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.

De novo prediction of explicit water molecule positions by a novel algorithm within the protein design software MUMBO.

By mediating interatomic interactions, water molecules play a major role in protein-protein, protein-DNA and protein-ligand interfaces, significantly affecting affinity and specificity. This notwithstanding, explicit water molecules are usually not considered in protein design software because of high computational costs. To challenge this situation, we analyzed the binding characteristics of 60,000 waters from high resolution crystal structures and used the observed parameters to implement the prediction of water molecules in the protein design and side chain-packing software MUMBO.

An antiviral targeting strategy based on the inducible interference with cytomegalovirus nuclear egress complex.

The nucleocytoplasmic capsid egress of herpesviruses like the human cytomegalovirus (HCMV) is based on a uniquely regulated process. The core nuclear egress complex (NEC) of HCMV, represented by the pUL50-pUL53 heterodimer, is able to oligomerize and thus to build hexameric lattices. Recently, we and others validated the NEC as a novel target for antiviral strategies.

Cross-Species Analysis of Innate Immune Antagonism by Cytomegalovirus IE1 Protein.

The human cytomegalovirus (CMV) immediate early 1 (IE1) protein has evolved as a multifunctional antagonist of intrinsic and innate immune mechanisms. In addition, this protein serves as a transactivator and potential genome maintenance protein. Recently, the crystal structures of the human and rat CMV IE1 (hIE1, rIE1) core domain were solved.

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.

Cytomegalovirus immediate-early 1 proteins form a structurally distinct protein class with adaptations determining cross-species barriers.

Restriction factors are potent antiviral proteins that constitute a first line of intracellular defense by blocking viral replication and spread. During co-evolution, however, viruses have developed antagonistic proteins to modulate or degrade the restriction factors of their host. To ensure the success of lytic replication, the herpesvirus human cytomegalovirus (HCMV) expresses the immediate-early protein IE1, which acts as an antagonist of antiviral, subnuclear structures termed PML nuclear bodies (PML-NBs).

A PROSS-designed extensively mutated estrogen receptor α variant displays enhanced thermal stability while retaining native allosteric regulation and structure.

Protein stability limitations often hamper the exploration of proteins as drug targets. Here, we show that the application of PROSS server algorithms to the ligand-binding domain of human estrogen receptor alpha (hERα) enabled the development of variant ER that comprises 24 amino acid substitutions and exhibits multiple improved characteristics. The protein displays enhanced production rates in E.

Exploring the Human Cytomegalovirus Core Nuclear Egress Complex as a Novel Antiviral Target: A New Type of Small Molecule Inhibitors.

Nuclear egress is an essential process in the replication of human cytomegalovirus (HCMV), as it enables the migration of newly formed viral capsids from the nucleus into the cytoplasm. Inhibition of the HCMV core nuclear egress complex (core NEC), composed of viral proteins pUL50 and pUL53, has been proposed as a potential new target for the treatment of HCMV infection and disease. Here, we present a new type of small molecule inhibitors of HCMV core NEC formation, which inhibit the pUL50-pUL53 interaction at nanomolar concentrations.

Crystal structures of adenylylated and unadenylylated P protein GlnK from Corynebacterium glutamicum.

P proteins are ubiquitous signaling proteins that are involved in the regulation of the nitrogen/carbon balance in bacteria, archaea, and some plants and algae. Signal transduction via P proteins is modulated by effector molecules and post-translational modifications in the P T-loop. Whereas the binding of ADP, ATP and the concomitant binding of ATP and 2-oxoglutarate (2OG) engender two distinct conformations of the T-loop that either favor or disfavor the interaction with partner proteins, the structural consequences of post-translational modifications such as phosphorylation, uridylylation and adenylylation are far less well understood.

Nuclear Egress Complexes of HCMV and Other Herpesviruses: Solving the Puzzle of Sequence Coevolution, Conserved Structures and Subfamily-Spanning Binding Properties.

