DNA-binding Helix Research Articles

Structural and dynamical aspect of DNA motif sequence specific binding of AP-1 transcription factor.

Activator protein-1 (AP-1) comprises one of the largest and most evolutionary conserved families of ubiquitous eukaryotic transcription factors that act as a pioneer factor. Diversity in DNA binding interaction of AP-1 through a conserved basic-zipper (bZIP) domain directs in-depth understanding of how AP-1 achieves its DNA binding selectivity and consequently gene regulation specificity. Here, we address the structural and dynamical aspects of the DNA target recognition process of AP-1 using microsecond-long atomistic simulations based on the structure of the human AP-1 FosB/JunD bZIP-DNA complex. Our results show the unique role of DNA shape features in selective base specific interactions, characteristic ion population, and solvation properties of DNA grooves to form the motif sequence specific AP-1-DNA complex. The TpG step at the two terminals of the AP-1 site plays an important role in the structural adjustment of DNA by modifying the helical twist in the AP-1 bound state. We addressed the role of intrinsic motion of the bZIP domain in terms of opening and closing gripper motions of DNA binding helices, in target site recognition and binding of AP-1 factors. Our observations suggest that binding to the cognate motif in DNA is mainly accompanied with the precise adjustment of closing gripper motion of DNA binding helices of the bZIP domain.

Stability and dynamics of extradenticle modulates its function

Extradenticle (EXD) is a partner protein of the HOX transcription factors and plays an important role in the development of Drosophila. It confers increased affinity and specificity of DNA-binding to the HOX proteins. However, the DNA-binding homeodomain of EXD has a significantly weaker affinity to DNA compared to the HOX homeodomains. Here, we show that a glycine residue (G290) in the middle of the EXD DNA-binding helix primarily results in this weaker binding. Glycine destabilizes helices. To probe its role in the stability and function of the protein, G290 was mutated to alanine. The intrinsic stability of the DNA-binding helix increased in the G290A mutant as observed by NMR studies and molecular dynamics (MD) simulation. Also, NMR dynamics and MD simulation show that dynamic motions present in the wild-type protein are quenched in the mutant. This in turn resulted in increased stability of the entire homeodomain (ΔΔGG→A of −2.6 kcal/mol). Increased protein stability resulted in three-fold better DNA-binding affinity of the mutant as compared to the wild-type protein. Molecular mechanics with generalized Born and surface area solvation (MMGBSA) analysis of our MD simulation on DNA-bound models of both wild-type and mutant proteins shows that the contribution to binding is enhanced for most of the interface residues in the mutant compared to the wild-type. Interestingly, the flexible N-terminal arm makes more stable contact with the DNA minor groove in the mutant. We found that the two interaction sites i.e. the DNA-binding helix and the unstructured N-terminal arm influence each other via the bound DNA. These results provide an interesting conundrum: alanine at position 290 enhances both the stability and the DNA-binding affinity of the protein, however, evolution prefers glycine at this position. We have provided several plausible explanations for this apparent conundrum. The function of the EXD as a HOX co-factor requires its ability to discriminate similar DNA sequences, which is most likely compromised by the quenching of its flexibility and enhancement of its stability and DNA-binding affinity.

Structural Dynamics of the Bacillus subtilis MntR Transcription Factor Is Locked by Mn2+ Binding.

Manganese (II) ions are essential for a variety of bacterial cellular processes. The transcription factor MntR is a metallosensor that regulates Mn2+ ion homeostasis in the bacterium Bacillus subtilis. Its DNA-binding affinity is increased by Mn2+ ion binding, allowing it to act as a transcriptional repressor of manganese import systems. Although experimentally well-researched, the molecular mechanism that regulates this process is still a puzzle. Computational simulations supported by circular dichroism (CD), differential scanning calorimetry (DSC) and native gel electrophoresis (native-PAGE) experiments were employed to study MntR structural and dynamical properties in the presence and absence of Mn2+ ions. The results of molecular dynamics (MD) simulations revealed that Mn2+ ion binding reduces the structural dynamics of the MntR protein and shifts the dynamic equilibrium towards the conformations adequate for DNA binding. Results of CD and DSC measurements support the computational results showing the change in helical content and stability of the MntR protein upon Mn2+ ion binding. Further, MD simulations show that Mn2+ binding induces polarization of the protein electrostatic potential, increasing the positive electrostatic potential of the DNA-binding helices in particular. In order to provide a deeper understanding of the changes in protein structure and dynamics due to Mn2+ binding, a mutant in which Mn2+ binding is mimicked by a cysteine bridge was constructed and also studied computationally and experimentally.

