Regulatory mechanism of the light-activable allosteric switch LOV-TAP for the control of DNA binding: a computer simulation study

The spatio-temporal control of gene expression is fundamental to elucidate cell proliferation and deregulation phenomena in living systems. Novel approaches based on light-sensitive multiprotein complexes have recently been devised, showing promising perspectives for the noninvasive and reversible modulation of the DNA-transcriptional activity in vivo. This has lately been demonstrated in a striking way through the generation of the artificial protein construct light-oxygen-voltage (LOV)-tryptophan-activated protein (TAP), in which the LOV-2-Jα photoswitch of phototropin1 from Avena sativa (AsLOV2-Jα) has been ligated to the tryptophan-repressor (TrpR) protein from Escherichia coli. Although tremendous progress has been achieved on the generation of such protein constructs, a detailed understanding of their functioning as opto-genetical tools is still in its infancy. Here, we elucidate the early stages of the light-induced regulatory mechanism of LOV-TAP at the molecular level, using the noninvasive molecular dynamics simulation technique. More specifically, we find that Cys450-FMN-adduct formation in the AsLOV2-Jα-binding pocket after photoexcitation induces the cleavage of the peripheral Jα-helix from the LOV core, causing a change of its polarity and electrostatic attraction of the photoswitch onto the DNA surface. This goes along with the flexibilization through unfolding of a hairpin-like helix-loop-helix region interlinking the AsLOV2-Jα- and TrpR-domains, ultimately enabling the condensation of LOV-TAP onto the DNA surface. By contrast, in the dark state the AsLOV2-Jα photoswitch remains inactive and exerts a repulsive electrostatic force on the DNA surface. This leads to a distortion of the hairpin region, which finally relieves its tension by causing the disruption of LOV-TAP from the DNA.

AMPA RECEPTOR ALLOSTERISM: MEASUREMENT OF THE CONFORMATIONAL CHANGES IN THE LIGAND BINDING DOMAIN OF A FUNCTIONAL RECEPTOR

Ionotropic glutamate receptors are important excitatory neurotransmitter receptors in the mammalian central nervous system that have been implicated in a number of neuropathologies such as epilepsy, ischemia, and amyotrophic lateral sclerosis. Glutamate binding to an extracellular ligand binding domain initiates a series of structural changes that leads to the formation of a cation selective transmembrane channel, which consequently closes due to desensitization of the receptor. The crystal structures of the AMPA subtype of the glutamate receptor have been particularly useful in providing initial insight into the conformational changes in the ligand binding domain; however, these structures are limited by crystallographic constraint. To gain a clear picture of how agonist binding is coupled to channel activation and desensitization, it is essential to study changes in the ligand binding domain in a dynamic, physiological state. In this dissertation, a technique called Luminescence Resonance Energy Transfer was used to determine the conformational changes associated with activation and desensitization in a functional AMPA receptor (ÄN*-AMPA) that contains the ligand binding domain and transmembrane segments; ÄN*-AMPA has been modified such that fluorophores can be introduced at specific sites to serve as a readout of cleft closure or to establish intersubunit distances. Previous structural studies of cleft closure of the isolated ligand binding domain in conjunction with functional studies of the full receptor suggest that extent of cleft closure correlates with extent of activation. Here, LRET has been used to show that a similar relationship between cleft closure and activation is observed in the “full length” receptor showing that the isolated ligand binding domain is a good model of the domain in the full length receptor for changes within a subunit. Similar LRET investigations were used to study intersubunit distances specifically to probe conformational changes between subunits within a dimer in the tetrameric receptor. These studies show that the dimer interface is coupled in the open state, and decoupled in the desensitized state, similar to the isolated ligand binding domain crystal structure studies. However, we show that the apo state dimer interface is not pre-formed as in the crystal structure, hence suggesting a mechanism for functional transitions within the receptor based on LRET distances obtained.

Crystal structures of progressive Ca2+ binding states of the Ca2+ sensor Ca2+ binding domain 1 (CBD1) from the CALX Na+/Ca2+ exchanger reveal incremental conformational transitions.

