Molecular Mechanism Of Coupling Research Articles

Enhancing aggregation of microalgae on polystyrene microplastics by high light: Processes, drivers, and environmental risk assessment

Microplastics (MPs) are emerging pollutants, causing potential threats to aquatic ecosystems and serious concern in aggregating with microalgae (critical primary producers). When entering water bodies, MPs are expected to sink below the water surface and disperse into varying water compartments with different light intensities. However, how light influences the aggregation processes of algal cells onto MPs and the associated molecular coupling mechanisms and derivative risks remain poorly understood. Herein, we investigated the aggregation behavior between polystyrene microplastics (mPS, 10 µm) and Chlorella pyrenoidosa under low (LL, 15 μmol·m−2·s−1), normal (NL, 55 μmol·m−2·s−1), and high light (HL, 150 μmol·m−2·s−1) conditions from integrated in vivo and in silico assays. The results indicated that under LL, the mPS particles primarily existed independently, whereas under NL and HL, C. pyrenoidosa tightly bounded to mPS by secreting more protein-rich extracellular polymeric substances. Infrared spectroscopy analysis and density functional theory calculation revealed that the aggregation formation was driven by non-covalent interaction involving van der Waals force and hydrogen bond. These processes subsequently enhanced the deposition and adherence capacity of mPS and relieved its phytotoxicity. Overall, our findings advance the practical and theoretical understanding of the ecological impacts of MPs in complex aquatic environments.

Mechanisms of Energy Transduction by Charge Translocating Membrane Proteins.

Life relies on the constant exchange of different forms of energy, i.e., on energy transduction. Therefore, organisms have evolved in a way to be able to harvest the energy made available by external sources (such as light or chemical compounds) and convert these into biological useable energy forms, such as the transmembrane difference of electrochemical potential (Δμ̃). Membrane proteins contribute to the establishment of Δμ̃ by coupling exergonic catalytic reactions to the translocation of charges (electrons/ions) across the membrane. Irrespectively of the energy source and consequent type of reaction, all charge-translocating proteins follow two molecular coupling mechanisms: direct- or indirect-coupling, depending on whether the translocated charge is involved in the driving reaction. In this review, we explore these two coupling mechanisms by thoroughly examining the different types of charge-translocating membrane proteins. For each protein, we analyze the respective reaction thermodynamics, electron transfer/catalytic processes, charge-translocating pathways, and ion/substrate stoichiometries.

Contact Ion Pairs of Phosphate Groups in Water: Two-Dimensional Infrared Spectroscopy of Dimethyl Phosphate and ab Initio Simulations.

The interaction of phosphate groups with ions in an aqueous environment has a strong impact on the structure and folding processes of DNA and RNA. The dynamic variety of ionic arrangements, including both contact pairs and water separated ions, and the molecular coupling mechanisms are far from being understood. In a combined experimental and theoretical approach, we address the properties of contact ion pairs of the prototypical system dimethyl phosphate with Na+, Ca2+, and Mg2+ ions in water. Linear and femtosecond two-dimensional infrared (2D-IR) spectroscopy of the asymmetric (PO2)- stretching vibration separates and characterizes the different species via their blue-shifted vibrational signatures and 2D-IR line shapes. Phosphate-magnesium contact pairs stand out as the most compact geometry while the contact pairs with Ca2+ and Na+ display a wider structural variation. Microscopic density functional theory simulations rationalize the observed frequency shifts and reveal distinct differences between the contact geometries.

Molecular mechanisms of coupling to voltage sensors in voltage-evoked cellular signals.

The voltage sensor domain (VSD) has long been studied as a unique domain intrinsic to voltage-gated ion channels (VGICs). Within VGICs, the VSD is tightly coupled to the pore-gate domain (PGD) in diverse ways suitable for its specific function in each physiological context, including action potential generation, muscle contraction and relaxation, hormone and neurotransmitter secretion, and cardiac pacemaking. However, some VSD-containing proteins lack a PGD. Voltage-sensing phosphatase contains a cytoplasmic phosphoinositide phosphatase with similarity to phosphatase and tensin homolog (PTEN). Hv1, a voltage-gated proton channel, also lacks a PGD. Within Hv1, the VSD operates as a voltage sensor, gate, and pore for both proton sensing and permeation. Hv1 has a C-terminal coiled coil that mediates dimerization for cooperative gating. Recent progress in the structural biology of VGICs and VSD proteins provides insights into the principles of VSD coupling conserved among these proteins as well as the hierarchy of protein organization for voltage-evoked cell signaling.