An integrated investigation of structural and pathway alteration caused by PIK3CA and TP53 mutations identified in cfDNA of metastatic breast cancer.

In peripheral blood, cell-free DNA (cfDNA) contains circulating tumor DNA (ctDNA), which indicates molecular abnormalities in metastatic breast tumor tissue. The sequencing of cfDNA of Metastatic Breast Cancer (MBC) patients allows assessment of therapy response and noninvasive treatment. In the proposed study, clinically significant alterations in PIK3CA and TP53 genes associated with MBC resulting in a missense substitution of His1047Arg and Arg282Trp from an next-generation sequencing-based multi-gene panel were reported in a cfDNA of a patient with MBC. To investigate the impact of the reported mutation, we used molecular docking, molecular dynamics simulation, network analysis, and pathway analysis. Molecular Docking analysis determined the distinct binding pattern revealing H1047R-ATP complex has a higher number of Hydrogen bonds (H-bonds) and binding affinity with a slight difference compared to the PIK3CA-ATP complex. Following, molecular dynamics simulation for 200 ns, of which H1047R-ATP complex resulted in the instability of PIK3CA. Similarly, for TP53 mutant R282W, the zinc-free state (apo) and zinc-bounded (holo) complexes were investigated for conformational change between apo and holo complexes, of which the holo complex mutant R282W was unstable. To validate the conformational change of PIK3CA and TP53, 80% mutation of H1047R in the kinase domain of p110α expressed ubiquitously in PIK3CA protein that alters PI3K pathway, while R282W mutation in DNA binding helix (H2) region of P53 protein inhibits the transcription factor in P53 pathway causing MBC. According to our findings, the extrinsic (hypoxia, oxidative stress, and acidosis); intrinsic factors (MYC amplification) in PIK3CA and TP53 mutations will provide potential insights for developing novel therapeutic methods for MBC therapy.

Взаимодействие MxyR Mycobacterium tuberculosis c ксиланами: необычные лиганды транскрипционных регуляторов семейства MarR

Among the repertoire of MarR family transcriptional regulators in Mycobacterium tuberculosis, gene rv3095 (mxyR) encodes Mycobacterial Xylan Regulator, MxyR. Gene mxyR is divergently oriented from a hydrolase (rv3094c), oxidoreductase (rv3093c) and an ABC transporter (rv3092c) and convergently oriented with xylanase (rv3096). Xylanase is commonly used by plant pathogenic microbes where they degrade xylan, the major component of hemicellulose. We have purified the transcriptional regulatory protein encoded by rv3095 and its molecular interactions were studied in detail using electrophoretic mobility shift assay. MxyR interacts with its upstream intergenic region mxyO with high specificity and at a dissociation constant of 5.01 ± 0.017 nM. Notably, this binding is attenuated by specific carbohydrate ligands such as xylan, L-Arabinose and D-Galactose with an IC50 values of 22.7 ± 1.02 ng/μL, 360.8 ± 24.25 ng/μL and 2320.0 ± 96.97 μg/μL, respectively. Consequently, it is evident that this association changes the conformation of the DNA binding helix α4 making the transcriptional regulator incompatible with binding to its cognate DNA, allowing xylanase and other genes to be transcribed. This study establishes the natural ligands of MxyR of M. tuberculosis providing insight on metabolic regulation of the carbohydrate, xylan.

Structural changes of antitoxin HigA from Shigella flexneri by binding of its cognate toxin HigB.

