Incorporation of CC steps into Z-DNA: interplay between B-Z junction and Z-DNA helical formation.

The left-handed DNA structure, Z-DNA, is believed to play important roles in gene expression and regulation. Z-DNA forms sequence-specifically with a preference for sequences rich in pyrimidine/purine dinucleotide steps. In vivo, Z-DNA is generated in the presence of negative supercoiling or upon binding proteins that absorb the high energetic cost of the B-to-Z transition, including the creation of distorted junctions between B-DNA and Z-DNA. To date, the sequence preferences for the B-to-Z transition have primarily been studied in the context of sequence repeats lacking B-Z junctions. Here, we develop a method for characterizing sequence-specific preferences for Z-DNA formation and B-Z junction localization within heterogeneous DNA duplexes that is based on combining 2-aminopurine fluorescence measurements with a new quantitative application of circular dichroism spectroscopy for determining the fraction of B- versus Z-DNA. Using this approach, we show that at least three consecutive CC dinucleotide steps, traditionally thought to disfavor Z-DNA, can be incorporated within heterogeneous Z-DNA containing B-Z junctions upon binding to the Zα domain of the RNA adenosine deaminase protein. Our results indicate that the incorporation of CC steps into Z-DNA is driven by favorable sequence-specific Z-Z and B-Z stacking interactions as well as by sequence-specific energetics that localize the distorted B-Z junction at flexible sites. Together, our results expose higher-order complexities in the Z-DNA code within heterogeneous sequences and suggest that Z-DNA can in principle propagate into a wider range of genomic sequence elements than previously thought.

Sequence-specific B-DNA flexibility modulates Z-DNA formation.

Conversion of right-handed B-DNA into left-handed Z-DNA is one of the largest structural transitions in biology that plays fundamental roles in gene expression and regulation. Z-DNA segments must form within genomes surrounded by a sea of B-DNA and require creation of energetically costly B/Z junctions. Here, we show using a combination of natural abundance NMR R(1ρ) carbon relaxation measurements and CD spectroscopy that sequence-specific B-DNA flexibility modulates the thermodynamic propensity to form Z-DNA and the location of B/Z junctions. We observe sequence-specific flexibility in B-DNA spanning fast (ps-ns) and slow (µs-ms) time scales localized at the site of B/Z junction formation. Further, our studies show that CG-repeats play an active role tuning this intrinsic B-DNA flexibility. Taken together, our results suggest that sequence-specific B-DNA flexibility may provide a mechanism for defining the length and location of Z-DNA in genomes.

Crystal structure of a junction between B-DNA and Z-DNA reveals two extruded bases.

Left-handed Z-DNA is a higher-energy form of the double helix, stabilized by negative supercoiling generated by transcription or unwrapping nucleosomes. Regions near the transcription start site frequently contain sequence motifs favourable for forming Z-DNA, and formation of Z-DNA near the promoter region stimulates transcription. Z-DNA is also stabilized by specific protein binding; several proteins have been identified with low nanomolar binding constants. Z-DNA occurs in a dynamic state, forming as a result of physiological processes then relaxing to the right-handed B-DNA. Each time a DNA segment turns into Z-DNA, two B-Z junctions form. These have been examined extensively, but their structure was unknown. Here we describe the structure of a B-Z junction as revealed by X-ray crystallography at 2.6 A resolution. A 15-base-pair segment of DNA is stabilized at one end in the Z conformation by Z-DNA binding proteins, while the other end remains B-DNA. Continuous stacking of bases between B-DNA and Z-DNA segments is found, with the breaking of one base pair at the junction and extrusion of the bases on each side (Fig. 1). These extruded bases may be sites for DNA modification.

Characterization of DNA-binding activity of Z alpha domains from poxviruses and the importance of the beta-wing regions in converting B-DNA to Z-DNA.