Nature and Value of Freely Dissolved EPS Ecosystem Services: Insight into Molecular Coupling Mechanisms for Regulating Metal Toxicity.

Extracellular polymeric substances (EPSs) dispersed in natural waters play a significant role in relieving impacts to microbial survival associated with heavy metal release, yet little is known about the association of freely dissolved EPS ecosystem services with metal transformation in natural waters. Here, we demonstrate that dispersive EPSs mitigate the metal toxicity to microbial cells through an associative coordination reaction. Microtitrimetry coupled with fluorescence spectroscopy ascribes the combination of freely dissolved EPSs from Escherichia coli (E. coli) with Cu2+/Cd2+ to a coordination reaction associated with chemical static quenching. Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and computational chemistry confirm that carboxyl residues in protein-like substances of the EPSs are responsible for the coordination. Frontier molecular orbitals (MOs) of a deprotonated carboxyl integrate with the occupied d orbitals of Cu2+ and/or d, s orbitals of Cd2+ to form metal-EPS complexes. Microcosmic systems show that because the metal-EPS complexes decrease cellular absorbability of metals, E. coli survivals increase by 4.3 times for Cu2+ and 1.6 times for Cd2+, respectively. Based on bonding energies for six metals-EPS coordination, an associative toxic effect further confirms that increased bonding energies facilitate retardation of metals in the EPS matrix, protecting against E. coli apoptosis.

Allosteric Modulation of Intact γ-Secretase Structural Dynamics

As a protease complex involved in the cleavage of amyloid precursor proteins that lead to the formation of amyloid β fibrils implicated in Alzheimer's disease, γ-secretase is an important target for developing therapeutics against Alzheimer's disease. γ-secretase is composed of four subunits: nicastrin (NCT) in the extracellular (EC) domain, presenilin-1 (PS1), anterior pharynx defective 1, and presenilin enhancer 2 in the transmembrane (TM) domain. NCT and PS1 play important roles in binding amyloid β precursor proteins and modulating PS1 catalytic activity. Yet, the molecular mechanisms of coupling between substrate/modulator binding and catalytic activity remain to be elucidated. Recent determination of intact human γ-secretase cryo-electron microscopy structure has opened the way for a detailed investigation of the structural dynamics of this complex. Our analysis, based on a membrane-coupled anisotropic network model, reveals two types of NCT motions, bending and twisting, with respect to PS1. These underlie the fluctuations between the “open” and “closed” states of the lid-like NCT with respect to a hydrophilic loop 1 (HL1) on PS1, thus allowing or blocking access of the substrate peptide (EC portion) to HL1 and to the neighboring helix TM2. In addition to this alternating access mechanism, fluctuations in the volume of the PS1 central cavity facilitate the exposure of the catalytic site for substrate cleavage. Druggability simulations show that γ-secretase presents several hot spots for either orthosteric or allosteric inhibition of catalytic activity, consistent with experimental data. In particular, a hinge region at the interface between the EC and TM domains, near the interlobe groove of NCT, emerges as an allo-targeting site that would impact the coupling between HL1/TM2 and the catalytic pocket, opening, to our knowledge, new avenues for structure-based design of novel allosteric modulators of γ-secretase protease activity.

Role of water and protein dynamics in proton pumping by respiratory complex I

Membrane bound respiratory complex I is the key enzyme in the respiratory chains of bacteria and mitochondria, and couples the reduction of quinone to the pumping of protons across the membrane. Recently solved crystal or electron microscopy structures of bacterial and mitochondrial complexes have provided significant insights into the electron and proton transfer pathways. However, due to large spatial separation between the electron and proton transfer routes, the molecular mechanism of coupling remains unclear. Here, based on atomistic molecular dynamics simulations performed on the entire structure of complex I from Thermus thermophilus, we studied the hydration of the quinone-binding site and the membrane-bound subunits. The data from simulations show rapid diffusion of water molecules in the protein interior, and formation of hydrated regions in the three antiporter-type subunits. An unexpected water-protein based connectivity between the middle of the Q-tunnel and the fourth proton channel is also observed. The protonation-state dependent dynamics of key acidic residues in the Nqo8 subunit suggest that the latter may be linked to redox-coupled proton pumping in complex I. We propose that in complex I the proton and electron transfer paths are not entirely separate, instead the nature of coupling may in part be ‘direct’.

