A helicase-tethered ORC flip enables bidirectional helicase loading

Replication origins are licensed by loading two Mcm2‑7 helicases around DNA in a head-to-head conformation poised to initiate bidirectional replication. This process requires ORC, Cdc6, and Cdt1. Although different Cdc6 and Cdt1 molecules load each helicase, whether two ORC proteins are required is unclear. Using colocalization single-molecule spectroscopy combined with FRET, we investigated interactions between ORC and Mcm2‑7 during helicase loading. In the large majority of events, we observed a single ORC molecule recruiting both Mcm2‑7/Cdt1 complexes via similar interactions that end upon Cdt1 release. Between first and second helicase recruitment, a rapid change in interactions between ORC and the first Mcm2-7 occurs. Within seconds, ORC breaks the interactions mediating first Mcm2-7 recruitment, releases from its initial DNA-binding site, and forms a new interaction with the opposite face of the first Mcm2-7. This rearrangement requires release of the first Cdt1 and tethers ORC as it flips over the first Mcm2-7 to form an inverted Mcm2‑7-ORC-DNA complex required for second-helicase recruitment. To ensure correct licensing, this complex is maintained until head-to-head interactions between the two helicases are formed. Our findings reconcile previous observations and reveal a highly-coordinated series of events through which a single ORC molecule can load two oppositely-oriented helicases.

A Helicase-tethered ORC Flip Enables Bidirectional Helicase Loading

Replication origins are licensed by loading two Mcm2-7 helicases around DNA in a head-to-head conformation poised to initiate bidirectional replication. This process requires ORC, Cdc6, and Cdt1. Although different Cdc6 and Cdt1 molecules load each helicase, whether two ORC proteins are required is unclear. Using colocalization single-molecule spectroscopy combined with FRET, we investigated interactions between ORC and Mcm2-7 during helicase loading. We demonstrate that a single ORC molecule can recruit both Mcm2-7/Cdt1 complexes via similar interactions that end upon Cdt1 release. Between the first and second helicase recruitment, we observe a rapid change in interactions between ORC and the first Mcm2-7. In quick succession ORC breaks the interactions mediating first Mcm2-7 recruitment, releases from its initial DNA-binding site, and forms a new interaction with the opposite face of the first Mcm2-7. This rearrangement requires release of the first Cdt1 and tethers ORC as it flips over the first Mcm2-7 to form an inverted Mcm2-7-ORC-DNA complex required for second-helicase recruitment. To ensure correct licensing, this complex is maintained until head-to-head interactions between the two helicases are formed. Our findings reconcile previous observations and reveal a highly-coordinated series of events through which a single ORC molecule can load two oppositely-oriented helicases.

Trajectory Stress and Angle Values Correlation to Deep Beams Dimensional Ratio

SNI 2847-2019 defines a deep beam as a structural component that is loaded on one side and supported on the opposite face, allowing compressive components such as struts to form between the loads and supports. It is also stated that the ratio of the net span of the beam to the height of the beam must match the standards (l/h) 4. The goal of this investigation is to determine the value of the stress’s correlation and the trajectory angle to the ratio of the l/beam h's with a span of 4 meters when subjected to a point load of P = 2,000 kN. In the analysis procedure, SAP v.14 is being used to determine the value of stresses and trajectory angles of variations l/h. The results obtained from this study is ratio of l / h of deep beam affects the magnitude of the stress and the angle of the trajectory. Increasingly the width of the beam has no significant effect on the resulting trajectory angle.

Genetic and structural basis for recognition of SARS-CoV-2 spike protein by a two-antibody cocktail

The SARS-CoV-2 pandemic has led to an urgent need to understand the molecular basis for immune recognition of SARS-CoV-2 spike (S) glycoprotein antigenic sites. To define the genetic and structural basis for SARS-CoV-2 neutralization, we determined the structures of two human monoclonal antibodies COV2-2196 and COV2-21301, which form the basis of the investigational antibody cocktail AZD7442, in complex with the receptor binding domain (RBD) of SARS-CoV-2. COV2-2196 forms an “aromatic cage” at the heavy/light chain interface using germline-encoded residues in complementarity determining regions (CDRs) 2 and 3 of the heavy chain and CDRs 1 and 3 of the light chain. These structural features explain why highly similar antibodies (public clonotypes) have been isolated from multiple individuals1–4. The structure of COV2-2130 reveals that an unusually long LCDR1 and HCDR3 make interactions with the opposite face of the RBD from that of COV2-2196. Using deep mutational scanning and neutralization escape selection experiments, we comprehensively mapped the critical residues of both antibodies and identified positions of concern for possible viral escape. Nonetheless, both COV2-2196 and COV2130 showed strong neutralizing activity against SARS-CoV-2 strain with recent variations of concern including E484K, N501Y, and D614G substitutions. These studies reveal germline-encoded antibody features enabling recognition of the RBD and demonstrate the activity of a cocktail like AZD7442 in preventing escape from emerging variant viruses.

Influence of friction in a case of impact simulation

This paper presents an analysis of two cases, simulating the impact of a cylindical projectile on a perfectly rigid plate. One case is run without friction, the second one is run taking into account a friction coeficient between the plate and the projectile. The authors used for the projectile the same material constitutive model for both cases, based on experimental data and model developed by Johnson and Cook. Here, the comparing criteria were the maximum value of von Mises stress, the velocity and acceleration of the central point on the opposite face to the contact face of the projectile. Introducing friction, the simulation is more realistic. Taking into account friction, the projectile is less deformed and there was no edge breakage, at the same time moment.