The E3L gene is essential for pathogenesis in vaccinia virus. The E3L gene product consists of an N-terminal Z alpha domain and a C-terminal double-stranded RNA (dsRNA) binding domain; the left-handed Z-DNA-binding activity of the Z alpha domain of E3L is required for viral pathogenicity in mice. E3L is highly conserved among poxviruses, including the smallpox virus, and it is likely that the orthologous Z alpha domains play similar roles. To better understand the biological function of E3L proteins, we have investigated the Z-DNA-binding behavior of five representative Z alpha domains from poxviruses. Using surface plasmon resonance (SPR), we have demonstrated that these viral Z alpha domains bind Z-DNA tightly. Ability of Z alpha(E3L) converting B-DNA to Z-DNA was measured by circular dichroism (CD). The extents to which these Z alphas can stabilize Z-DNA vary considerably. Mutational studies demonstrate that residues in the loop of the beta-wing play an important role in this stabilization. Notably the Z alpha domain of vaccinia E3L acquires ability to convert B-DNA to Z-DNA by mutating amino acid residues in this region. Differences in the host cells of the various poxviruses may require different abilities to stabilize Z-DNA; this may be reflected in the observed differences in behavior in these Zalpha proteins.

A left-handed RNA double helix bound by the Z alpha domain of the RNA-editing enzyme ADAR1.

The A form RNA double helix can be transformed to a left-handed helix, called Z-RNA. Currently, little is known about the detailed structural features of Z-RNA or its involvement in cellular processes. The discovery that certain interferon-response proteins have domains that can stabilize Z-RNA as well as Z-DNA opens the way for the study of Z-RNA. Here, we present the 2.25 A crystal structure of the Zalpha domain of the RNA-editing enzyme ADAR1 (double-stranded RNA adenosine deaminase) complexed to a dUr(CG)(3) duplex RNA. The Z-RNA helix is associated with a unique solvent pattern that distinguishes it from the otherwise similar conformation of Z-DNA. Based on the structure, we propose a model suggesting how differences in solvation lead to two types of Z-RNA structures. The interaction of Zalpha with Z-RNA demonstrates how the interferon-induced isoform of ADAR1 could be targeted toward selected dsRNAs containing purine-pyrimidine repeats, possibly of viral origin.

A peptide with alternating lysines can act as a highly specific Z-DNA binding domain.

Many nucleic acid binding proteins use short peptide sequences to provide specificity in recognizing their targets, which may be either a specific sequence or a conformation. Peptides containing alternating lysine have been shown to bind to poly(dG-d5meC) in the Z conformation, and stabilize the higher energy form [H. Takeuchi, N. Hanamura, H. Hayasaka and I. Harada (1991) FEBS Lett., 279, 253-255 and H. Takeuchi, N. Hanamura and I. Harada (1994) J. Mol. Biol., 236, 610-617.]. Here we report the construction of a Z-DNA specific binding protein, with the peptide KGKGKGK as a functional domain and a leucine zipper as a dimerization domain. The resultant protein, KGZIP, induces the Z conformation in poly(dG-d5meC) and binds to Z-DNA stabilized by bromination with high affinity and specificity. The binding of KGZIP is sufficient to convert poly(dG-d5meC) from the B to the Z form, as shown by circular dichroism. The sequence KGKGKGK is found in many proteins, although no functional role has been established. KGZIP also has potential for engineering other Z-DNA specific proteins for future studies of Z-DNA in vitro and in vivo.

Versatile and on-demand biologics co-production in yeast.

Current limitations to on-demand drug manufacturing can be addressed by technologies that streamline manufacturing processes. Combining the production of two or more drugs into a single batch could not only be useful for research, clinical studies, and urgent therapies but also effective when combination therapies are needed or where resources are scarce. Here we propose strategies to concurrently produce multiple biologics from yeast in single batches by multiplexing strain development, cell culture, separation, and purification. We demonstrate proof-of-concept for three biologics co-production strategies: (i) inducible expression of multiple biologics and control over the ratio between biologic drugs produced together; (ii) consolidated bioprocessing; and (iii) co-expression and co-purification of a mixture of two monoclonal antibodies. We then use these basic strategies to produce drug mixtures as well as to separate drugs. These strategies offer a diverse array of options for on-demand, flexible, low-cost, and decentralized biomanufacturing applications without the need for specialized equipment.

Rational cytokine design for increased lifetime and enhanced potency using pH-activated "histidine switching".