Herpesviruses uniquely express two essential nuclear egress-regulating proteins forming a heterodimeric nuclear egress complex (core NEC). These core NECs serve as hexameric lattice-structured platforms for capsid docking and recruit viral and cellular NEC-associated factors that jointly exert nuclear lamina as well as membrane-rearranging functions (multicomponent NEC). The regulation of nuclear egress has been profoundly analyzed for murine and human cytomegaloviruses (CMVs) on a mechanistic basis, followed by the description of core NEC crystal structures, first for HCMV, then HSV-1, PRV and EBV.

The CD83 Molecule - An Important Immune Checkpoint.

The CD83 molecule has been identified to be expressed on numerous activated immune cells, including B and T lymphocytes, monocytes, dendritic cells, microglia, and neutrophils. Both isoforms of CD83, the membrane-bound as well as its soluble form are topic of intensive research investigations. Several studies revealed that CD83 is not a typical co-stimulatory molecule, but rather plays a critical role in controlling and resolving immune responses.

Recombinant protein production and purification of SiiD, SiiE and SiiF - Components of the SPI4-encoded type I secretion system from Salmonella Typhimurium.

In humans, Salmonella enterica infections are responsible for a plethora of medical conditions. These include intestinal inflammation and typhoid fever. The initial contact between Salmonella and polarized epithelial cells is established by the SPI4-encoded type I secretion system (T1SS), which secretes SiiE, a giant non-fimbrial adhesin.

The complex formed between a synthetic RNA aptamer and the transcription repressor TetR is a structural and functional twin of the operator DNA-TetR regulator complex.

RNAs play major roles in the regulation of gene expression. Hence, designer RNA molecules are increasingly explored as regulatory switches in synthetic biology. Among these, the TetR-binding RNA aptamer was selected by its ability to compete with operator DNA for binding to the bacterial repressor TetR.

High-resolution crystal structures of two prototypical β- and γ-herpesviral nuclear egress complexes unravel the determinants of subfamily specificity.

Herpesviruses uniquely express two essential nuclear egress-regulating proteins forming a heterodimeric basic structure of the nuclear egress complex (core NEC). These core NECs serve as a hexameric lattice-structured platform for capsid docking and recruit viral and cellular NEC-associated factors that jointly exert nuclear lamina- and membrane-rearranging functions (multicomponent NEC). Here, we report the X-ray structures of β- and γ-herpesvirus core NECs obtained through an innovative recombinant expression strategy based on NEC-hook::NEC-groove protein fusion constructs.

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.

NewBG: A surrogate corticosteroid-binding globulin with an unprecedentedly high ligand release efficacy.

The introduction of ligand-binding sites into proteins and the engineering of molecular allosteric coupling pathways are topical issues in protein design. Here, we show that these issues can be addressed concurrently, using the serpin human α1-antichymotrypsin (ACT) as a model. We have introduced up to 15 amino acid substitutions into ACT, converting it into a surrogate corticosteroid-binding globulin (CBG), thereby creating a new binding globulin (NewBG).

PRISEs (progesterone 5β-reductase and/or iridoid synthase-like 1,4-enone reductases): Catalytic and substrate promiscuity allows for realization of multiple pathways in plant metabolism.

PRISEs (progesterone 5β-reductase and/or iridoid synthase-like 1,4-enone reductases) are involved in cardenolide and iridoid biosynthesis. We here investigated a PRISE (rAtSt5βR) from Arabidopsis thaliana, a plant producing neither cardenolides nor iridoids. The structure of rAtSt5βR was elucidated with X-ray crystallography and compared to the known structures of PRISEs from Catharanthus roseus (rCrISY) and Digitalis lanata (rDlP5βR).

Design of an allosterically modulated doxycycline and doxorubicin drug-binding protein.

