Assessment of machine-learning predictions for the Mediator complex subunit MED25 ACID domain interactions with transactivation domains.

The human Mediator complex subunit MED25 binds transactivation domains (TADs) present in various cellular and viral proteins using two binding interfaces, named H1 and H2, which are found on opposite sides of its ACID domain. Here, we use and compare deep learning methods to characterize human MED25-TAD interfaces and assess the predicted models to published experimental data. For the H1 interface, AlphaFold produces predictions with high-reliability scores that agree well with experimental data, while the H2 interface predictions appear inconsistent, preventing reliable binding modes.

NMR Spectroscopy of the Main Protease of SARS-CoV-2 and Fragment-Based Screening Identify Three Protein Hotspots and an Antiviral Fragment.

The main protease (3CLp) of the SARS-CoV-2, the causative agent for the COVID-19 pandemic, is one of the main targets for drug development. To be active, 3CLp relies on a complex interplay between dimerization, active site flexibility, and allosteric regulation. The deciphering of these mechanisms is a crucial step to enable the search for inhibitors.

Crystal structure of human Mediator subunit MED23.

The Mediator complex transduces regulatory information from enhancers to promoters and performs essential roles in the initiation of transcription in eukaryotes. Human Mediator comprises 26 subunits forming three modules termed Head, Middle and Tail. Here we present the 2.

Solution Structure of the N-Terminal Domain of Mediator Subunit MED26 and Molecular Characterization of Its Interaction with EAF1 and TAF7.

MED26 is a subunit of Mediator, a large complex central to the regulation of gene transcription by RNA Polymerase II. MED26 plays a role in the switch between the initiation and elongation phases of RNA Polymerase II-mediated transcription process. Regulation of these steps requires successive binding of MED26 N-terminal domain (NTD) to TATA-binding protein-associated factor 7 (TAF7) and Eleven-nineteen lysine-rich in leukemia-Associated Factor 1 (EAF1).

1H, 15N and 13C assignments of the N-terminal domain of the Mediator complex subunit MED26.

MED26 is a subunit of the Mediator, a very large complex involved in regulation of gene transcription by RNA Polymerase II. MED26 regulates the switch between initiation and elongation phases of the transcription. This function requires interaction of its N-terminal domain (NTD) with several protein partners implicated in transcriptional regulation.

Structure of UBE2Z Enzyme Provides Functional Insight into Specificity in the FAT10 Protein Conjugation Machinery.

FAT10 conjugation, a post-translational modification analogous to ubiquitination, specifically requires UBA6 and UBE2Z as its activating (E1) and conjugating (E2) enzymes. Interestingly, these enzymes can also function in ubiquitination. We have determined the crystal structure of UBE2Z and report how the different domains of this E2 enzyme are organized.

Characterization of ERM transactivation domain binding to the ACID/PTOV domain of the Mediator subunit MED25.

The N-terminal acidic transactivation domain (TAD) of ERM/ETV5 (ERM38-68), a PEA3 group member of Ets-related transcription factors, directly interacts with the ACID/PTOV domain of the Mediator complex subunit MED25. Molecular details of this interaction were investigated using nuclear magnetic resonance (NMR) spectroscopy. The TAD is disordered in solution but has a propensity to adopt local transient secondary structure.

Conserved Omp85 lid-lock structure and substrate recognition in FhaC.

Omp85 proteins mediate translocation of polypeptide substrates across and into cellular membranes. They share a common architecture comprising substrate-interacting POTRA domains, a C-terminal 16-stranded β-barrel pore and two signature motifs located on the inner barrel wall and at the tip of the extended L6 loop. The observation of two distinct conformations of the L6 loop in the available Omp85 structures previously suggested a functional role of conformational changes in L6 in the Omp85 mechanism.

A Salmonella type three secretion effector/chaperone complex adopts a hexameric ring-like structure.