We describe a method for the rational design of more effective therapeutic proteins using amino acid substitutions that reduce receptor binding affinity in intracellular endosomal compartments, thereby leading to increased recycling in the ligand-sorting process and consequently resulting in longer half-life in extracellular medium. We demonstrate this approach for granulocyte colony-stimulating factor by using computationally predicted histidine substitutions that switch protonation states between cell-surface and endosomal pH. Molecular modeling of binding electrostatics indicates two different single-histidine mutants that fulfill our design requirements; experimental assays demonstrate that each mutant indeed exhibits an order-of-magnitude increase in medium half-life along with enhanced potency due to increased endocytic recycling.

Production of Functional Anti-Ebola Antibodies in Pichia pastoris.

The 2013-2016 Ebola outbreak highlighted the limited treatment options and lack of rapid response strategies for emerging pathogen outbreaks. Here, we propose an efficient development cycle using glycoengineered Pichia pastoris to produce monoclonal antibody cocktails against pathogens. To enable rapid genetic engineering of P. pastoris, we introduced a genomic landing pad for reliable recombinase-mediated DNA integration. We then created strains expressing each of the three monoclonal antibodies that comprise the ZMapp cocktail, and demonstrated that the secreted antibodies bind to the Ebola virus glycoprotein by immunofluorescence assay. We anticipate that this approach could accelerate the production of therapeutics against future pathogen outbreaks.

The crystal structure of the Zbeta domain of the RNA-editing enzyme ADAR1 reveals distinct conserved surfaces among Z-domains.

The Zalpha domains represent a growing subfamily of the winged helix-turn-helix (HTH) domain family whose members share a remarkable ability to bind specifically to Z-DNA and/or Z-RNA. They have been found exclusively in proteins involved in interferon response and, while their importance in determining pox viral pathogenicity has been demonstrated, their actual target and biological role remain obscure. Cellular proteins containing Zalpha domains bear a second homologous domain termed Zbeta, which appears to lack the ability to bind left-handed nucleic acids. Here, we present the crystal structure of the Zbeta domain from the human double-stranded RNA adenosine deaminase ADAR1 at 0.97 A, determined by single isomorphous replacement including anomalous scattering. Zbeta maintains a winged-HTH fold with the addition of a C-terminal helix. Mapping of the Zbeta conservation profile on the Zbeta surface reveals a new conserved surface formed partly by the terminal helix 4, involved in metal binding and dimerization and absent from Zalpha domains. Our results show how two domains similar in fold may have evolved into different functional entities even in the context of the same protein.

Parsing the effects of binding, signaling, and trafficking on the mitogenic potencies of granulocyte colony-stimulating factor analogues.

The pharmacodynamic potency of a therapeutic cytokine interacting with a cell-surface receptor can be attributed primarily to three central properties: [1] cytokine/receptor binding affinity, [2] cytokine/receptor endocytic trafficking dynamics, and [3] cytokine/receptor signaling. Thus, engineering novel or second-generation cytokines requires an understanding of the contribution of each of these to the overall cell response. We describe here an efficient method toward this goal in demonstrated application to the clinically important cytokine granulocyte colony-stimulating factor (GCSF) with a chemical analogue and a number of genetic mutants. Using a combination of simple receptor-binding and dose-response proliferation assays we construct an appropriately scaled plot of relative mitogenic potency versus ligand concentration normalized by binding affinity. Analysis of binding and proliferation data in this manner conveniently indicates which of the cytokine properties-binding, trafficking, and/or signaling-are contributing substantially to altered potency effects. For the GCSF analogues studied here, two point mutations as well as a poly(ethylene glycol) chemical conjugate were found to have increased potencies despite comparable or slightly lower affinities, and trafficking was predicted to be the responsible mechanism. A third point mutant exhibiting comparable binding affinity but reduced potency was predicted to have largely unchanged trafficking properties. Surprisingly, another mutant possessing an order-of-magnitude weaker binding affinity displayed enhanced potency, and increased ligand half-life was predicted to be responsible for this net beneficial effect. Each of these predictions was successfully demonstrated by subsequent measurements of depletion of these five analogues from cell culture medium. Thus, for the GCSF system we find that ligand trafficking dynamics can play a major role in regulating mitogenic potency. Our results demonstrate that cytokine analogues can exhibit pharmacodynamic behaviors across a diverse spectrum of "binding-potency space" and that our analysis through normalization can efficiently elucidate hypotheses for the underlying mechanisms for further dedicated testing. We have also extended the Black-Leff model of pharmacological agonism to include trafficking effects along with binding and signaling, and this model provides a framework for parsing the effects of these factors on pharmacodynamic potency.