In toxin-antitoxin systems, many antitoxin proteins that neutralize their cognate toxin proteins also bind to DNA to repress transcription, and the DNA-binding affinity of the antitoxin is affected by its toxin. We solved crystal structures of the antitoxin HigA (apo-SfHigA) and its complex with the toxin HigB (SfHigBA) from Shigella flexneri. The apo-SfHigA shows a distinctive V-shaped homodimeric conformation with sequestered N-domains having a novel fold. SfHigBA appears as a heterotetramer formed by N-terminal dimerization of SfHigB-bound SfHigA molecules. The conformational change in SfHigA upon SfHigB binding is mediated by rigid-body movements of its C-domains, which accompanied an overall conformational change from wide V-shaped to narrow V-shaped dimer. Consequently, the two putative DNA-binding helices (α7 in each subunit) are repositioned to a conformation more compatible with canonical homodimeric DNA-binding proteins containing HTH motifs. Collectively, this study demonstrates a conformational change in an antitoxin protein, which occurs upon toxin binding and is responsible for regulating antitoxin DNA binding.

T4 DNA ligase structure reveals a prototypical ATP-dependent ligase with a unique mode of sliding clamp interaction.

DNA ligases play essential roles in DNA replication and repair. Bacteriophage T4 DNA ligase is the first ATP-dependent ligase enzyme to be discovered and is widely used in molecular biology, but its structure remained unknown. Our crystal structure of T4 DNA ligase bound to DNA shows a compact α-helical DNA-binding domain (DBD), nucleotidyl-transferase (NTase) domain, and OB-fold domain, which together fully encircle DNA. The DBD of T4 DNA ligase exhibits remarkable structural homology to the core DNA-binding helices of the larger DBDs from eukaryotic and archaeal DNA ligases, but it lacks additional structural components required for protein interactions. T4 DNA ligase instead has a flexible loop insertion within the NTase domain, which binds tightly to the T4 sliding clamp gp45 in a novel α-helical PIP-box conformation. Thus, T4 DNA ligase represents a prototype of the larger eukaryotic and archaeal DNA ligases, with a uniquely evolved mode of protein interaction that may be important for efficient DNA replication.

De novo missense variants in MEIS2 recapitulate the microdeletion phenotype of cardiac and palate abnormalities, developmental delay, intellectual disability and dysmorphic features.

Gross deletions involving the MEIS2 gene have been described in a small number of patients with overlapping phenotypes of atrial or ventricular septal defects, cleft palate, and variable developmental delays and intellectual disability. Non-specific dysmorphic features were noted in some patients, including broad forehead with high anterior hairline, arched eyebrows, thin or tented upper lip, and short philtrum. Recently, a patient with a de novo single amino acid deletion, c.998_1000delGAA (p.Arg333del), and a patient with a de novo nonsense variant, (c.611C>G, p.Ser204*), were reported with a similar, but apparently more severe phenotypes. Clinical whole exome sequencing (WES) performed at our clinical molecular diagnostic laboratory identified four additional patients with predicted damaging de novo MEIS2 missense variants. Our patients' features closely resembled those previously reported in patients with gross deletions, but also included some less commonly reported features, such as autism spectrum disorder, hearing loss, and short stature, as well as features that may be unique to nucleotide-level variants, such as hypotonia, failure to thrive, gastrointestinal, skeletal, limb, and skin abnormalities. All of the observed missense variants, Pro302Leu, Gln322Leu, Arg331Lys, and Val335Ala, are located in the functionally important MEIS2 homeodomain. Pro302Leu is found in the region between helix 1 and helix 2, while the other three are located in the DNA-binding helix 3. To our knowledge, these are the first described de novo missense variants in MEIS2, expanding the known mutation spectrum of the newly recognized human disorder caused by aberrations in this gene.

A New Family of HEAT-Like Repeat Proteins Lacking a Critical Substrate Recognition Motif Present in Related DNA Glycosylases.