Many bacterial pathogens use type three secretion systems (T3SS) to inject virulence factors, named effectors, directly into the cytoplasm of target eukaryotic cells. Most of the T3SS components are conserved among plant and animal pathogens, suggesting a common mechanism of recognition and secretion of effectors. However, no common motif has yet been identified for effectors allowing T3SS recognition.

Structure of the secretion domain of HxuA from Haemophilus influenzae.

Haemophilus influenzae HxuA is a cell-surface protein with haem-haemopexin binding activity which is key to haem acquisition from haemopexin and thus is one of the potential sources of haem for this microorganism. HxuA is secreted by its specific transporter HxuB. HxuA/HxuB belongs to the so-called two-partner secretion systems (TPSs) that are characterized by a conserved N-terminal domain in the secreted protein which is essential for secretion.

The structural organization of the N-terminus domain of SopB, a virulence factor of Salmonella, depends on the nature of its protein partners.

The TTSS is used by Salmonella and many bacterial pathogens to inject virulence factors directly into the cytoplasm of target eukaryotic cells. Once translocated these so-called effector proteins hijack a vast array of crucial cellular functions to the benefit of the bacteria. In the bacterial cytoplasm, some effectors are stabilized and maintained in a secretion competent state by interaction with specific type III chaperones.

The DivJ, CbrA and PleC system controls DivK phosphorylation and symbiosis in Sinorhizobium meliloti.

Sinorhizobium meliloti is a soil bacterium that invades the root nodules it induces on Medicago sativa, whereupon it undergoes an alteration of its cell cycle and differentiates into nitrogen-fixing, elongated and polyploid bacteroid with higher membrane permeability. In Caulobacter crescentus, a related alphaproteobacterium, the principal cell cycle regulator, CtrA, is inhibited by the phosphorylated response regulator DivK. The phosphorylation of DivK depends on the histidine kinase DivJ, while PleC is the principal phosphatase for DivK.

The Mediator complex subunit MED25 is targeted by the N-terminal transactivation domain of the PEA3 group members.

PEA3, ERM and ER81 belong to the PEA3 subfamily of Ets transcription factors and play important roles in a number of tissue-specific processes. Transcriptional activation by PEA3 subfamily factors requires their characteristic amino-terminal acidic transactivation domain (TAD). However, the cellular targets of this domain remain largely unknown.

Structural insights into ChpT, an essential dimeric histidine phosphotransferase regulating the cell cycle in Caulobacter crescentus.

Two-component and phosphorelay signal-transduction proteins are crucial for bacterial cell-cycle regulation in Caulobacter crescentus. ChpT is an essential histidine phosphotransferase that controls the activity of the master cell-cycle regulator CtrA by phosphorylation. Here, the 2.

Purification of SUMO-1 modified IκBα and complex formation with NF-κB.

Covalent modification of proteins with SUMO (Small Ubiquitin-like MOdifier) affects many cellular processes, including transcriptional regulation, DNA repair and signal transduction. Although hundreds of SUMO targets have been identified, many biological outcomes of protein sumoylation remain poorly understood. In particular, biochemical and structural analysis can only be easily conducted if highly pure sumoylated substrates are available.

NMR structure of the human Mediator MED25 ACID domain.

MED25 (ARC92/ACID1) is a 747 residues subunit specific to higher eukaryote Mediator complex, an essential component of the RNA polymerase II general transcriptional machinery. MED25 is a target of the Herpes simplex virus transactivator protein VP16. MED25 interacts with VP16 through a central MED25 PTOV (Prostate tumour overexpressed)/ACID (Activator interacting domain) domain of unknown structure.

Solution structure of the N-terminal transactivation domain of ERM modified by SUMO-1.

ERM is a member of the PEA3 group of the Ets transcription factor family that plays important roles in development and tumorigenesis. The PEA3s share an N-terminal transactivation domain (TADn) whose activity is inhibited by small ubiquitin-like modifier (SUMO). However, the consequences of sumoylation and its underlying molecular mechanism remain unclear.