Alternate rRNA secondary structures as regulators of translation.

Structural dynamics of large molecular assemblies are intricately linked to function. For ribosomes, macromolecular changes occur especially during mRNA translation and involve participation of ribosomal RNA. Without suitable probes specific to RNA secondary structure, however, elucidation of more subtle dynamic ribosome structure-function relationships, especially in vivo, remains challenging. Here we report that the Z-DNA- and Z-RNA-binding domain Zα, derived from the human RNA editing enzyme ADAR1-L, binds with high stability to specific rRNA segments of Escherichia coli and human ribosomes. Zα impaired in Z-RNA recognition does not associate with ribosomes. Notably, Zα(ADAR1)-ribosome interaction blocks translation in vitro and in vivo, with substantial physiological consequences. Our study shows that ribosomes can be targeted by a protein that specifically recognizes an alternate rRNA secondary structure, and suggests a new mechanism of translational regulation on the ribosome.

A PKR-like eukaryotic initiation factor 2alpha kinase from zebrafish contains Z-DNA binding domains instead of dsRNA binding domains.

The double-stranded RNA (dsRNA)-dependent protein kinase (PKR) is induced as part of the IFN response in mammals and acts to shut down protein synthesis by the phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha). In fish, a PKR-like kinase activity has been detected, but the enzyme responsible has eluded characterization. Here, we describe a PKR-like kinase from zebrafish. Phylogenetic analysis shows that the C-terminal kinase domain is more closely related to the kinase domain of PKR than to any of the other three known eIF2alpha kinases. Surprisingly, instead of the two dsRNA binding domains found at the N terminus of PKR, there are two Zalpha domains. Zalpha domains specifically bind dsDNA and RNA in the left-handed Z conformation, often with high affinity. They have been found previously in two other IFN-inducible proteins, the dsRNA editing enzyme, ADAR1, and Z-DNA binding protein 1 (ZBP1), as well as in the poxvirus virulence factor, E3L. This previously undescribed kinase, designated PKZ (protein kinase containing Z-DNA binding domains), is transcribed constitutively at low levels and is highly induced after injection of poly(inosinic)-poly(cytidylic) acid, which simulates viral infection. Binding of Z-DNA by the Zalpha domain of PKZ was demonstrated by circular dichroism. PKZ inhibits translation in transfected cells; site-directed mutagenesis indicates that this inhibition depends on its catalytic activity. Identification of a gene combining Zalpha domains with a PKR-like kinase domain strengthens the hypothesis that the ability to bind left-handed nucleic acid plays a role in the host response to viruses.

Evidence that vaccinia virulence factor E3L binds to Z-DNA in vivo: Implications for development of a therapy for poxvirus infection.

The E3L gene product found in all poxviruses is required for the lethality of mice in vaccinia virus infection. Both the C-terminal region, consisting of a double-stranded RNA-binding motif, and the N-terminal region (vZ(E3L)), which is similar to the Zalpha family of Z-DNA-binding proteins, are required for infection. It has recently been demonstrated that the function of the N-terminal domain depends on its ability to bind Z-DNA; Z-DNA-binding domains from unrelated mammalian proteins fully complement an N-terminal deletion of E3L. Mutations that decrease affinity for Z-DNA have similar effects in decreasing pathogenicity. Compounds that block the Z-DNA-binding activity of E3L may also limit infection by the poxvirus. Here we show both an in vitro and an in vivo assay with the potential to be used in screening for such compounds. Using a conformation-specific yeast one-hybrid assay, we compared the results for Z-DNA binding of vZ(E3L) with those for human Zbeta(ADAR1), a peptide that has similarity to the Zalpha motif but does not bind Z-DNA, and with a mutant of hZbeta(ADAR1), which binds Z-DNA. The results suggest that this system can be used for high-throughput screening.