DNA glycosylases are important repair enzymes that eliminate a diverse array of aberrant nucleobases from the genomes of all organisms. Individual bacterial species often contain multiple paralogs of a particular glycosylase, yet the molecular and functional distinctions between these paralogs are not well understood. The recently discovered HEAT-like repeat (HLR) DNA glycosylases are distributed across all domains of life and are distinct in their specificity for cationic alkylpurines and mechanism of damage recognition. Here, we describe a number of phylogenetically diverse bacterial species with two orthologs of the HLR DNA glycosylase AlkD. One ortholog, which we designate AlkD2, is substantially less conserved. The crystal structure of Streptococcus mutans AlkD2 is remarkably similar to AlkD but lacks the only helix present in AlkD that penetrates the DNA minor groove. We show that AlkD2 possesses only weak DNA binding affinity and lacks alkylpurine excision activity. Mutational analysis of residues along this DNA binding helix in AlkD substantially reduced binding affinity for damaged DNA, for the first time revealing the importance of this structural motif for damage recognition by HLR glycosylases.

Insights into Homodimerization 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. Because of this all ETS TFs have a core GGA binding site, and thus ETS TFs are found to redundantly regulate the same genes. However, each ETS TF has unique targets as well. One of the prevailing hypotheses explaining this duality is protein-protein interactions, including homodimerization, that allows each TF distinct behavior. The goal of this work was to study the effect of homodimerization on ETS1 specificity. We made mutations in the promoter of MMP3, a gene tightly regulated by ETS1. This promoter has two head-to-head ETS1 binding sites. Binding assays show that both sites are needed for DNA-binding; ETS1 is unable to bind as a monomer in this context. In a crosslinking assay, we also show that ETS1's ability to parse binding sites is based on its free monomeric state, as a mutation made in ETS1's DNA binding helix perturbs its ability to differentiate specific and non-specific targets, apparently by preventing direct contact with the binding site, but also by disrupting autoinhibition, allowing ETS1 to dimerize in the absence of DNA. Here we show that, on the MMP3 site, ETS1 must relieve its autoinhibitory state to bind DNA and must bind as a dimer. As non-specific DNA can induce dimerization, our results indicate how ETS1 finds binding sites on a larger scale. We have also shown that autoinhibition, which was previously thought to only prevent off-target DNA binding, also functions to prevent inappropriate dimerization.

The Crystal Structures of Apo and cAMP-Bound GlxR from Corynebacterium glutamicum Reveal Structural and Dynamic Changes upon cAMP Binding in CRP/FNR Family Transcription Factors

The cyclic AMP-dependent transcriptional regulator GlxR from Corynebacterium glutamicum is a member of the super-family of CRP/FNR (cyclic AMP receptor protein/fumarate and nitrate reduction regulator) transcriptional regulators that play central roles in bacterial metabolic regulatory networks. In C. glutamicum, which is widely used for the industrial production of amino acids and serves as a non-pathogenic model organism for members of the Corynebacteriales including Mycobacterium tuberculosis, the GlxR homodimer controls the transcription of a large number of genes involved in carbon metabolism. GlxR therefore represents a key target for understanding the regulation and coordination of C. glutamicum metabolism. Here we investigate cylic AMP and DNA binding of GlxR from C. glutamicum and describe the crystal structures of apo GlxR determined at a resolution of 2.5 Å, and two crystal forms of holo GlxR at resolutions of 2.38 and 1.82 Å, respectively. The detailed structural analysis and comparison of GlxR with CRP reveals that the protein undergoes a distinctive conformational change upon cyclic AMP binding leading to a dimer structure more compatible to DNA-binding. As the two binding sites in the GlxR homodimer are structurally identical dynamic changes upon binding of the first ligand are responsible for the allosteric behavior. The results presented here show how dynamic and structural changes in GlxR lead to optimization of orientation and distance of its two DNA-binding helices for optimal DNA recognition.