Na(+)/Ca(2+) exchangers (NCX) constitute a major Ca(2+) export system that facilitates the re-establishment of cytosolic Ca(2+) levels in many tissues. Ca(2+) interactions at its Ca(2+) binding domains (CBD1 and CBD2) are essential for the allosteric regulation of Na(+)/Ca(2+) exchange activity. The structure of the Ca(2+)-bound form of CBD1, the primary Ca(2+) sensor from canine NCX1, but not the Ca(2+)-free form, has been reported, although the molecular mechanism of Ca(2+) regulation remains unclear. Here, we report crystal structures for three distinct Ca(2+) binding states of CBD1 from CALX, a Na(+)/Ca(2+) exchanger found in Drosophila sensory neurons. The fully Ca(2+)-bound CALX-CBD1 structure shows that four Ca(2+) atoms bind at identical Ca(2+) binding sites as those found in NCX1 and that the partial Ca(2+) occupancy and apoform structures exhibit progressive conformational transitions, indicating incremental regulation of CALX exchange by successive Ca(2+) binding at CBD1. The structures also predict that the primary Ca(2+) pair plays the main role in triggering functional conformational changes. Confirming this prediction, mutagenesis of Glu(455), which coordinates the primary Ca(2+) pair, produces dramatic reductions of the regulatory Ca(2+) affinity for exchange current, whereas mutagenesis of Glu(520), which coordinates the secondary Ca(2+) pair, has much smaller effects. Furthermore, our structures indicate that Ca(2+) binding only enhances the stability of the Ca(2+) binding site of CBD1 near the hinge region while the overall structure of CBD1 remains largely unaffected, implying that the Ca(2+) regulatory function of CBD1, and possibly that for the entire NCX family, is mediated through domain interactions between CBD1 and the adjacent CBD2 at this hinge.

REPSA: A method for determining the sequence specificity of DNA binding ligands

We developed a novel combinatorial method termed restriction endonuclease protection selection and amplification (REPSA) to identify consensus binding sites of DNA-binding ligands. REPSA uses a unique enzymatic selection based on the inhibition of cleavage by a type IIS restriction endonuclease, an enzyme that cleaves DNA at a site distal from its recognition sequence. Sequences bound by a ligand are protected from cleavage while unprotected sequences are cleaved. This enzymatic selection occurs in solution under mild conditions and is dependant only on the DNA-binding ability of the ligand. Thus, REPSA is useful for a broad range of ligands including all classes of DNA-binding ligands, weakly binding ligands, mixed populations of ligands, and unknown ligands. Here I describe REPSA and the application of this method to select the consensus DNA-binding sequences of three representative DNA-binding ligands; a nucleic acid (triplex-forming single-stranded DNA), a protein (the TATA-binding protein), and a small molecule (Distamycin A). These studies generated new information regarding the specificity of these ligands in addition to establishing their DNA-binding sequences. ^

Low dose irinotecan improves advanced lupus nephritis in mice potentially by changing DNA relaxation and anti–double-stranded DNA binding

Objective Albeit clear advances in the treatment of SLE, many patients still present with refractory lupus nephritis requiring new treatment strategies for this disease. Here we determined whether reduced doses of the topoisomerase I inhibitor irinotecan, which is known as chemotherapeutic agent, were able to suppress SLE in NZB/W F1 mice. We further evaluated the potential mechanism how irinotecan influenced the course of SLE. Methods NZB/W F1 mice were treated with low dose irinotecan either from week 24 of age or from established glomerulonephritis defined by a proteinuria ≥grade 3+. Binding of anti-dsDNA antibodies was measured by ELISA; and DNA relaxation was visualized by gel electrophoresis. Results Significantly reduced irinotecan dosages improved lupus nephritis and prolonged survival in NZB/W F1 mice. The lowest dose successfully used for the treatment of established murine lupus nephritis was more than 50 times lower than the dose usually applied for chemotherapy in humans. As a mechanism, low dose irinotecan reduced B cell activity; however, the levels of B cell activity in irinotecan-treated mice were similar to those in Balb/c mice of the same age suggesting that irinotecan did not induce a clear immunosuppression. In addition, incubation of double-stranded (ds) DNA with topoisomerase I increased binding of murine and human anti-dsDNA antibodies showing for the first time that relaxed DNA is more susceptible to anti-dsDNA antibody binding. This effect was reversed by addition of the topoisomerase I inhibitor camptothecin. Conclusion Our results propose topoisomerase I inhibitors as a novel and targeted therapy for SLE. © 2014 American College of Rheumatology.

