A Novel RNA Aptamer as Synthetic Inducer of DasR Controlled Transcription.

Progress in the synthetic biology field is driven by the development of new tools for synthetic circuit engineering. Traditionally, the focus has relied on protein-based designs. In recent years, the use of RNA-based tools has tremendously increased, due to their versatile functionality and applicability. A promising class of molecules is RNA aptamers, small, single-stranded RNA molecules that bind to a target molecule with high affinity and specificity. When targeting bacterial repressors, RNA aptamers allow one to add a new layer to an established protein-based regulation. In the present study, we selected an RNA aptamer binding the bacterial repressor DasR, preventing its binding to its operator sequence and activating DasR-controlled transcription in vivo. This was made possible only by the combination of an in vitro selection and subsequent in vivo screening. Next-generation sequencing of the selection process proved the importance of the in vivo screening for the discovery of aptamers functioning in the cell. Mutational and biochemical studies led to the identification of the minimal necessary binding motif. Taken together, the resulting combination of bacterial repressor and RNA aptamer enlarges the synthetic biology toolbox by adding a new level of regulation.

The crystal structure of the varicella-zoster Orf24-Orf27 nuclear egress complex spotlights multiple determinants of herpesvirus subfamily specificity.

Varicella-zoster virus (VZV) is a human pathogen from the α-subfamily of herpesviruses. The VZV Orf24-Orf27 complex represents the essential viral core nuclear egress complex (NEC) that orchestrates the egress of the preassembled virus capsids from the nucleus. While previous studies have primarily emphasized that the architecture of core NEC complexes is highly conserved among herpesviruses, the present report focuses on subfamily-specific structural and functional features that help explain the differences in the autologous versus nonautologous interaction patterns observed for NEC formation across herpesviruses. Here, we describe the crystal structure of the Orf24-Orf27 complex at 2.1 Å resolution. Coimmunoprecipitation and confocal imaging data show that Orf24-Orf27 complex formation displays some promiscuity in a herpesvirus subfamily-restricted manner. At the same time, analysis of thermodynamic parameters of NEC formation of three prototypical α-, ß-, and γ herpesviruses, i.e., VZV, human cytomegalovirus (HCMV), and Epstein-Barr virus (EBV), revealed highly similar binding affinities for the autologous interaction with specific differences in enthalpy and entropy. Computational alanine scanning, structural comparisons, and mutational data highlight intermolecular interactions shared among α-herpesviruses that are clearly distinct from those seen in ß- and γ-herpesviruses, including a salt bridge formed between Orf24-Arg167 and Orf27-Asp126. This interaction is located outside of the hook-into-groove interface and contributes significantly to the free energy of complex formation. Combined, these data explain distinct properties of specificity and permissivity so far observed in herpesviral NEC interactions. These findings will prove valuable in attempting to target multiple herpesvirus core NECs with selective or broad-acting drug candidates.

Structural and functional characterization of SiiA, an auxiliary protein from the SPI4-encoded type 1 secretion system from Salmonella enterica.

Salmonella invasion is mediated by a concerted action of the Salmonella pathogenicity island 4 (SPI4)-encoded type one secretion system (T1SS) and the SPI1-encoded type three secretion system (T3SS-1). The SPI4-encoded T1SS consists of five proteins (SiiABCDF) and secretes the giant adhesin SiiE. Here, we investigated structure-function relationships in SiiA, a non-canonical T1SS subunit. We show that SiiA consists of a membrane domain, an intrinsically disordered periplasmic linker region and a folded globular periplasmic domain (SiiA-PD). The crystal structure of SiiA-PD displays homology to that of MotB and other peptidoglycan (PG)-binding domains. SiiA-PD binds PG in vitro, albeit at an acidic pH, only. Mutation of Arg162 impedes PG binding of SiiA and reduces Salmonella invasion efficacy. SiiA forms a complex with SiiB at the inner membrane (IM), and the observed SiiA-MotB homology is paralleled by a predicted SiiB-MotA homology. We show that, similar to MotAB, SiiAB translocates protons across the IM. Mutating Asp13 in SiiA impairs proton translocation. Overall, SiiA shares numerous properties with MotB. However, MotAB uses the proton motif force (PMF) to energize the bacterial flagellum, it remains to be shown how usage of the PMF by SiiAB assists T1SS function and Salmonella invasion.

