The Effect of Active Site Mutations on the Homodimeric Behavior of the PvuII Restriction Endonuclease

The PvuII restriction endonuclease, a homodimer of two 18 kDa subunits, belongs to the type II family of restriction enzymes. Located in the active site of PvuII, Tyrosine 94 has previously been shown to be involved in the metal ion binding by the enzyme. The profile of the Ca2+ dependence of the DNA binding to the Y94F variant is shown to be clearly biphasic. The application of a sequential binding model yielded a coupling energy (ΔGcoop) at -0.54 for the upper phase and -1.15 kcal/mole for the lower phase. The similar metal binding pattern between the Y94F and the WT PvuII for Mg2+, Ca2+, Tb3+ and Eu3+ in the absence of DNA is also shown. Through 1H-15N HSQC spectroscopy and chemical denaturation of the Y94F variant the conformational impact of Tyr94 is confirmed. The Y94F slightly repositions the metal ions in the active site of PvuII affecting the intra and/or inter-subunit interactions among the metal binding sites. The Single chain (SC) PvuII bearing a covalent linker between the two subunits is utilized in the exploration of the modes of cooperativity among the metal binding sites. The heterodimeric WT|E68A-SC PvuII was prepared and studied in parallel to the WT-SC homodimer. Global analysis of DNA binding isotherms at different Ca2+ concentrations for the WT|E68A-SC variant returned an intra-subunit ΔGcoop at ?1.7 and -2.3 kcal/mole in the absence and presence of DNA, respectively. Combined with similar analysis for the WT-SC variant, the inter-subunit ΔGcoop values are shown at -1.1 and -3.1 kcal/mole. It is shown that the effect of Ca2+ ions on DNA binding is greater than the effect of the DNA on the affinity for Ca2+ ions. The cleavage of plasmid DNA under single turnover conditions reveals a similar dependence of the nicking and linearization rates on the concentration of Mg2+ ions for the WT-SC and the WT|E68A-SC PvuII. The series of events leading to the linear product (DNA association, nicking, release of the intermediate, re-association and linearization) with Mg2+ ions in one PvuII subunit is not slower than the synchronized double strand cleavage with both PvuII subunits bearing Mg2+ ions

Intermembrane crosstalk drives inner membrane protein organization in Escherichia coli

Gram-negative bacteria depend on energised protein complexes that connect the two membranes of the cell envelope. However, β-barrel outer-membrane proteins (OMPs) and α-helical inner-membrane proteins (IMPs) display quite different organisation. OMPs cluster into islands that restrict their lateral mobility, while IMPs generally diffuse throughout the cell. Here, using live cell imaging of Escherichia coli, we demonstrate that when transient, energy-dependent transmembrane connections are formed, IMPs become subjugated by the inherent organisation of OMPs and that such connections impact IMP function. We show that while establishing a translocon for import, the colicin ColE9 sequesters the IMPs of the proton motive force (PMF)-linked Tol-Pal complex into islands mirroring those of colicin-bound OMPs. Through this imposed organisation, the bacteriocin subverts the outer-membrane stabilising role of Tol-Pal, blocking its recruitment to cell division sites and slowing membrane constriction. The ordering of IMPs by OMPs via an energised inter-membrane bridge represents an emerging functional paradigm in cell envelope biology

A chicken bioreactor for efficient production of functional cytokines

The global market for protein drugs has the highest compound annual growth rate of any pharmaceutical class but their availability, especially outside of the US market, is compromised by the high cost of manufacture and validation compared to traditional chemical drugs. Improvements in transgenic technologies allow valuable proteins to be produced by genetically-modified animals; several therapeutic proteins from such animal bioreactors are already on the market after successful clinical trials and regulatory approval. Chickens have lagged behind mammals in bioreactor development, despite a number of potential advantages, due to the historic difficulty in producing transgenic birds, but the production of therapeutic proteins in egg white of transgenic chickens would substantially lower costs across the entire production cycle compared to traditional cell culture-based production systems. This could lead to more affordable treatments and wider markets, including in developing countries and for animal health applications. Here we report the efficient generation of new transgenic chicken lines to optimize protein production in eggs. As proof-of-concept, we describe the expression, purification and functional characterization of three pharmaceutical proteins, the human cytokine interferon α2a and two species-specific Fc fusions of the cytokine CSF1. Our work optimizes and validates a transgenic chicken system for the cost-effective production of pure, high quality, biologically active protein for therapeutics and other applications