Mononuclear Tetraamineplatinum(II) Complexes: Synthesis, Anticancer Activity, DNA Binding, and Cellular Uptake

The synthesis of three bis[(tert-butoxy)carbonyl]-protected (tetramine)dichloroplatinum complexes 2a – c of formula cis-[PtCl2(LL)] and of their cationic deprotected analogs 3a – c and their evaluation with respect to in vitro cytotoxicity, intramolecular stability, DNA binding, and cellular uptake is reported. The synthesis comprises the complexation of K2[PtCl4] with di-N-protected tetramines 1a – c to give 2a – c and subsequent acidolysis, yielding 3a – c. The cytotoxicity of the complexes is in direct relation to the length of the polyamine. Complexes 3a – c display a significant higher affinity for CT DNA as well as for cellular DNA in A2780 cells than cisplatin.

DNA binding properties of oligodeoxynucleotides containing pyrrolidino C-nucleosides

We have incorporated pyrrolidino-C-nucleosides (pyrrolidino-pseudonucleosides) containing the base uracil and N-1-methyl uracil into oligodeoxynucleotides and compared their thermal duplex and triplex stabilities with unmodified or pseudouridine-containing oligodeoxynucleotides. We find relative destabilizations of triplex formation by ca. -13 to -1 degrees C per modification (relative to thymidine) in a strongly sequence dependent mode. Duplex formation is less destabilizing and more homogeneous with -4 to -2 degrees C per modification

POU domain transcription factor Brn3b is critical for the normal development and survival of retinal ganglion cells

The POU domain transcription factor Brn3b/POU4F2 plays a critical role regulating gene expression in mouse retinal ganglion cells (RGCs). Previous investigations have shown that Brn3b is not required for initial cell fate specification or migration; however, it is essential for normal RGC differentiation. In contrast to wild type axons, the mutant neurites were phenotypically different: shorter, rougher, disorganized, and poorly fasciculated. Wild type axons stained intensely with axon specific marker tau-1, while mutant projections were weakly stained and the mutant projections showed strong labeling with dendrite specific marker MAP2. Brn-3b mutant axonal projections contained more microtubules and fewer neurofilaments, a dendritic characteristic, than the wild type. The mutant neurites also exhibited significantly weaker staining of neurofilament low-molecular-weight (NF-L) in the axon when compared to the wild type, and NF-L accumulation in the neuron cell body. The absence of Brn-3b results in an inability to form normal axons and enhanced apoptosis in RGCs, suggesting that Brn-3b may control a set of genes involved in axon formation. ^ Brn3b contains several distinct sequence motifs: a glycine/serine rich region, two histidine rich regions, and a fifteen amino acid conserved sequence shared by all Brn3 family members in the N-terminus and a POU specific and POU homeodomain in the C-terminus. Brn3b activates a Luciferase reporter over 25 fold in cell culture when binding to native brn3 binding sites upstream of a minimal promoter. When fused to the Gal4 DNA Binding domain (DBD) and driven by either a strong (CMV) or weaker (pAHD) promoter, the N-terminal of Brn3b is capable of similar activation when binding to Gal4 UAS sites, indicating a presumptive activator of transcription. Both full length Brn3b or the C-terminus fused to the Gal4DBD and driven by pCMV repressed a Luciferase reporter downstream of UAS binding sites. Lower levels of expression of the fusion protein driven by pADH resulted in an alleviation of repression. This repression appears to be a limitation of this system of transcriptional analysis and a potential pitfall in conventional pCMV based transfection assays. ^

Elucidation of the role of 53BP1 in DNA double strand break (DSB) repair and tumor suppression