State-Dependent Lipid Interactions Couple the Conformations of the Voltage-Sensing and Pore-Gate Domains

Coupling between the voltage-sensing domain (VSD) and pore-gate domain (PGD) is required for the voltage-dependent gating of ion channels, but the molecular mechanisms of coupling are unclear. Previous studies have identified protein-protein interactions that are important for coupling, while structural and recent functional data demonstrate that membrane lipids also play a role. In a recent study of Kv7.1, we found that phosphatidylinositol 4,5-bisphosphate (PIP2) binding at the VSD-PD interface is required to couple the activated-state of the VSD to the open-state of the PD. We devised a method to directly measure PIP2 mediated coupling and an allosteric framework for describing such coupling. These advances provide new tools to investigate the mechanisms of VSD-PGD coupling. We also identified a putative PIP2 binding site and found that mutations of residues within this site reduce PIP2-mediated coupling. Paradoxically, a set of mutations near the PIP2 binding site increased the macroscopic current. Using a combined computational and experimental approach to study these gain of function mutations, we now identify a PIP2-interaction that is preferred by the resting-closed channel. Using the KCNE1 accessory subunit as an experimental tool, we are able to resolve the functional effects of this resting-closed state interaction. These results allow us to propose a novel mechanism for voltage-dependent gating in which repositioning of cofactor lipids at the VSD-PD represents a critical step in the transitions between resting-closed and activated-open states. This model can be used to explain the phenomena of cooperativity and concerted motion in voltage-gated channels.

Arginine Movement in Synergistic Coupling of Substrate and Ion Binding to Glutamate Transporter Homologue

Glutamate transporters are membrane pumps, which catalyze reuptake of the neurotransmitter from the brain synapses driven primarily by symport of sodium ions. A bacterial homologue, GltPh is a sodium/aspartate symporter, for which the crystal structures of sodium and substrate bound states have been determined. The thermodynamic studies have demonstrated that binding of sodium ions and aspartate to the transporter are coupled, such that the affinity for the substrate is much higher in the presence of the ions and vice versa. However, the ions are not directly coordinated by the substrate and the molecular mechanism of coupling remains largely unknown. Arginine 397 interacts with the side chain carboxylate of the substrate in the transporter crystal structures, is highly conserved among glutamate transporters, and has been shown in functional studies to be a key residue for substrate binding in mammalian homologues. To probe how the formation of the substrate-binding site affects sodium binding, we generated R397A mutant. We show that, as expected, R397A mutant has a low substrate affinity even in the presence of 100 mM sodium, measured by the isothermal titration calorimetry. Remarkably, in the absence of aspartate the sodium binding affinity for R397A is increased by ∼10 fold, compared to the wild type transporter, as measured by a fluorescence assay in vitro. Furthermore, substrate does not significantly increase the affinity of the mutant transporter for the ions. We suggest that arginine 397 in the wild type GltPh interferes with the sodium binding site or sites in the absence of the substrate and that its re-orientation upon substrate coordination relieves this interference. We hypothesize that these events are key to the mechanism of the allosteric coupling between substrate and ion binding.

Coupling of Ci-VSP Modules Requires a Combination of Structure and Electrostatics within the Linker

The voltage-sensitive phosphatase Ci-VSP consists of an intracellular phosphatase domain (PD) coupled to a transmembrane voltage-sensor domain (VSD). Depolarization triggers the selective dephosphorylation of phosphoinositides. However, the molecular mechanisms of coupling are still elusive. To clarify the role of the VSD-PD linker as a putative partner for electrostatic interactions with the membrane, we carried out a cysteine-scanning mutagenesis of the whole motif M240-K257. Upon coexpression with PI(4,5)P2-sensitive KCNQ2/KCNQ3 channels in Xenopus oocytes, we identified four positions (A242C, R245C, K252C, and Y255C) with a completely abrogated PD activity. Because the mutation effect occurred periodically, we hypothesize that α-helical elements exist within the linker, with a gap near position S249. The combination of these results with the analysis of transient sensing currents of the VSD revealed distinct roles for the N-terminal (M240-S249) and C-terminal (Q250-K257) linker motifs in the VSD-PD coupling. According to our functional results, the computational structure prediction of the Q239-D258 fragment confirmed α-helical structures within the linker, with a short β-turn around S249 in the activated conformation. Remarkably, the position K252 may be a candidate for interacting with the PD rather than for binding to the membrane. This provides the first insight (to our knowledge) into the direct intervention of the linker in the VSD-PD coupling process.