ELGAR—a European Laboratory for Gravitation and Atom-interferometric Research

Gravitational waves (GWs) were observed for the first time in 2015, one century after Einstein predicted their existence. There is now growing interest to extend the detection bandwidth to low frequency. The scientific potential of multi-frequency GW astronomy is enormous as it would enable to obtain a more complete picture of cosmic events and mechanisms. This is a unique and entirely new opportunity for the future of astronomy, the success of which depends upon the decisions being made on existing and new infrastructures. The prospect of combining observations from the future space-based instrument LISA together with third generation ground based detectors will open the way toward multi-band GW astronomy, but will leave the infrasound (0.1–10 Hz) band uncovered. GW detectors based on matter wave interferometry promise to fill such a sensitivity gap. We propose the European Laboratory for Gravitation and Atom-interferometric Research (ELGAR), an underground infrastructure based on the latest progress in atomic physics, to study space–time and gravitation with the primary goal of detecting GWs in the infrasound band. ELGAR will directly inherit from large research facilities now being built in Europe for the study of large scale atom interferometry and will drive new pan-European synergies from top research centers developing quantum sensors. ELGAR will measure GW radiation in the infrasound band with a peak strain sensitivity of $3.3{\times}1{0}^{-22}/\sqrt{\text{Hz}}$ at 1.7 Hz. The antenna will have an impact on diverse fundamental and applied research fields beyond GW astronomy, including gravitation, general relativity, and geology.AB acknowledges support from the ANR (project EOSBECMR), IdEx Bordeaux—LAPHIA (project OE-TWR), theQuantERA ERA-NET (project TAIOL) and the Aquitaine Region (projets IASIG3D and USOFF).XZ thanks the China Scholarships Council (No. 201806010364) program for financial support. JJ thanks ‘AssociationNationale de la Recherche et de la Technologie’ for financial support (No. 2018/1565).SvAb, NG, SL, EMR, DS, and CS gratefully acknowledge support by the German Space Agency (DLR) with funds provided by the Federal Ministry for Economic Affairs and Energy (BMWi) due to an enactment of the German Bundestag under Grants No. DLR∼50WM1641 (PRIMUS-III), 50WM1952 (QUANTUS-V-Fallturm), and 50WP1700 (BECCAL), 50WM1861 (CAL), 50WM2060 (CARIOQA) as well as 50RK1957 (QGYRO)SvAb, NG, SL, EMR, DS, and CS gratefully acknowledge support by ‘Niedersächsisches Vorab’ through the ‘Quantum- and Nano-Metrology (QUANOMET)’ initiative within the project QT3, and through ‘Förderung von Wissenschaft und Technik in Forschung und Lehre’ for the initial funding of research in the new DLR-SI Institute, the CRC 1227 DQ-mat within the projects A05 and B07DS gratefully acknowledges funding by the Federal Ministry of Education and Research (BMBF) through the funding program Photonics Research Germany under contract number 13N14875.RG acknowledges Ville de Paris (Emergence programme HSENS-MWGRAV), ANR (project PIMAI) and the Fundamental Physics and Gravitational Waves (PhyFOG) programme of Observatoire de Paris for support. We also acknowledge networking support by the COST actions GWverse CA16104 and AtomQT CA16221 (Horizon 2020 Framework Programme of the European Union).The work was also supported by the German Space Agency (DLR) with funds provided by the Federal Ministry for Economic Affairs and Energy (BMWi) due to an enactment of the German Bundestag under Grant Nos.∼50WM1556, 50WM1956 and 50WP1706 as well as through the DLR Institutes DLR-SI and DLR-QT.PA-S, MN, and CFS acknowledge support from contracts ESP2015-67234-P and ESP2017-90084-P from the Ministry of Economy and Business of Spain (MINECO), and from contract 2017-SGR-1469 from AGAUR (Catalan government).SvAb, NG, SL, EMR, DS, and CS gratefully acknowledge support by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy—EXC-2123 QuantumFrontiers—390837967 (B2) andCRC1227 ‘DQ-mat’ within projects A05, B07 and B09.LAS thanks Sorbonne Universités (Emergence project LORINVACC) and Conseil Scientifique de l'Observatoire de Paris for funding.This work was realized with the financial support of the French State through the ‘Agence Nationale de la Recherche’ (ANR) in the frame of the ‘MRSEI’ program (Pre-ELGAR ANR-17-MRS5-0004-01) and the ‘Investissement d'Avenir’ program (Equipex MIGA: ANR-11-EQPX-0028, IdEx Bordeaux—LAPHIA: ANR-10-IDEX-03-02).Peer Reviewe