The protein p53 binding protein one (53BP1) was discovered in a yeast two-hybrid screen that used the DNA binding domain of p53 as bait. Cloning of full-length 53BP1 showed that this protein contains several protein domains which help make up the protein, which include two tandem BRCT domains and a amino-terminal serine/glutamine cluster domain (SCD). These are two protein domains are often seen in factors that are involved in the cellular response to DNA damage and control of cell cycle checkpoints and we hypothesize that 53BP1 is involved in the cellular response to DNA damage. In support of this hypothesis we observe that 53BP1 is phosphorylated and undergoes a dramatic nuclear re-localization in response to DNA damaging agents. 53BP1 also interacts with several factors that are important in the cellular response to DNA damage, such as the BRCA1 tumor suppressor, ATM and Rad3 related (ATR), and the phosphorylated version of the histone variant H2AX. Mice deficient in 53BP1 display increased sensitivity ionizing radiation (IR), a DNA damaging agent that introduces DNA double strand breaks (DSBs). In addition, 53BP1-deficient mice do not properly undergo the process of class switch recombination (CSR). We also observe that when a defect in 53BP1 is combined with a defect in p53; the resulting mice have an increased rate of formation of spontaneous tumors, notably the formation of B and T lineage lymphomas. The T lineage tumors arise by two distinct mechanisms: one driven by defects in cell cycle regulation and a second driven by defects in the ability to repair DNA DSBs. The B lineage tumors arise by the inability to repair DNA damage and over-expression of the oncogene c-myc. ^ With these observations, we conclude that not only does 53BP1 function in the cellular response to DNA damage, but it also works in concert with p53 to suppress tumor formation. ^

Probing the Saccharomyces cerevisiae centromeric DNA (CEN DNA)–binding factor 3 (CBF3) kinetochore complex by using atomic force microscopy

Yeast centromeric DNA (CEN DNA) binding factor 3 (CBF3) is a multisubunit protein complex that binds to the essential CDEIII element in CEN DNA. The four CBF3 proteins are required for accurate chromosome segregation and are considered to be core components of the yeast kinetochore. We have examined the structure of the CBF3–CEN DNA complex by atomic force microscopy. Assembly of CBF3–CEN DNA complexes was performed by combining purified CBF3 proteins with a DNA fragment that includes the CEN region from yeast chromosome III. Atomic force microscopy images showed DNA molecules with attached globular bodies. The contour length of the DNA containing the complex is ≈9% shorter than the DNA alone, suggesting some winding of DNA within the complex. The measured location of the single binding site indicates that the complex is located asymmetrically to the right of CDEIII extending away from CDEI and CDEII, which is consistent with previous data. The CEN DNA is bent ≈55° at the site of complex formation. A significant fraction of the complexes are linked in pairs, showing three to four DNA arms, with molecular volumes approximately three times the mean volumes of two-armed complexes. These multi-armed complexes indicate that CBF3 can bind two DNA molecules together in vitro and, thus, may be involved in holding together chromatid pairs during mitosis.

Activities and response to DNA damage of latent and active sequence-specific DNA binding forms of mouse p53

The mouse p53 protein generated by alternative splicing (p53as) has amino acid substitutions at its C terminus that result in constitutively active sequence-specific DNA binding (active form), whereas p53 protein itself binds inefficiently (latent form) unless activated by C-terminal modification. Exogenous p53as expression activated transcription of reporter plasmids containing p53 binding sequences and inhibited growth of mouse and human cells lacking functional endogenous p53. Inducible p53as in stably transfected p53 null fibroblasts increased p21WAF1/Cip-1/Sdi and decreased bcl-2 protein steady-state levels. Endogenous p53as and p53 proteins differed in response to cellular DNA damage. p53 protein was induced transiently in normal keratinocytes and fibroblasts whereas p53as protein accumulation was sustained in parallel with induction of p21WAF1/Cip-1/Sdi protein and mRNA, in support of p53as transcriptional activity. Endogenous p53 and p53as proteins in epidermal tumor cells responded to DNA damage with different kinetics of nuclear accumulation and efficiencies of binding to a p53 consensus DNA sequence. A model is proposed in which C-terminally distinct p53 protein forms specialize in functions, with latent p53 forms primarily for rapid non-sequence-specific binding to sites of DNA damage and active p53 forms for sustained regulation of transcription and growth.