A role for Z-DNA binding in vaccinia virus pathogenesis.

The N-terminal domain of the E3L protein of vaccinia virus has sequence similarity to a family of Z-DNA binding proteins of defined three-dimensional structure and it is necessary for pathogenicity in mice. When other Z-DNA-binding domains are substituted for the similar E3L domain, the virus retains its lethality after intracranial inoculation. Mutations decreasing Z-DNA binding in the chimera correlate with decreases in viral pathogenicity, as do analogous mutations in wild-type E3L. A chimeric virus incorporating a related protein that does not bind Z-DNA is not pathogenic, but a mutation that creates Z-DNA binding makes a lethal virus. The ability to bind the Z conformation is thus essential to E3L activity. This finding may allow the design of a class of antiviral agents, including agents against variola (smallpox), which has an almost identical E3L.

Crystallization of the Zalpha domain of the human editing enzyme ADAR1 complexed with a DNA-RNA chimeric oligonucleotide in the left-handed Z-conformation.

The Zalpha domain of human double-stranded RNA adenosine deaminase (ADAR1) has been crystallized with a hexanucleotide containing alternating deoxyribose and ribose furanose sugars. Solution circular dichroism experiments show that this double-stranded chimera (dCrG)(3) initially adopts the right-handed A-conformation. However, addition of stoichiometric amounts of Zalpha causes a rapid transition to the Z-conformation. Raman spectroscopy of crystals of the Zalpha-(dCrG)(3) complex confirm that the chimeric oligonucleotide is stabilized in the Z-conformation. A complete data set has been obtained at 2.5 A resolution. The Zalpha-(dCrG)(3) crystals belong to the tetragonal I422 space group, with unit-cell parameters a = b = 104.2, c = 117.6 A. Work is under way to solve the structure by molecular replacement.

The solution structure of the N-terminal domain of E3L shows a tyrosine conformation that may explain its reduced affinity to Z-DNA in vitro.

The N-terminal domain of the vaccinia virus protein E3L (Z alpha(E3L)) is essential for full viral pathogenicity in mice. It has sequence similarity to the high-affinity human Z-DNA-binding domains Z alpha(ADAR1) and Z alpha(DLM1). Here, we report the solution structure of Z alpha(E3L) and the chemical shift map of its interaction surface with Z-DNA. The global structure and the Z-DNA interaction surface of Z alpha(E3L) are very similar to the high-affinity Z-DNA-binding domains Z alpha(ADAR1) and Z alpha(DLM1). However, the key Z-DNA contacting residue Y48 of Z alpha(E3L) adopts a different side chain conformation in unbound Z alpha(E3L), which requires rearrangement for binding to Z-DNA. This difference suggests a molecular basis for the significantly lower in vitro affinity of Z alpha(E3L) to Z-DNA compared with its homologues.

A poxvirus protein forms a complex with left-handed Z-DNA: crystal structure of a Yatapoxvirus Zalpha bound to DNA.

A conserved feature of poxviruses is a protein, well characterized as E3L in vaccinia virus, that confers IFN resistance on the virus. This protein comprises two domains, an N-terminal Z-DNA-binding protein domain (Zalpha) and a C-terminal double-stranded RNA-binding domain. Both are required for pathogenicity of vaccinia virus in mice infected by intracranial injection. Here, we describe the crystal structure of the Zalpha domain from the E3L-like protein of Yaba-like disease virus, a Yatapoxvirus, in a complex with Z-DNA, solved at a 2.0-A resolution. The DNA contacting surface of Yaba-like disease virus Zalpha(E3L) closely resembles that of other structurally defined members of the Zalpha family, although some variability exists in the beta-hairpin region. In contrast to the Z-DNA-contacting surface, the nonbinding surface of members of the Zalpha family are unrelated; this surface may effect protein-specific interactions. The presence of the conserved and tailored Z-DNA-binding surface, which interacts specifically with the zigzag backbone and syn base diagnostic of the Z-form, reinforces the importance to poxvirus infection of the ability of this protein to recognize the Z-conformation.