Crystal Structure of the Extracellular Domain of the Human Dendritic Cell Surface Marker CD83.

CD83 is a type-I membrane protein and an efficient marker for identifying mature dendritic cells. Whereas membrane-bound, full-length CD83 co-stimulates the immune system, a soluble variant (sCD83), consisting of the extracellular domain only, displays strong immune-suppressive activities. Besides a prediction that sCD83 adopts a V-set Ig-like fold, however, little is known about the molecular architecture of CD83 and the mechanism by which CD83 exerts its function on dendritic cells and additional immune cells. Here, we report the crystal structure of human sCD83 up to a resolution of 1.7Å solved in three different crystal forms. Interestingly, ß-strands C', C″, and D that are typical for V-set Ig-domains could not be traced in sCD83. Mass spectrometry analyses, limited proteolysis experiments, and bioinformatics studies show that the corresponding segment displays enhanced main-chain accessibility, extraordinary low sequence conservation, and a predicted high disorder propensity. Chimeric proteins with amino acid swaps in this segment show unaltered immune-suppressive activities in a TNF-α assay when compared to wild-type sCD83. This strongly indicates that this segment does not participate in the biological activity of CD83. The crystal structure of CD83 shows the recurrent formation of dimers and trimers in the various crystal forms and reveals strong structural similarities between sCD83 and B7 family members and CD48, a signaling lymphocyte activation molecule family member. This suggests that CD83 exerts its immunological activity by mixed homotypic and heterotypic interactions as typically observed for proteins present in the immunological synapse.

Identification of a Novel TGF-ß-Binding Site in the Zona Pellucida C-terminal (ZP-C) Domain of TGF-ß-Receptor-3 (TGFR-3).

The zona pellucida (ZP) domain is present in extracellular proteins such as the zona pellucida proteins and tectorins and participates in the formation of polymeric protein networks. However, the ZP domain also occurs in the cytokine signaling co-receptor transforming growth factor ß (TGF-ß) receptor type 3 (TGFR-3, also known as betaglycan) where it contributes to cytokine ligand recognition. Currently it is unclear how the ZP domain architecture enables this dual functionality. Here, we identify a novel major TGF-ß-binding site in the FG loop of the C-terminal subdomain of the murine TGFR-3 ZP domain (ZP-C) using protein crystallography, limited proteolysis experiments, surface plasmon resonance measurements and synthetic peptides. In the murine 2.7 Å crystal structure that we are presenting here, the FG-loop is disordered, however, well-ordered in a recently reported homologous rat ZP-C structure. Surprisingly, the adjacent external hydrophobic patch (EHP) segment is registered differently in the rat and murine structures suggesting that this segment only loosely associates with the remaining ZP-C fold. Such a flexible and temporarily-modulated association of the EHP segment with the ZP domain has been proposed to control the polymerization of ZP domain-containing proteins. Our findings suggest that this flexibility also extends to the ZP domain of TGFR-3 and might facilitate co-receptor ligand interaction and presentation via the adjacent FG-loop. This hints that a similar C-terminal region of the ZP domain architecture possibly regulates both the polymerization of extracellular matrix proteins and cytokine ligand recognition of TGFR-3.

Mouse ApoM displays an unprecedented seven-stranded lipocalin fold: folding decoy or alternative native fold?

Mouse apolipoprotein M (m-apoM) displays a 79% sequence identity to human apolipoprotein M (h-apoM). Both proteins are apolipoproteins associated with high-density lipoproteins, with similar anticipated biological functions. The structure of h-apoM has recently been determined by X-ray crystallography, which revealed that h-apoM displays, as expected, a lipocalin-like fold characterized by an eight-stranded ß­barrel that encloses an internal fatty-acid-binding site. Surprisingly, this is not true for m-apoM. After refolding from inclusion bodies, the crystal structure of m-apoM (reported here at 2.5 Å resolution) displays a novel yet unprecedented seven-stranded ß-barrel structure. The fold difference is not caused by a mere deletion of a single ß-strand; instead, ß-strands E and F are removed and replaced by a single ß-strand A' formed from residues from the N-terminus. Molecular dynamics simulations suggest that m-apoM is able to adopt both a seven-stranded barrel structure and an eight-stranded barrel structure in solution, and that both folds are comparably stable. Thermal unfolding simulations identify the position where ß-strand exchange occurs as the weak point of the ß-barrel. We wonder whether the switch in topology could have a biological function and could facilitate ligand release, since it goes hand in hand with a narrowing of the barrel diameter. Possibly also, the observed conformation represents an on-pathway or off-pathway folding intermediate of apoM. The difference in fold topology is quite remarkable, and the fold promiscuity observed for m-apoM might possibly provide a glimpse at potential cross-points during the evolution of ß-barrels.