Structural insight into DNA binding and oligomerization of the multifunctional Cox protein of bacteriophage P2

The Cox protein from bacteriophage P2 is a small multifunctional DNA-binding protein. It is involved in site-specific recombination leading to P2 prophage excision and functions as a transcriptional repressor of the P2 Pc promoter. Furthermore, it transcriptionally activates the unrelated, defective prophage P4 that depends on phage P2 late gene products for lytic growth. In this article, we have investigated the structural determinants to understand how P2 Cox performs these different functions. We have solved the structure of P2 Cox to 2.4 Å resolution. Interestingly, P2 Cox crystallized in a continuous oligomeric spiral with its DNA-binding helix and wing positioned outwards. The extended C-terminal part of P2 Cox is largely responsible for the oligomerization in the structure. The spacing between the repeating DNA-binding elements along the helical P2 Cox filament is consistent with DNA binding along the filament. Functional analyses of alanine mutants in P2 Cox argue for the importance of key residues for protein function. We here present the first structure from the Cox protein family and, together with previous biochemical observations, propose that P2 Cox achieves its various functions by specific binding of DNA while wrapping the DNA around its helical oligomer.

Differentiation Induction In Acute Myeloid Leukemia Using Site-Specific DNA-Targeting

Hoxa9 and Meis1 are overexpressed in >70% of acute myeloid leukemia (AML) and associated with poor prognosis and survival. Hoxa9 and Meis1 interact with DNA and PBX to achieve transcription of differentiation-blocking genes. We tested transcriptional repression at Hoxa9-PBX-Meis1 genomic binding sites to induce differentiation in a model of human AMLWe designed a DNA-recognition strategy based on the known structure of the Hoxa9-PBX-DNA complex by fusing the DNA binding helices of Hoxa9 and PBX to create concise homeodomain fusion proteins that target the Hoxa9-PBX DNA recognition sequence. To confer transcription-repressing properties to the proteins, we attached a transcriptional repressor (sin3 interacting) domain and ectopically expressed this protein in Hoxa9-Meis1 immortalized murine progenitors. Introduction of this transcription repressor protein significantly enabled cell differentiation versus control (51.2% Mac-1high Gr-1high cells versus 11.3% for control). Multiple gene transcripts indicative of differentiation, such as GCSFR, myeloperoxidase, neutrophil elastase, and the calcium binding protein, S100A8, were also elevated in repressor-expressing cells. Furthermore, direct transcriptional targets of Hoxa9 (e.g. SOX2, CD34, FOXP1, FLT3R, DNAJC10) were down regulated in repressor-expressing cells. Importantly, a mutant repressor lacking the DNA-interacting amino acids did not affect transcription of Hoxa9 targets, demonstrating on-target specificity. Repressor-expressing cells also exhibited lower surface expression of c-Kit and Flt3 receptors and when transplanted into mice resulted in a significant increase in disease latency with a 94 day median latency versus 62 day latency for the control group (p value = 0.002).Our results demonstrate that site-specific DNA-targeting using homeodomain fusion proteins can enable AML cell differentiation and significantly increase disease latency. Disclosures:Scadden:Fate Therapeutics: Consultancy, Equity Ownership.

A Strategy for Antagonizing Quorum Sensing

Quorum-sensing bacteria communicate via small molecules called autoinducers to coordinate collective behaviors. Because quorum sensing controls virulence factor expression in many clinically relevant pathogens, membrane-permeable quorum sensing antagonists that prevent population-wide expression of virulence genes offer a potential route to novel antibacterial therapeutics. Here, we report a strategy for inhibiting quorum-sensing receptors of the widespread LuxR family. Structure-function studies with natural and synthetic ligands demonstrate that the dimeric LuxR-type transcription factor CviR from Chromobacterium violaceum is potently antagonized by molecules that bind in place of the native acylated homoserine lactone autoinducer, provided that they stabilize a closed conformation. In such conformations, each of the two DNA-binding domains interacts with the ligand-binding domain of the opposing monomer. Consequently, the DNA-binding helices are held apart by ∼60 Å, twice the ∼30 Å separation required for operator binding. This approach may represent a general strategy for the inhibition of multidomain proteins.

