Binding-site Occlusion Research Articles

MCM2-7 loading-dependent ORC release ensures genome-wide origin licensing

Origin recognition complex (ORC)-dependent loading of the replicative helicase MCM2-7 onto replication origins in G1-phase forms the basis of replication fork establishment in S-phase. However, how ORC and MCM2-7 facilitate genome-wide DNA licensing is not fully understood. Mapping the molecular footprints of budding yeast ORC and MCM2-7 genome-wide, we discovered that MCM2-7 loading is associated with ORC release from origins and redistribution to non-origin sites. Our bioinformatic analysis revealed that origins are compact units, where a single MCM2-7 double hexamer blocks repetitive loading through steric ORC binding site occlusion. Analyses of A-elements and an improved B2-element consensus motif uncovered that DNA shape, DNA flexibility, and the correct, face-to-face spacing of the two DNA elements are hallmarks of ORC-binding and efficient helicase loading sites. Thus, our work identified fundamental principles for MCM2-7 helicase loading that explain how origin licensing is realised across the genome.

Slc11 Synapomorphy: A Conserved 3D Framework Articulating Carrier Conformation Switch.

Transmembrane carriers of the Slc11 family catalyze proton (H+)-dependent uptake of divalent metal ions (Me2+) such as manganese and iron-vital elements coveted during infection. The Slc11 mechanism of high-affinity Me2+ cell import is selective and conserved between prokaryotic (MntH) and eukaryotic (Nramp) homologs, though processes coupling the use of the proton motive force to Me2+ uptake evolved repeatedly. Adding bacterial piracy of Nramp genes spread in distinct environmental niches suggests selective gain of function that may benefit opportunistic pathogens. To better understand Slc11 evolution, Alphafold (AF2)/Colabfold (CF) 3D predictions for bacterial sequences from sister clades of eukaryotic descent (MCb and MCg) were compared using both native and mutant templates. AF2/CF model an array of native MCb intermediates spanning the transition from outwardly open (OO) to inwardly open (IO) carriers. In silico mutagenesis targeting (i) a set of (evolutionarily coupled) sites that may define Slc11 function (putative synapomorphy) and (ii) residues from networked communities evolving during MCb transition indicates that Slc11 synapomorphy primarily instructs a Me2+-selective conformation switch which unlocks carrier inner gate and contributes to Me2+ binding site occlusion and outer gate locking. Inner gate opening apparently proceeds from interaction between transmembrane helix (h) h5, h8 and h1a. MCg1 xenologs revealed marked differences in carrier shape and plasticity, owing partly to an altered intramolecular H+ network. Yet, targeting Slc11 synapomorphy also converted MCg1 IO models to an OO state, apparently mobilizing the same residues to control gates. But MCg1 response to mutagenesis differed, with extensive divergence within this clade correlating with MCb-like modeling properties. Notably, MCg1 divergent epistasis marks the emergence of the genus Bordetella-Achromobacter. Slc11 synapomorphy localizes to the 3D areas that deviate least among MCb and MCg1 models (either IO or OO) implying that it constitutes a 3D network of residues articulating a Me2+-selective carrier conformation switch which is maintained in fast-evolving clades at the cost of divergent epistatic interactions impacting carrier shape and dynamics.

Nanomechanical sensor for rapid and ultrasensitive detection of tumor markers in serum using nanobody.

Early cancer diagnosis requires ultrasensitive detection of tumor markers in blood. To this end, we develop a novel microcantilever immunosensor using nanobodies (Nbs) as receptors. As the smallest antibody (Ab) entity comprising an intact antigen-binding site, Nbs achieve dense receptor layers and short distances between antigen-binding regions and sensor surfaces, which significantly elevate the generation and transmission of surface stress. Owing to the inherent thiol group at the C-terminus, Nbs are covalently immobilized on microcantilever surfaces in directed orientation via one-step reaction, which further enhances the stress generation. For microcantilever-based nanomechanical sensor, these advantages dramatically increase the sensor sensitivity. Thus, Nb-functionalized microcantilevers can detect picomolar concentrations of tumor markers with three orders of magnitude higher sensitivity, when compared with conventional Ab-functionalized microcantilevers. This proof-of-concept study demonstrates an ultrasensitive, label-free, rapid, and low-cost method for tumor marker detection. Moreover, interestingly, we find Nb inactivation on sensor interfaces when using macromolecule blocking reagents. The adsorption-induced inactivation is presumably caused by the change of interfacial properties, due to binding site occlusion upon complex coimmobilization formations. Our findings are generalized to any coimmobilization methodology for Nbs and, thus, for the construction of high-performance immuno-surfaces.Electronic Supplementary MaterialSupplementary material (experimental section, HER2 detection using anti-HER2-mAb-functionalized microcantilevers) is available in the online version of this article at 10.1007/s12274-021-3588-4.