Direct interaction of Gβγ with a C-terminal Gβγ-binding domain of the Ca2+ channel α1 subunit is responsible for channel inhibition by G protein-coupled receptors

Several classes of voltage-gated Ca2+ channels (VGCCs) are inhibited by G proteins activated by receptors for neurotransmitters and neuromodulatory peptides. Evidence has accumulated to indicate that for non-L-type Ca2+ channels the executing arm of the activated G protein is its βγ dimer (Gβγ). We report below the existence of two Gβγ-binding sites on the A-, B-, and E-type α1 subunits that form non-L-type Ca2+ channels. One, reported previously, is in loop 1 connecting transmembrane domains I and II. The second is located approximately in the middle of the ca. 600-aa-long C-terminal tails. Both Gβγ-binding regions also bind the Ca2+ channel β subunit (CCβ), which, when overexpressed, interferes with inhibition by activated G proteins. Replacement in α1E of loop 1 with that of the G protein-insensitive and Gβγ-binding-negative loop 1 of α1C did not abolish inhibition by G proteins, but the exchange of the α1E C terminus with that of α1C did. This and properties of α1E C-terminal truncations indicated that the Gβγ-binding site mediating the inhibition of Ca2+ channel activity is the one in the C terminus. Binding of Gβγ to this site was inhibited by an α1-binding domain of CCβ, thus providing an explanation for the functional antagonism existing between CCβ and G protein inhibition. The data do not support proposals that Gβγ inhibits α1 function by interacting with the site located in the loop I–II linker. These results define the molecular mechanism by which presynaptic G protein-coupled receptors inhibit neurotransmission.

Structural mimicry of a native protein by a minimized binding domain

The affinity between molecules depends both on the nature and presentation of the contacts. Here, we observe coupling of functional and structural elements when a protein binding domain is evolved to a smaller functional mimic. Previously, a 38-residue form of the 59-residue B-domain of protein A, termed Z38, was selected by phage display. Z38 contains 13 mutations and binds IgG only 10-fold weaker than the native B-domain. We present the solution structure of Z38 and show that it adopts a tertiary structure remarkably similar to that observed for the first two helices of B-domain in the B-domain/Fc complex [Deisenhofer, J. (1981) Biochemistry 20, 2361–2370], although it is significantly less stable. Based on this structure, we have improved on Z38 by designing a 34-residue disulfide-bonded variant (Z34C) that has dramatically enhanced stability and binds IgG with 9-fold higher affinity. The improved stability of Z34C led to NMR spectra with much greater chemical shift dispersion, resulting in a more precisely determined structure. Z34C, like Z38, has a structure virtually identical to the equivalent region from native protein A domains. The well-defined hydrophobic core of Z34C reveals key structural features that have evolved in this small, functional domain. Thus, the stabilized two-helix peptide, about half the size and having one-third of the remaining residues altered, accurately mimics both the structure and function of the native domain.

Inhibition of RNA polymerase II transcription in human cells by synthetic DNA-binding ligands

Sequence-specific DNA-binding small molecules that can permeate human cells potentially could regulate transcription of specific genes. Multiple cellular DNA-binding transcription factors are required by HIV type 1 for RNA synthesis. Two pyrrole–imidazole polyamides were designed to bind DNA sequences immediately adjacent to binding sites for the transcription factors Ets-1, lymphoid-enhancer binding factor 1, and TATA-box binding protein. These synthetic ligands specifically inhibit DNA-binding of each transcription factor and HIV type 1 transcription in cell-free assays. When used in combination, the polyamides inhibit virus replication by >99% in isolated human peripheral blood lymphocytes, with no detectable cell toxicity. The ability of small molecules to target predetermined DNA sequences located within RNA polymerase II promoters suggests a general approach for regulation of gene expression, as well as a mechanism for the inhibition of viral replication.