Molecular basis of a redox switch: molecular dynamics simulations and surface plasmon resonance provide insight into reduced and oxidised angiotensinogen.
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ABSTRACT:
SUBMITTER: Crowther JM
PROVIDER: S-EPMC7612104 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Blood pressure is critically controlled by angiotensins, which are vasopressor peptides specifically released by the enzyme renin from the tail of angiotensinogen-a non-inhibitory member of the serpin family of protease inhibitors. Although angiotensinogen has long been regarded as a passive substrate, the crystal structures solved here to 2.1 Å resolution show that the angiotensin cleavage site is inaccessibly buried in its amino-terminal tail. The conformational rearrangement that makes this site accessible for proteolysis is revealed in our 4.4 Å structure of the complex of human angiotensinogen with renin. The co-ordinated changes involved are seen to be critically linked by a conserved but labile disulphide bridge. Here we show that the reduced unbridged form of angiotensinogen is present in the circulation in a near 40:60 ratio with the oxidized sulphydryl-bridged form, which preferentially interacts with receptor-bound renin. We propose that this redox-responsive transition of angiotensinogen to a form that will more effectively release angiotensin at a cellular level contributes to the modulation of blood pressure. Specifically, we demonstrate the oxidative switch of angiotensinogen to its more active sulphydryl-bridged form in the maternal circulation in pre-eclampsia-the hypertensive crisis of pregnancy that threatens the health and survival of both mother and child.
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Project description:BackgroundPQS (PseudomonasQuinolone Signal) and its precursor HHQ are signal molecules of the P. aeruginosa quorum sensing system. They explicate their role in mammalian pathogenicity by binding to the receptor PqsR that induces virulence factor production and biofilm formation. The enzyme PqsD catalyses the biosynthesis of HHQ.ResultsEnzyme kinetic analysis and surface plasmon resonance (SPR) biosensor experiments were used to determine mechanism and substrate order of the biosynthesis. Comparative analysis led to the identification of domains involved in functionality of PqsD. A kinetic cycle was set up and molecular dynamics (MD) simulations were used to study the molecular bases of the kinetics of PqsD. Trajectory analysis, pocket volume measurements, binding energy estimations and decompositions ensured insights into the binding mode of the substrates anthraniloyl-CoA and β-ketodecanoic acid.ConclusionsEnzyme kinetics and SPR experiments hint at a ping-pong mechanism for PqsD with ACoA as first substrate. Trajectory analysis of different PqsD complexes evidenced ligand-dependent induced-fit motions affecting the modified ACoA funnel access to the exposure of a secondary channel. A tunnel-network is formed in which Ser317 plays an important role by binding to both substrates. Mutagenesis experiments resulting in the inactive S317F mutant confirmed the importance of this residue. Two binding modes for β-ketodecanoic acid were identified with distinct catalytic mechanism preferences.
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Project description:The small heat shock proteins (sHSPs) are a virtually ubiquitous and diverse group of molecular chaperones that can bind and protect unfolding proteins from irreversible aggregation. It has been suggested that intrinsic disorder of the N-terminal arm (NTA) of sHSPs is important for substrate recognition. To investigate conformations of the NTA that could recognize substrates we performed replica exchange molecular dynamics simulations. Behavior at normal and stress temperatures of the dimeric building blocks of dodecameric HSPs from wheat (Ta16.9) and pea (Ps18.1) were compared because they display high sequence similarity, but Ps18.1 is more efficient in binding specific substrates. In our simulations, the NTAs of the dimer are flexible and dynamic; however, rather than exhibiting highly extended conformations they retain considerable α-helical character and contacts with the conserved α-crystallin domain (ACD). Network analysis and clustering methods reveal that there are two major conformational forms designated either "open" or "closed" based on the relative position of the two NTAs and their hydrophobic solvent accessible surface area. The equilibrium constant for the closed to open transition is significantly different for Ta16.9 and Ps18.1, with the latter showing more open conformations at elevated temperature correlated with its more effective chaperone activity. In addition, the Ps18.1 NTAs have more hydrophobic solvent accessible surface than those of Ta16.9. NTA hydrophobic patches are comparable in size to the area buried in many protein-protein interactions, which would enable sHSPs to bind early unfolding intermediates. Reduced interactions of the Ps18.1 NTAs with each other and with the ACD contribute to the differences in dynamics and hydrophobic surface area of the two sHSPs. These data support a major role for the conformational equilibrium of the NTA in substrate binding and indicate features of the NTA that contribute to sHSP chaperone efficiency.
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Project description:Transferrin is an attractive candidate for drug delivery due to its ability to cross the blood brain barrier. However, in order to be able to use it for therapeutic purposes, it is important to investigate how its stability depends on different formulation conditions. Combining high-throughput thermal and chemical denaturation studies with small angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations, it was possible to connect the stability of transferrin with its conformational changes. Lowering pH induces opening of the transferrin N-lobe, which results in a negative effect on the stability. Presence of NaCl or arginine at low pH enhances the opening and has a negative impact on the overall protein stability. Statement of Significance:Protein-based therapeutics have become an essential part of medical treatment. They are highly specific, have high affinity and fewer off-target effects. However, stabilization of proteins is critical, time-consuming, and expensive, and it is not yet possible to predict the behavior of proteins under different conditions. The current work is focused on a molecular understanding of the stability of human serum transferrin; a protein which is abundant in blood serum, may pass the blood brain barrier and therefore with high potential in drug delivery. Combination of high throughput unfolding techniques and structural studies, using small angle X-ray scattering and molecular dynamic simulations, allows us to understand the behavior of transferrin on a molecular level.