Dynamical Allosterism in the Mechanism of Action of DNA Mismatch Repair Protein MutS

The multidomain protein Thermus aquaticus MutS and its prokaryotic and eukaryotic homologs recognize DNA replication errors and initiate mismatch repair. MutS actions are fueled by ATP binding and hydrolysis, which modulate its interactions with DNA and other proteins in the mismatch-repair pathway. The DNA binding and ATPase activities are allosterically coupled over a distance of ∼70 Å, and the molecular mechanism of coupling has not been clarified. To address this problem, all-atom molecular dynamics simulations of ∼150 ns including explicit solvent were performed on two key complexes—ATP-bound and ATP-free MutS⋅DNA(+T bulge). We used principal component analysis in fluctuation space to assess ATP ligand-induced changes in MutS structure and dynamics. The molecular dynamics-calculated ensembles of thermally accessible structures showed markedly small differences between the two complexes. However, analysis of the covariance of dynamical fluctuations revealed a number of potentially significant interresidue and interdomain couplings. Moreover, principal component analysis revealed clusters of correlated atomic fluctuations linking the DNA and nucleotide binding sites, especially in the ATP-bound MutS⋅DNA(+T) complex. These results support the idea that allosterism between the nucleotide and DNA binding sites in MutS can occur via ligand-induced changes in motion, i.e., dynamical allosterism.

Metabotropic Glutamate Receptors as Novel Therapeutic Targets on Visceral Sensory Pathways

Metabotropic glutamate receptors (mGluR) have a diverse range of structures and molecular coupling mechanisms. There are eight mGluR subtypes divided into three major groups. Group I (mGluR1 and 5) is excitatory; groups II (mGluR2 and 3) and III (mGluR 4, 6, and 7) are inhibitory. All mGluR are found in the mammalian nervous system but some are absent from sensory neurons. The focus here is on mGluR in sensory pathways from the viscera, where they have been explored as therapeutic targets. Group I mGluR are activated by endogenous glutamate or constitutively active without agonist. Constitutive activity can be exploited by inverse agonists to reduce neuronal excitability without synaptic input. This is promising for reducing activation of nociceptive afferents and pain using mGluR5 negative allosteric modulators. Many inhibitory mGluR are also expressed in visceral afferents, many of which markedly reduce excitability. Their role in visceral pain remains to be determined, but they have shown promise in inhibition of the triggering of gastro-esophageal reflux, via an action on mechanosensory gastric afferents. The extent of reflux inhibition is limited, however, and may not reach a clinically useful level. On the other hand, negative modulation of mGluR5 has very potent actions on reflux inhibition, which has produced the most likely candidates so far as therapeutic drugs. These act probably outside the central nervous system, and may therefore provide a generous therapeutic window. There are many unanswered questions about mGluR along visceral afferent pathways, the answers to which may reveal many more therapeutic candidates.

Voltage and proton gradient sensing in Hv1 proton channels

Hv1 voltage-gated proton channels appear to conduct H+ through a voltage sensor domain (VSD) that is homologous to that found in voltage-dependent cation channels and phosphatases. A conserved S4 transmembrane helix that contains a series of at least three Arg residues is integral to the voltage sensing function of all VSD proteins. In contrast to other VSD-containing proteins, voltage-gated proton channels possess an additional unique biophysical property: coupling of the transmembrane pH gradient to voltage dependent activation. For both native voltage-gated H+ currents and expressed Hv1 channels, the apparent voltage threshold for H+ current activation (Vthr) shifts linearly ∼40 mV per log([H+]) over at least five pH units. The molecular mechanism of coupling between voltage and the pH gradients represents one of the central mysteries of proton channel function. What constitutes the pH sensor in proton channels and how does it interact with the voltage sensor?DeCoursey and colleagues previously proposed a model for H+ channel gating wherein protonation of discrete sites that are alternatively exposed to either the extra- or intra-cellular milieu regulates the voltage-dependence of channel opening (Cherny et al., 1996); the required first step in this model is deprotonation of an extracellular H+ binding site. In order to identify residues that mediate pH-dependent regulation of voltage sensitivity in Hv1, we performed site-directed mutagenesis to convert each of the candidate H+ acceptors in the Hv1 VSD to either neutral (alanine or asparagine), basic (arginine) or H+-titratable (histidine) amino acids. Mutant channels were expressed in HEK-293 cells and Vthr was determined under a variety of imposed pH gradients using whole-cell voltage clamp. Surprisingly, charge-neutralizing mutations failed to abrogate pH gradient sensing in Hv1. Our findings are interpreted in the context of the Cherny and DeCoursey model for proton channel gating.