Computational design of a chain-specific tetracycline repressor heterodimer.

The specificity and selectivity of protein-protein interactions are of central importance for many biological processes, including signal transduction and transcription control. We used the in-house side-chain packing program MUMBO to computationally design a chain-specific heterodimeric variant of the bacterial transcription regulator tetracycline repressor (TetR), called T-A(A)B. Our goal was to engineer two different TetR chain variants, A and B, that no longer interact as AA or BB homodimers but selectively recombine to form heterodimers. Although 56 residues from each chain contribute to a dimer interface as large as 2200 Å(2) in wild-type TetR, the substitution of only three residues in one chain and two residues in a second chain sufficed for generating specificity in a T-A(A)B heterodimer variant. The design was corroborated in vivo by a cell-based transcription assay, and in vitro by CD spectroscopy and X-ray crystallography. Crystal structure analyses showed that while selectivity in the B chain is achieved entirely through van der Waals repulsion, the best selectivity in the A chain is obtained for the variant with the lowest number of atoms in the interface, thus possibly leading to underpacking of the dimer interface. This results in a marked decrease in thermal stability and a drastic reduction in the solubility of the T-A(A)A(A) homodimer in comparison to the designed T-A(A)B heterodimer variant.

Vpx proteins of SIVmac239 and HIV-2ROD interact with the cytoskeletal protein alpha-actinin 1.

vpx genes of human immunodeficiency virus type 2 (HIV-2) and immunodeficiency viruses from macaques (SIVmac), sooty mangabeys (SIVsm) and red-capped mangabeys (SIVrcm) encode a 112 aa protein that is packed into virion particles via interaction with the p6 domain of p55(gag). Vpx localizes to the nucleus when expressed in the absence of other viral proteins. Moreover, Vpx is necessary for efficient nuclear import of the pre-integration complex (PIC) and critical for virus replication in quiescent cells, such as terminally differentiated macrophages and memory T cells. Vpx does not contain sequence elements that are homologous to previously characterized nuclear localization signals (NLSs). Therefore, it is likely that Vpx-dependent import of the PIC is mediated by interaction of Vpx with cellular proteins that do not belong to the classical import pathways. By using a yeast two-hybrid screen, alpha-actinin 1, a cytoskeletal protein, was identified to interact with SIVmac239 Vpx. Interestingly, deletion of the proline-rich C-terminal domain (aa 101-112) of Vpx, which is important for nuclear localization, resulted in loss of interaction with alpha-actinin 1. These findings suggest that the interaction with alpha-actinin 1 may play an important role in the transport of Vpx to the nucleus and in Vpx-mediated nuclear import of the PIC.

Negative feedback loop of Wnt signaling through upregulation of conductin/axin2 in colorectal and liver tumors.

Activation of Wnt signaling through beta-catenin/TCF complexes is a key event in the development of various tumors, in particular colorectal and liver tumors. Wnt signaling is controlled by the negative regulator conductin/axin2/axil, which induces degradation of beta-catenin by functional interaction with the tumor suppressor APC and the serine/threonine kinase GSK3beta. Here we show that conductin is upregulated in human tumors that are induced by beta-catenin/Wnt signaling, i.e., high levels of conductin protein and mRNA were found in colorectal and liver tumors but not in the corresponding normal tissues. In various other tumor types, conductin levels did not differ between tumor and normal tissue. Upregulation of conductin was also observed in the APC-deficient intestinal tumors of Min mice. Inhibition of Wnt signaling by a dominant-negative mutant of TCF downregulated conductin but not the related protein, axin, in DLD1 colorectal tumor cells. Conversely, activation of Wnt signaling by Wnt-1 or dishevelled increased conductin levels in MDA MB 231 and Neuro2A cells, respectively. In time course experiments, stabilization of beta-catenin preceded the upregulation of conductin by Wnt-1. These results demonstrate that conductin is a target of the Wnt signaling pathway. Upregulation of conductin may constitute a negative feedback loop that controls Wnt signaling activity.