EBPβ·Myb X-ray structure reported by others. The distinction is that, in addition to a C/EBPβ leucine zipper interaction similar to that with the Myb DBD, there is an interaction between a carboxyl-tail extension of one of the C/EBPβ leucine zipper monomers with Spi1. This is consistent with our previous report that the carboxyl-tail of C/EBPβ is critical for association with arginine 232 within a pocket at one end of the Spi1 DNA recognition helix. L-arginine, a known anti-inflammatory, was computationally identified to bind in this interaction pocket. In vivo studies of LPS-activated THP-1 macrophages treated with L-arginine decreased IL1B transcription in parallel with a reduction in C/EBPβ association with Spi1 on the IL1B promoter. No significant change in direct binding of Spi1 and C/EBPβ to DNA was observed. These results suggest that L-arginine inhibits IL1B gene expression by competitive inhibition of C/EBPβ binding to Spi1 in the absence of any effect on direct DNA binding for either factor, and support the predicted structure of the C/EBPβ·Spi1 interaction.

DNA-binding mechanism of the Hippo pathway transcription factor TEAD4.

TEA domain (TEAD) family transcription factors are key regulators in development, tissue homeostasis and cancer progression. TEAD4 acts as a critical downstream effector of the evolutionarily conserved Hippo signaling pathway. The well-studied oncogenic protein YAP forms a complex with TEAD4 to regulate gene transcription; so does the tumor suppressor VGLL4. Although it is known that TEAD proteins can bind promoter regions of target genes through the TEA domain, the specific and detailed mechanism of DNA recognition by the TEA domain remains partially understood. Here, we report the crystal structure of TEAD4 TEA domain in complex with a muscle-CAT DNA element. The structure revealed extensive interactions between the TEA domain and the DNA duplex involving both the major and minor grooves of DNA helix. The DNA recognition helix, α3 helix, determines the specificity of the TEA domain binding to DNA sequence. Structure-guided biochemical analysis identified two major binding sites on the interface of the TEA domain-DNA complex. Mutation of TEAD4 at either site substantially decreases its occupancy on the promoter region of target genes, and largely impaired YAP-induced TEAD4 transactivation and target gene transcription, leading to inhibition of growth and colony formation of gastric cancer cell HGC-27. Collectively, our work provides a structural basis for understanding the regulatory mechanism of TEAD-mediated gene transcription.

Crystal structures of the NO sensor NsrR reveal how its iron-sulfur cluster modulates DNA binding

NsrR from Streptomyces coelicolor (Sc) regulates the expression of three genes through the progressive degradation of its [4Fe–4S] cluster on nitric oxide (NO) exposure. We report the 1.95 Å resolution crystal structure of dimeric holo-ScNsrR and show that the cluster is coordinated by the three invariant Cys residues from one monomer and, unexpectedly, Asp8 from the other. A cavity map suggests that NO displaces Asp8 as a cluster ligand and, while D8A and D8C variants remain NO sensitive, DNA binding is affected. A structural comparison of holo-ScNsrR with an apo-IscR-DNA complex shows that the [4Fe–4S] cluster stabilizes a turn between ScNsrR Cys93 and Cys99 properly oriented to interact with the DNA backbone. In addition, an apo ScNsrR structure suggests that Asn97 from this turn, along with Arg12, which forms a salt-bridge with Asp8, are instrumental in modulating the position of the DNA recognition helix region relative to its major groove.

Structured and disordered regions cooperatively mediate DNA-binding autoinhibition of ETS factors ETV1, ETV4 and ETV5.

Autoinhibition enables spatial and temporal regulation of cellular processes by coupling protein activity to surrounding conditions, often via protein partnerships or signaling pathways. We report the molecular basis of DNA-binding autoinhibition of ETS transcription factors ETV1, ETV4 and ETV5, which are often overexpressed in prostate cancer. Inhibitory elements that cooperate to repress DNA binding were identified in regions N- and C-terminal of the ETS domain. Crystal structures of these three factors revealed an α-helix in the C-terminal inhibitory domain that packs against the ETS domain and perturbs the conformation of its DNA-recognition helix. Nuclear magnetic resonance spectroscopy demonstrated that the N-terminal inhibitory domain (NID) is intrinsically disordered, yet utilizes transient intramolecular interactions with the DNA-recognition helix of the ETS domain to mediate autoinhibition. Acetylation of selected lysines within the NID activates DNA binding. This investigation revealed a distinctive mechanism for DNA-binding autoinhibition in the ETV1/4/5 subfamily involving a network of intramolecular interactions not present in other ETS factors. These distinguishing inhibitory elements provide a platform through which cellular triggers, such as protein–protein interactions or post-translational modifications, may specifically regulate the function of these oncogenic proteins.