Protein flexibility directs DNA recognition by the papillomavirus E2 proteins

Although DNA flexibility is known to play an important role in DNA–protein interactions, the importance of protein flexibility is less well understood. Here, we show that protein dynamics are important in DNA recognition using the well-characterized human papillomavirus (HPV) type 6 E2 protein as a model system. We have compared the DNA binding properties of the HPV 6 E2 DNA binding domain (DBD) and a mutant lacking two C-terminal leucine residues that form part of the hydrophobic core of the protein. Deletion of these residues results in increased specific and non-specific DNA binding and an overall decrease in DNA binding specificity. Using 15N NMR relaxation and hydrogen/deuterium exchange, we demonstrate that the mutation results in increased flexibility within the hydrophobic core and loop regions that orient the DNA binding helices. Stopped-flow kinetic studies indicate that increased flexibility alters DNA binding by increasing initial interactions with DNA but has little or no effect on the structural rearrangements that follow this step. Taken together these data demonstrate that subtle changes in protein dynamics have a major influence on protein–DNA interactions.

Thermodynamics of Cooperative DNA Recognition at a Replication Origin and Transcription Regulatory Site

Binding cooperativity guides the formation of protein−nucleic acid complexes, in particular those that are highly regulated such as replication origins and transcription sites. Using the DNA binding domain of the origin binding and transcriptional regulator protein E2 from human papillomavirus type 16 as model, and through isothermal titration calorimetry analysis, we determined a positive, entropy-driven cooperativity upon binding of the protein to its cognate tandem double E2 site. This cooperativity is associated with a change in DNA structure, where the overall B conformation is maintained. Two homologous E2 domains, those of HPV18 and HPV11, showed that the enthalpic−entropic components of the reaction and DNA deformation can diverge. Because the DNA binding helix is almost identical in the three domains, the differences must lie dispersed throughout this unique dimeric β-barrel fold. This is in surprising agreement with previous results for this domain, which revealed a strong coupling between global dynamics and DNA recognition.

Characterization of the Bacteroides CTnDOT Regulatory Protein RteC

Excision of the Bacteroides conjugative transposon CTnDOT is stimulated by tetracycline. It was shown previously that a gene, rteC, is necessary for tetracycline-stimulated transcriptional regulation of the orf2c operon, which contains the excision genes. The protein encoded by this gene, RteC, did not have primary amino acid sequence homology to any known proteins in the databases. Accordingly, we sought structural homologs of RteC. A three-dimensional structure prediction by Robetta suggested that RteC might have two domains and that the C-terminal domain might have a winged helix motif. Based on the Robetta prediction, the human transcriptional factors E2F-4 and DP2 were identified as the most likely structural homologs of RteC. We made alanine substitutions within the putative DNA binding helix 3 region of RteC. Assays of orf2c::uidA activation by alanine mutants indicated that residues 174, 175, 178, 180, and 184 in helix 3 might contact the upstream region of P(E). The upstream region of orf2c contained two inverted-repeat half sites. Mutational analysis of these half sites showed that both half sites are important for activity. Thus, we have identified the DNA binding portion of RteC and the DNA site to which it binds.

Crystal structure of the P2 C-repressor: a binder of non-palindromic direct DNA repeats

As opposed to the vast majority of prokaryotic repressors, the immunity repressor of temperate Escherichia coli phage P2 (C) recognizes non-palindromic direct repeats of DNA rather than inverted repeats. We have determined the crystal structure of P2 C at 1.8 Å. This constitutes the first structure solved from the family of C proteins from P2-like bacteriophages. The structure reveals that the P2 C protein forms a symmetric dimer oriented to bind the major groove of two consecutive turns of the DNA. Surprisingly, P2 C has great similarities to binders of palindromic sequences. Nevertheless, the two identical DNA-binding helixes of the symmetric P2 C dimer have to bind different DNA sequences. Helix 3 is identified as the DNA-recognition motif in P2 C by alanine scanning and the importance for the individual residues in DNA recognition is defined. A truncation mutant shows that the disordered C-terminus is dispensable for repressor function. The short distance between the DNA-binding helices together with a possible interaction between two P2 C dimers are proposed to be responsible for extensive bending of the DNA. The structure provides insight into the mechanisms behind the mutants of P2 C causing dimer disruption, temperature sensitivity and insensitivity to the P4 antirepressor.

Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance

The self-assembling MexA-MexB-OprM efflux pump system, encoded by the mexO operon, contributes to facile resistance of Pseudomonas aeruginosa by actively extruding multiple antimicrobials. MexR negatively regulates the mexO operon, comprising two adjacent MexR binding sites, and is as such highly targeted by mutations that confer multidrug resistance (MDR). To understand how MDR mutations impair MexR function, we studied MexR-wt as well as a selected set of MDR single mutants distant from the proposed DNA-binding helix. Although DNA affinity and MexA-MexB-OprM repression were both drastically impaired in the selected MexR-MDR mutants, MexR-wt bound its two binding sites in the mexO with high affinity as a dimer. In the MexR-MDR mutants, secondary structure content and oligomerization properties were very similar to MexR-wt despite their lack of DNA binding. Despite this, the MexR-MDR mutants showed highly varying stabilities compared with MexR-wt, suggesting disturbed critical interdomain contacts, because mutations in the DNA-binding domains affected the stability of the dimer region and vice versa. Furthermore, significant ANS binding to MexR-wt in both free and DNA-bound states, together with increased ANS binding in all studied mutants, suggest that a hydrophobic cavity in the dimer region already shown to be involved in regulatory binding is enlarged by MDR mutations. Taken together, we propose that the biophysical MexR properties that are targeted by MDR mutations-stability, domain interactions, and internal hydrophobic surfaces-are also critical for the regulation of MexR DNA binding.

Zinc Finger Transcription Factor Zn3-Sp1 Reactions with Cd2+

Cadmium is a major environmental pollutant that causes kidney failure including the inability to resorb nutrients such as glucose. In a mouse kidney cell culture model, Cd(2+) inhibits Na(+)-dependent glucose uptake mediated by SGLT transporters. This defect has been traced to the down-regulation of SGLT mRNA synthesis mediated by the zinc-finger transcription factor, Zn(3)-Sp1. Incubation of Cd(2+) with Zn(2+)-Sp1 inhibited its capacity to bind to GC1, its binding site in the SGLT1 promoter. The extent of reaction was reduced as increasing concentrations of Zn(2+) are simultaneously present in the reaction mixture. The results are consistent with a Cd(2+)-Zn(2+) exchange reaction that inactivates the DNA binding function of the protein. The equilibrium constant for this reaction was calculated as 14 +/- 3 and 7 +/- 4 for the reactions measured by the binding to GC1 and an analogous SGLT2 promoter site. Sequential addition of Cd(2+) and Zn(2+) to Zn(3)-Sp1 failed to inhibit the reduction in DNA binding seen with Cd(2+) alone, indicating that substitution of Zn(2+) by Cd(2+) was followed by a second reaction that failed to respond to Zn(2+). Buffers for the DNA binding reaction (electrophoretic mobility shift assay) contain EDTA and Cd-EDTA is active in the same concentration range as Cd(2+). During the standard 15 min incubation, Cd(2+) down-regulates Zn(3)-Sp1 but is inactive against the adduct, Zn(3)-Sp1.GC1. Kinetic studies demonstrated that with 5 muM Cd(2+), Zn(3)-Sp1 was about 75% inactivated in 15 min, whereas, Zn(3)-Sp1.GC1 was slowly dissociated with 50% still remaining after 60 min. In contrast, Zn(3)-Sp1 bound to a cognate consensus site resisted any reaction over 60 min. An adduct of Zn(3)-Sp1.(polydI-dC) was just as reactive with Cd(2+) as Zn(3)-Sp1. Reexamination of the NMR structure of Zn- and Cd-finger peptides related to Sp1 fingers has revealed subtle changes in conformation of the metalbinding site and DNA-binding helix that occur when Cd(2+) is substituted by Zn(2+).