Single-molecule force spectroscopy of mycobacterial adhesin-adhesin interactions.

The heparin-binding hemagglutinin (HBHA) is one of the few virulence factors identified for Mycobacterium tuberculosis. It is a surface-associated adhesin that expresses a number of different activities, including mycobacterial adhesion to nonphagocytic cells and microbial aggregation. Previous evidence indicated that HBHA is likely to form homodimers or homopolymers via a predicted coiled-coil region located within the N-terminal portion of the molecule.

Expression and purification in high yield of a functionally active recombinant human Type I inositol(1,4,5)P3 5-phosphatase.

Inositol polyphosphates are the most widespread second messenger molecules in eukaryotic cells. Human Type I inositol 1,4,5-triphosphate (Ins(1,4,5)P(3)) 5-phosphatase removes the D-5 position phosphate from soluble Ins(1,4,5)P(3,) a key event in cell signaling particularly in Ca(2+) homeostasis. In this study, the cDNA encoding human Type I Ins(1,4,5)P(3) 5-phosphatase was subcloned into a modified pMAL expression vector.

Characterization of the Arabidopsis thaliana Arath;CDC25 dual-specificity tyrosine phosphatase.

CDC25 enzymes are dual-specificity phosphatases involved in the regulation of the cell cycle. No CDC25 enzymes have been described in higher plant organisms. We report here the characterization of an Arabidopsis thaliana CDC25 enzyme, constituted by a sole catalytic domain and devoid of the N-terminal regulatory region found in the human CDC25.

A small CDC25 dual-specificity tyrosine-phosphatase isoform in Arabidopsis thaliana.

The dual-specificity CDC25 phosphatases are critical positive regulators of cyclin-dependent kinases (CDKs). Even though an antagonistic Arabidopsis thaliana WEE1 kinase has been cloned and tyrosine phosphorylation of its CDKs has been demonstrated, no valid candidate for a CDC25 protein has been reported in higher plants. We identify a CDC25-related protein (Arath;CDC25) of A.

Function of the transcriptional regulating protein of 132 kDa (TReP-132) on human P450scc gene expression.

Cytochrome P450scc catalyzes the important first step in the steroid synthesis pathway; however, it is clear that additional factors regulating the temporal and spacial specific expression of the CYP11A1 gene remain to be identified. To isolate novel transcription factors that regulate this gene, a cis-acting element of the 5'-flanking region from nucleotides -155 to -131 (-155/-131) was used to screen a human placental lambda gt11 cDNA expression library, and an interacting clone was isolated. The open reading frame of the cDNA encodes several domains that are characteristic of transcription factors including an acidic region, a region rich in prolines and three zinc-finger motifs.

Visualization of TGN to endosome trafficking through fluorescently labeled MPR and AP-1 in living cells.

We have stably expressed in HeLa cells a chimeric protein made of the green fluorescent protein (GFP) fused to the transmembrane and cytoplasmic domains of the mannose 6-phosphate/insulin like growth factor II receptor in order to study its dynamics in living cells. At steady state, the bulk of this chimeric protein (GFP-CI-MPR) localizes to the trans-Golgi network (TGN), but significant amounts are also detected in peripheral, tubulo-vesicular structures and early endosomes as well as at the plasma membrane. Time-lapse videomicroscopy shows that the GFP-CI-MPR is ubiquitously detected in tubular elements that detach from the TGN and move toward the cell periphery, sometimes breaking into smaller tubular fragments.

The transcriptional regulating protein of 132 kDa (TReP-132) enhances P450scc gene transcription through interaction with steroidogenic factor-1 in human adrenal cells.

The human P450scc gene is regulated by the tissue-specific orphan nuclear receptor, steroidogenic factor-1 (SF-1), which plays a key role in several physiologic processes including steroid synthesis, adrenal and gonadal development, and sexual differentiation. Several studies have demonstrated the interaction of SF-1 with different proteins. However, it is clear that additional factors not yet identified are involved with SF-1 to regulate different target genes.