The structure of unliganded sterol carrier protein 2 from Yarrowia lipolytica unveils a mechanism for binding site occlusion

Isolated or as a part of multidomain proteins, Sterol Carrier Protein 2 (SCP2) exhibits high affinity and broad specificity for different lipidic and hydrophobic compounds. A wealth of structural information on SCP2 domains in all forms of life is currently available; however, many aspects of its ligand binding activity are poorly understood. ylSCP2 is a well-characterized single domain SCP2 from the yeast Yarrowia lipolytica. Herein, we report the X-ray structure of unliganded ylSCP2 refined to 2.0 Å resolution. Comparison with the previously solved liganded ylSCP2 structure unveiled a novel mechanism for binding site occlusion. The liganded ylSCP2 binding site is a large cavity with a volume of more than 800 Å3. In unliganded ylSCP2 the binding site is reduced to about 140 Å3. The obliteration is caused by a swing movement of the C-terminal α helix 5 and a subtle compaction of helices 2–4. Previous pairwise comparisons were between homologous SCP2 domains with a uncertain binding status. The reported unliganded ylSCP2 structure allows for the first time a fully controlled comparative analysis of the conformational effects of ligand occupation dispelling several doubts regarding the architecture of SCP2 binding site.

Mechanism of extracellular ion exchange and binding-site occlusion in a sodium/calcium exchanger.

Na+/Ca2+ exchangers utilize the Na+ electrochemical gradient across the plasma membrane to extrude intracellular Ca2+, and play a central role in Ca2+ homeostasis. Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state. This analysis defines the binding mode and relative affinity of these ions, establishes the structural basis for the anticipated 3Na+:1Ca2+ exchange stoichiometry, and reveals the conformational changes at the onset of the alternating-access transport mechanism. An independent analysis of the dynamics and conformational free-energy landscape of NCX_Mj in different ion-occupancy states, based on enhanced-sampling molecular-dynamics simulations, demonstrates that the crystal structures reflect mechanistically relevant, interconverting conformations. These calculations also reveal the mechanism by which the outward-to-inward transition is controlled by the ion-occupancy state, thereby explaining the emergence of strictly-coupled Na+/Ca2+ antiport.

A variant riboswitch aptamer class for S-adenosylmethionine common in marine bacteria

Riboswitches that sense S-adenosylmethionine (SAM) are widely distributed throughout a variety of bacterial lineages. Four classes of SAM-binding riboswitches have been reported to date, constituting the most diverse collection of riboswitch classes that sense the same compound. Three of these classes, termed SAM-I, SAM-II, and SAM-III represent unique structures that form distinct binding pockets for the ligand. SAM-IV riboswitches carry different conserved sequence and structural features compared to other SAM riboswitches, but nucleotides and substructures corresponding to the ligand binding pocket are identical to SAM-I aptamers. In this article, we describe a fifth class of SAM binding aptamer, which we have termed SAM-V. SAM-V was discovered by analyzing GC-rich intergenic regions preceding metabolic genes in the marine alpha-proteobacterium "Candidatus Pelagibacter ubique." Although the motif is nearly unrepresented in cultured bacteria whose genomes have been completely sequenced, SAM-V is prevalent in marine metagenomic sequences. The consensus sequence and structure of SAM-V show some similarities to that of the SAM-II riboswitch, and it is likely that the two aptamers form similar ligand binding pockets. In addition, we identified numerous examples of a tandem SAM-II/SAM-V aptamer architecture. In this arrangement, the SAM-II aptamer is always positioned 5' of the SAM-V aptamer and the SAM-II aptamer is followed by a predicted intrinsic transcription terminator stem. The SAM-V aptamer, however, appears to use a ribosome binding site occlusion mechanism for genetic regulation. This tandem riboswitch arrangement exhibits an architecture that can potentially control both the transcriptional and translational stages of gene expression.

Transcriptional repression of human hepatitis B virus genes by a bZIP family member, E4BP4.

Box alpha is an essential element of both the upstream regulatory sequence of the core promoter and the second enhancer, which positively regulate the transcription of human hepatitis B virus (HBV) genes. In this paper, we describe the cloning and characterization of a box alpha binding protein, E4BP4. E4BP4 is a bZIP type of transcription factor. Overexpression of E4BP4 represses the stimulating activity of box alpha in the upstream regulatory sequence of the core promoter and the second enhancer in differentiated human hepatoma cell lines. E4BP4 can also suppress the transcription of HBV genes and the production of HBV virions in a transient-transfection system that mimics the viral infection in vivo. Expression of an E4BP4 antisense transcript can, instead, elevate the transcription of the core promoter. A low abundance of E4BP4 protein and mRNA in differentiated human hepatoma cell lines is detected, and E4BP4 is not a major component of box alpha binding proteins in untransfected differentiated human hepatoma cell lines. C/EBPalpha and C/EBPbeta, in contrast, are major components of the box alpha binding activity present in nuclear extracts. E4BP4 has a stronger binding affinity towards box alpha than the endogenous box alpha binding activity present in nuclear extracts. Structure and function analysis of E4BP4 reveals that DNA binding activity is sufficient to confer the negative regulatory function of E4BP4. These results indicate that binding site occlusion is the mechanism whereby E4BP4 suppresses transcription in HBV.