Rational identification of natural organic compounds to target the intermolecular interaction between Foxm and DNA in colorectal cancer

The oncogenic transcription factor forkhead box M (Foxm) is overexpressed in human colorectal cancer (CRC). Targeting the protein interaction with its cognate DNA has been established as an attractive approach to anti-CRC chemotherapy. State-of-the-art molecular dynamics (MD) simulations revealed that the Foxm adopts considerably different conformations to interact with and without its DNA partner; the holo conformation is tightly packed as a typical globulin configuration, whereas the apo form is locally unstructured that exhibits intrinsic disorder in DNA recognition helix, indicating that DNA binding can help the Foxm refolding. With this finding, the MD equilibrium structure of DNA-free Foxm was utilized to perform molecular docking virtual screening against a natural organic compound library. Consequently, six hit compounds were identified as potential small-molecule mediators of Foxm–DNA interaction; their binding affinities (KD) to Foxm DNA-binding domain were then determined to range between 3.8 and 230μM by using isothermal titration calorimetry. These compounds were suggested to recognize and stabilize the apo conformation of Foxm, thus shifting the binding reaction equilibrium of Foxm from DNA-bound to DNA-free states to disrupt the formation of Foxm–DNA adduct.

A Role for Autoinhibition in Preventing Dimerization of the Transcription Factor ETS1

ETS1 is the archetype of the ETS transcription factor (TF) family. ETS TFs share a DNA-binding domain, the ETS domain. All ETS TFs recognize a core GGA(A/T) binding site, and thus ETS TFs are found to redundantly regulate the same genes. However, each ETS TF has unique targets as well. One prevailing hypotheses explaining this duality is that protein-protein interactions, including homodimerization, allow each ETS TF to display distinct behavior. The behavior of ETS1 is further regulated by autoinhibition. Autoinhibition apparently modulates ETS1 DNA binding affinity, but the mechanism of this inhibition is not completely understood. We sought to characterize the relationship between DNA binding and ETS1 homodimer formation. We find that ETS1 interrogates DNA and forms dimers even when the DNA does not contain an ETS recognition sequence. Mutational studies also link nonspecific DNA backbone contacts with dimer formation, in addition to providing a new role for the recognition helix of ETS1 in maintaining the autoinhibited state. Finally, in showing that residues in the DNA recognition helix affect autoinhibition, we define a new function of ETS1 autoinhibition: maintenance of a monomeric state in the absence of DNA. The conservation of relevant amino acid residues across all ETS TFs indicates that the mechanisms of nonspecific DNA interrogation and protein oligomer formation elucidated here may be common to all ETS proteins that autoinhibit.

Structures of the Ets Protein DNA-binding Domains of Transcription Factors Etv1, Etv4, Etv5, and Fev: DETERMINANTS OF DNA BINDING AND REDOX REGULATION BY DISULFIDE BOND FORMATION

Ets transcription factors, which share the conserved Ets DNA-binding domain, number nearly 30 members in humans and are particularly involved in developmental processes. Their deregulation following changes in expression, transcriptional activity, or by chromosomal translocation plays a critical role in carcinogenesis. Ets DNA binding, selectivity, and regulation have been extensively studied; however, questions still arise regarding binding specificity outside the core GGA recognition sequence and the mode of action of Ets post-translational modifications. Here, we report the crystal structures of Etv1, Etv4, Etv5, and Fev, alone and in complex with DNA. We identify previously unrecognized features of the protein-DNA interface. Interactions with the DNA backbone account for most of the binding affinity. We describe a highly coordinated network of water molecules acting in base selection upstream of the GGAA core and the structural features that may account for discrimination against methylated cytidine residues. Unexpectedly, all proteins crystallized as disulfide-linked dimers, exhibiting a novel interface (distant to the DNA recognition helix). Homodimers of Etv1, Etv4, and Etv5 could be reduced to monomers, leading to a 40-200-fold increase in DNA binding affinity. Hence, we present the first indication of a redox-dependent regulatory mechanism that may control the activity of this subset of oncogenic Ets transcription factors.

Structural basis of DNA sequence recognition by the response regulator PhoP

The PhoP-PhoR two-component system plays a key role in regulating virulence of Mycobacterium tuberculosis. The response regulator PhoP is a transcription regulator, and it regulates expression of more than 100 genes. PhoP belongs to the OmpR/PhoB subfamily of response regulators. Despite extensive research in recent years, the molecular mechanism of DNA sequence recognition by this large subfamily of response regulators is not fully understood, especially the role of the N-terminal regulatory domain on DNA binding of the effector domain. Here we present a crystal structure of the full-length PhoP in complex with a direct-repeat DNA sequence. PhoP binds to DNA as a dimer. The two effector domains bind in tandem, each interacting with a half site of the direct repeat DNA. The DNA recognition helix inserts into the major groove, reading the sequence of a 7-bp motif. The wing residues interact with the downstream sequence, with a conserved arginine side chain inserting into the minor groove. Surprisingly, the regulatory domain also forms a tandem arrangement, instead of the anticipated symmetric dimer. The regulatory domain of the upstream protomer interacts with both domains of the downstream protomer. The structural elements of the alpha4-beta5-alpha5 face, which often found to be involved in dimer interface of the regulatory domain, play important roles in the interactions between the protomers. The crystal structure explains why PhoP recognizes direct repeats of two 7-bp motifs with a strict spacing of 4 bp and the highly cooperative binding of the two monomers. Detailed analysis of the structure along with analysis of the DNA sequence requirements and ITC measurements of protein-DNA binding interactions will be presented.