Consequences of inducing intrinsic disorder in a high-affinity protein-protein interaction

The kinetic and thermodynamic consequences of intrinsic disorder in protein-protein recognition are controversial. We address this by inducing one partner of the high-affinity colicin E3 rRNase domain-Im3 complex (Kd ≈ 10(-12) M) to become an intrinsically disordered protein (IDP). Through a variety of biophysical measurements, we show that a single alanine mutation at Tyr507 within the hydrophobic core of the isolated colicin E3 rRNase domain causes the enzyme to become an IDP (E3 rRNase(IDP)). E3 rRNase(IDP) binds stoichiometrically to Im3 and forms a structure that is essentially identical to the wild-type complex. However, binding of E3 rRNase(IDP) to Im3 is 4 orders of magnitude weaker than that of the folded rRNase, with thermodynamic parameters reflecting the disorder-to-order transition on forming the complex. Critically, pre-steady-state kinetic analysis of the E3 rRNase(IDP)-Im3 complex demonstrates that the decrease in affinity is mostly accounted for by a drop in the electrostatically steered association rate. Our study shows that, notwithstanding the advantages intrinsic disorder brings to biological systems, this can come at severe kinetic and thermodynamic cost

Structure and function of the escherichia coli Tol-Pal stator protein TolR

TolR is a 15-kDa inner membrane protein subunit of the Tol-Pal complex in Gram-negative bacteria, and its function is poorly understood. Tol-Pal is recruited to cell division sites where it is involved in maintaining the integrity of the outer membrane. TolR is related to MotB, the peptidoglycan (PG)-binding stator protein from the flagellum, suggesting it might serve a similar role in Tol-Pal. The only structure thus far reported for TolR is of the periplasmic domain from Haemophilus influenzae in which N- and C-terminal residues had been deleted (TolR(62–133), Escherichia coli numbering). H. influenzae TolR(62–133) is a symmetrical dimer with a large deep cleft at the dimer interface. Here, we present the 1.7-Å crystal structure of the intact periplasmic domain of E. coli TolR (TolR(36–142)). E. coli TolR(36–142) is also dimeric, but the architecture of the dimer is radically different from that of TolR(62–133) due to the intertwining of its N and C termini. TolR monomers are rotated ∼180° relative to each other as a result of this strand swapping, obliterating the putative PG-binding groove seen in TolR(62–133). We found that removal of the strand-swapped regions (TolR(60–133)) exposes cryptic PG binding activity that is absent in the full-length domain. We conclude that to function as a stator in the Tol-Pal complex dimeric TolR must undergo large scale structural remodeling reminiscent of that proposed for MotB, where the N- and C-terminal sequences unfold in order for the protein to both reach and bind the PG layer ∼90 Å away from the inner membrane