Determinants for forming a supramolecular myelin-like proteolipid lattice

Myelin protein P2 is a peripheral membrane protein of the fatty acid binding protein family. It functions in the formation and maintenance of the peripheral nerve myelin sheath, and several P2 mutations causing human Charot-Marie-Tooth neuropathy have been reported. Here, electron cryomicroscopy of myelin-like proteolipid multilayers revealed a three-dimensionally ordered lattice of P2 molecules between stacked lipid bilayers, visualizing its possible assembly at the myelin major dense line. A single layer of P2 is inserted between two bilayers in a tight intermembrane space of ∼3 nm, implying direct interactions between P2 and two membrane surfaces. Further details on lateral protein organization were revealed through X-ray diffraction from bicelles stacked by P2. Surface mutagenesis of P2 coupled to structural and functional experiments revealed a role for both the portal region and the opposite face of P2 in membrane interactions. Atomistic molecular dynamics simulations of P2 on myelin-like and model membrane surfaces suggested that Arg88 is an important residue for P2-membrane interactions, in addition to the helical lid domain on the opposite face of the molecule. Negatively charged myelin lipid headgroups anchor P2 stably on the bilayer surface. Membrane binding may be accompanied by opening of the P2 β barrel structure and ligand exchange with the apposing lipid bilayer. Our results provide an unprecedented view into an ordered, multilayered biomolecular membrane system induced by the presence of a peripheral membrane protein from human myelin. This is an important step towards deciphering the 3-dimensional assembly of a mature myelin sheath at the molecular level.

Architecture of Bacterial Promoters; The case of the Escherichia coli ogt Promoter

<p class="1Body">All bacteria utilize RNA polymerase enzyme and transcription activator proteins to regulate gene expression in response to internal or external stress. Some bacterial promoters are regulated with only one transcription factor whilst two or more transcription activators regulate some other promoters. NarL is a transcription activator protein that activates the <em>E. coli yeaR</em> and <em>ogt</em> promoters in response to nitrate and nitrite induction in absence of oxygen. In the present study we have studied <em>ogt1052</em> promoter, which is a derivative of <em>ogt</em> promoter containing only one NarL binding site very close to -35 element. Therefore, it is considered as class II activator dependent promoter just as <em>yeaR</em> promoter. A molecular structure of <em>ogt1052</em> promoter was proposed which suggests that NarL binding site is located in opposite face of DNA that contains Alpha-CTD and sigma domain 4 of RNA polymerase enzyme required for promoter recognition. The aim of the present study was to study and test the suggested molecular model by creating point mutations at -35 element and deletion of one base pair in spacer region, to test whether sigma domain 4 is necessary to bind -35 hexamer in order to start transcription initiation, and to test whether NarL activates the promoter by interaction with Alpha-CTD in the opposite face of the DNA. Based on the result achieved, <em>ogt1052</em> promoter is a class I promoter “dressed” like a class II promoter.</p>

An antibody that prevents serpin polymerisation acts by inducing a novel allosteric behaviour

Serpins are important regulators of proteolytic pathways with an antiprotease activity that involves a conformational transition from a metastable to a hyperstable state. Certain mutations permit the transition to occur in the absence of a protease; when associated with an intermolecular interaction, this yields linear polymers of hyperstable serpin molecules, which accumulate at the site of synthesis. This is the basis of many pathologies termed the serpinopathies. We have previously identified a monoclonal antibody (mAb4B12) that, in single-chain form, blocks α1-antitrypsin (α1-AT) polymerisation in cells. Here, we describe the structural basis for this activity. The mAb4B12 epitope was found to encompass residues Glu32, Glu39 and His43 on helix A and Leu306 on helix I. This is not a region typically associated with the serpin mechanism of conformational change, and correspondingly the epitope was present in all tested structural forms of the protein. Antibody binding rendered β-sheet A — on the opposite face of the molecule — more liable to adopt an ‘open’ state, mediated by changes distal to the breach region and proximal to helix F. The allosteric propagation of induced changes through the molecule was evidenced by an increased rate of peptide incorporation and destabilisation of a preformed serpin–enzyme complex following mAb4B12 binding. These data suggest that prematurely shifting the β-sheet A equilibrium towards the ‘open’ state out of sequence with other changes suppresses polymer formation. This work identifies a region potentially exploitable for a rational design of ligands that is able to dynamically influence α1-AT polymerisation.

Architecture and RNA binding of the human negative elongation factor

Transcription regulation in metazoans often involves promoter-proximal pausing of RNA polymerase (Pol) II, which requires the 4-subunit negative elongation factor (NELF). Here we discern the functional architecture of human NELF through X-ray crystallography, protein crosslinking, biochemical assays, and RNA crosslinking in cells. We identify a NELF core subcomplex formed by conserved regions in subunits NELF-A and NELF-C, and resolve its crystal structure. The NELF-AC subcomplex binds single-stranded nucleic acids in vitro, and NELF-C associates with RNA in vivo. A positively charged face of NELF-AC is involved in RNA binding, whereas the opposite face of the NELF-AC subcomplex binds NELF-B. NELF-B is predicted to form a HEAT repeat fold, also binds RNA in vivo, and anchors the subunit NELF-E, which is confirmed to bind RNA in vivo. These results reveal the three-dimensional architecture and three RNA-binding faces of NELF.