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Project description:Here, we report the 1.53-Å crystal structure of the enzyme 7-cyano-7-deazaguanine reductase (QueF) from Vibrio cholerae, which is responsible for the complete reduction of a nitrile (CN) bond to a primary amine (H(2)C-NH(2)). At present, this is the only example of a biological pathway that includes reduction of a nitrile bond, establishing QueF as particularly noteworthy. The structure of the QueF monomer resembles two connected ferrodoxin-like domains that assemble into dimers. Ligands identified in the crystal structure suggest the likely binding conformation of the native substrates NADPH and 7-cyano-7-deazaguanine. We also report on a series of numerical simulations that have shed light on the mechanism by which this enzyme affects the transfer of four protons (and electrons) to the 7-cyano-7-deazaguanine substrate. In particular, the simulations suggest that the initial step of the catalytic process is the formation of a covalent adduct with the residue Cys194, in agreement with previous studies. The crystal structure also suggests that two conserved residues (His233 and Asp102) play an important role in the delivery of a fourth proton to the substrate.
| S-EPMC3366508 | biostudies-literature
Project description:The correlation of electron transfer with molecular conductance (g: electron transport through single molecules) by Nitzan and others has contributed to a fundamental understanding of single-molecule electronic materials. When an unsymmetric, dipolar molecule spans two electrodes, the possibility exists for different conductance values at equal, but opposite electrode biases. In the device configuration, these molecules serve as rectifiers of the current and the efficiency of the device is given by the rectification ratio (RR = gforward/greverse). Experimental determination of the RR is challenging since the orientation of the rectifying molecule with respect to the electrodes and with respect to the electrode bias direction is difficult to establish. Thus, while two different values of g can be measured and a RR calculated, one cannot easily assign each conductance value as being aligned with or opposed to the molecular dipole, and calculations are often required to resolve the uncertainty. Herein, we describe the properties of two isomeric, triplet ground state biradical molecules that serve as constant-bias analogs of single-molecule electronic devices. Through established theoretical relationships between g and electronic coupling, H2, and between H2 and magnetic exchange coupling, J (g ? H2 ? J), we use the ratio of experimental J-values for our two isomers to calculate a RR for an unsymmetric bridge molecule with known geometry relative to the two radical fragments of the molecule and at a spectroscopically-defined potential bias. Our experimental results are compared with device transport calculations.
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Project description:AimsThe ferric uptake regulator (Fur) is the main transcriptional regulator of genes involved in iron homeostasis in most prokaryotes. FurA from Anabaena sp. PCC 7120 contains five cysteine residues, four of them arranged in two redox-active CXXC motifs. The protein needs not only metal but also reducing conditions to remain fully active in vitro. Through a mutational study of the cysteine residues present in FurA, we have investigated their involvement in metal and DNA binding.ResultsResidue C101 that belongs to a conserved CXXC motif plays an essential role in both metal and DNA binding activities in vitro. Substitution of C101 by serine impairs DNA and metal binding abilities of FurA. Isothermal titration calorimetry measurements show that the redox state of C101 is responsible for the protein ability to coordinate the metal corepressor. Moreover, the redox state of C101 varies with the presence or absence of C104 or C133, suggesting that the environments of these cysteines are mutually interdependent.InnovationWe propose that C101 is part of a thiol/disulfide redox switch that determines FurA ability to bind the metal corepressor.ConclusionThis mechanism supports a novel feature of a Fur protein that emerges as a regulator, which connects the response to changes in the intracellular redox state and iron management in cyanobacteria. Antioxid. Redox Signal. 00, 000-000.
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Project description:The L,L-diaminopimelate aminotransferase (DapL) pathway, a recently discovered variant of the lysine biosynthetic pathway, is an attractive pipeline to identify targets for the development of novel antibiotic compounds. DapL is a homodimer that catalyzes the conversion of tetrahydrodipicolinate to L,L-diaminopimelate in a single transamination reaction. The penultimate and ultimate products of the lysine biosynthesis pathway, meso-diaminopimelate and lysine, are key components of the Gram-negative and Gram-positive bacterial peptidoglycan cell wall. Humans are not able to synthesize lysine, and DapL has been identified in 13% of bacteria whose genomes have been sequenced and annotated to date, thus it is an attractive target for the development of narrow spectrum antibiotics through the prevention of both lysine biosynthesis and peptidoglycan crosslinking. To address the common lack of structural information when conducting compound screening experiments and provide support for the use of modeled structures, our analyses utilized inferred structures from related homologous enzymes. Using a comprehensive and comparative molecular dynamics (MD) software package-DROIDS (Detecting Relative Outlier Impacts in Dynamic Simulations) 2.0, we investigated the binding dynamics of four previously identified antagonistic ligands of DapL from Verrucomicrobium spinosum, a non-pathogenic relative of Chlamydia trachomatis. Here, we present putative docking positions of the four ligands and provide confirmatory comparative molecular dynamics simulations supporting the conformations. The simulations performed in this study can be applied to evaluate putative targets to predict compound effectiveness prior to in vivo and in vitro experimentation. Moreover, this approach has the potential to streamline the process of antibiotic development.
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