The allosteric interplay between distant functional sites present in a single protein provides for one of the most important regulatory mechanisms in biological systems. While the design of ligand-binding sites into proteins remains challenging, this holds even truer for the coupling of a newly engineered binding site to an allosteric mechanism that regulates the ligand affinity. Here it is shown how computational design algorithms enabled the introduction of doxycycline- and doxorubicin-binding sites into the serine proteinase inhibitor (serpin) family member α1-antichymotrypsin.

Hydration Structure and Dynamics of Inhibitor-Bound HIV-1 Protease.

Water is essential in many biological processes, and the hydration structure plays a critical role in facilitating protein folding, dynamics, and ligand binding. A variety of biophysical spectroscopic techniques have been used to probe the water solvating proteins, often complemented with molecular dynamics (MD) simulations to resolve the spatial and dynamic features of the hydration shell, but comparing relative water structure is challenging. In this study 1 μs MD simulations were performed to identify and characterize hydration sites around HIV-1 protease bound to an inhibitor, darunavir (DRV).

Near-native, site-specific and purification-free protein labeling for quantitative protein interaction analysis by MicroScale Thermophoresis.

MicroScale Thermophoresis (MST) is a frequently used method for the quantitative characterization of intermolecular interactions with several advantages over other technologies. One of these is its capability to determine equilibrium constants in solution including complex biological matrices such as cell lysates. MST requires one binding partner to be fluorescent, which is typically achieved by labeling target proteins with a suitable fluorophore.

The human cytomegalovirus nuclear egress complex unites multiple functions: Recruitment of effectors, nuclear envelope rearrangement, and docking to nuclear capsids.

Background: Nuclear replication represents a common hallmark of herpesviruses achieved by a number of sequentially unrolled regulatory processes. A rate-limiting step is provided by nucleo-cytoplasmic capsid export, for which a defined multiregulatory protein complex, namely, the nuclear egress complex (NEC), is assembled comprising both viral and cellular components. The NEC regulates at least 3 aspects of herpesviral nuclear replication: (1) multimeric recruitment of NEC-associated effector proteins, (2) reorganization of the nuclear lamina and membranes, and (3) the docking to nuclear capsids.

Structural and functional dissection reveals distinct roles of Ca2+-binding sites in the giant adhesin SiiE of Salmonella enterica.

The giant non-fimbrial adhesin SiiE of Salmonella enterica mediates the first contact to the apical site of epithelial cells and enables subsequent invasion. SiiE is a 595 kDa protein composed of 53 repetitive bacterial immunoglobulin (BIg) domains and the only known substrate of the SPI4-encoded type 1 secretion system (T1SS). The crystal structure of BIg50-52 of SiiE revealed two distinct Ca2+-binding sites per BIg domain formed by conserved aspartate or glutamate residues.

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.

Similarities in the structure of the transcriptional repressor AmtR in two different space groups suggest a model for the interaction with GlnK.

AmtR belongs to the TetR family of transcription regulators and is a global nitrogen regulator that is induced under nitrogen-starvation conditions in Corynebacterium glutamicum. AmtR regulates the expression of transporters and enzymes for the assimilation of ammonium and alternative nitrogen sources, for example urea, amino acids etc. The recognition of operator DNA by homodimeric AmtR is not regulated by small-molecule effectors as in other TetR-family members but by a trimeric adenylylated P-type signal transduction protein named GlnK.

The Human Cytomegalovirus IE1 Protein Antagonizes PML Nuclear Body-Mediated Intrinsic Immunity via the Inhibition of PML De Novo SUMOylation.

Unlabelled: PML nuclear bodies (NBs) are accumulations of cellular proteins embedded in a scaffold-like structure built by SUMO-modified PML/TRIM19. PML and other NB proteins act as cellular restriction factors against human cytomegalovirus (HCMV); however, this intrinsic defense is counteracted by the immediate early protein 1 (IE1) of HCMV. IE1 directly interacts with the PML coiled-coil domain via its globular core region and disrupts NB foci by inducing a loss of PML SUMOylation.