Evaluation of a proposed mechanism of ligand-gated ion channel activation in the GABA A and glycine receptors

Ligand-gated ion channels (LGICs) mediate rapid chemical neurotransmission in the mammalian brain. This gene superfamily includes the nicotinic acetylcholine (nAChR), GABA A, 5-hydroxytryptamine type 3, and glycine receptors. Upon agonist binding these receptors undergo a rapid allosteric transition from the closed to open state. The molecular mechanism of coupling between agonist binding and channel gating remains poorly understood, in part due to the lack of a high-resolution structure of the entire receptor. Miyazawa, Fujiyoshi, and Unwin published a 4 Å resolution structure of the nAChR, and proposed that a single residue—valine 44 in Loop 2 of the extracellular domain—functions as a critical determinant of a “pin-into-socket” mechanism for receptor activation in nAChR. Here we examined whether this proposed “pin-into-socket” mechanism also contributes to channel activation in the GABA A and glycine receptors. We mutated residues corresponding to nAChR valine 44 in the GABA A (α 1 histidine 56 and β 2 valine 53) and glycine (α 1 threonine 54) receptors. The results obtained in this study do not support a simple “pin-into-socket” mechanism of activation for the activation of GABA A and glycine receptors. This conclusion is consistent with other recent reports in which mutations of residues distributed throughout several loops of nAChR, GABA A and glycine receptors had large effects on gating behavior.

Evolutionary epitopes of Hsp90 and p23: implications for their interaction.

The amino-terminal domain (N-domain) of Hsp90 represents the ATP binding site and is important for interaction with its cochaperone, p23. Whereas some evidence suggests that p23 may bind to this domain in an ATP-dependent manner and that this process requires the dimerization of two N-domains, the interaction sites between them and the molecular mechanism of coupling these two events to p23 binding remain unsolved. As a first step toward establishing the interaction mechanism, we used the evolutionary tracing (ET) method [Lichtarge, O., Bourne, H. R., and Cohen, F. E. (1996) J. Mol. Biol. 257, 342-358] to identify the putative functional surfaces of Hsp90 and p23, and combined with protein-protein docking techniques, to predict their binding interface. Both evolutionarily privileged surfaces of Hsp90 and p23 identified by ET appear on this putative interface. An analysis of the complex model produced using the ET results combined with available experimental data highlights a putative conformational pathway in the ATP binding domain of Hsp90, where a series of conformational changes transfer the ATP-induced N-domain dimerization signal for the binding of p23. In this pathway, the closure of "lid" may result in reorientation of the helix alpha1 and the following loop (residues 10-27 in yeast Hsp90), which will expose more hydrophobic surface, and thus triggers the dimerization of N-domain.

A model for coupling of H(+) and substrate fluxes based on "time-sharing" of a common binding site.

Both prokaryotic and eukaryotic cells contain an array of membrane transport systems maintaining the cellular homeostasis. Some of them (primary pumps) derive energy from redox reactions, ATP hydrolysis, or light absorption, whereas others (ion-coupled transporters) utilize ion electrochemical gradients for active transport. Remarkable progress has been made in understanding the molecular mechanism of coupling in some of these systems. In many cases carboxylic residues are essential for either binding or coupling. Here we suggest a model for the molecular mechanism of coupling in EmrE, an Escherichia coli 12-kDa multidrug transporter. EmrE confers resistance to a variety of toxic cations by removing them from the cell interior in exchange for two protons. EmrE has only one membrane-embedded charged residue, Glu-14, which is conserved in more than 50 homologous proteins. We have used mutagenesis and chemical modification to show that Glu-14 is part of the substrate-binding site. Its role in proton binding and translocation was shown by a study of the effect of pH on ligand binding, uptake, efflux, and exchange reactions. The studies suggest that Glu-14 is an essential part of a binding site, which is common to substrates and protons. The occupancy of this site by H(+) and substrate is mutually exclusive and provides the basis of the simplest coupling for two fluxes.

Phase-sensitive observables as a route to understanding molecular continua

We consider the origin and implications of the phase lag, an observable in two-pathway excitation schemes whose recent measurement raised both interest and controversy. A closed-form expression is derived which illustrates the various sources of a nonvanishing phase lag, distinguishes their roles and exposes their unifying feature. Several formally interesting and experimentally relevant limits of the general form are considered and the potential application of phase-sensitive measurements as a route to understanding molecular coupling mechanisms is illustrated.

Function of protonatable residues in the flagellar motor of Escherichia coli: a critical role for Asp 32 of MotB.

Rotation of the bacterial flagellar motor is powered by a transmembrane gradient of protons or, in some species, sodium ions. The molecular mechanism of coupling between ion flow and motor rotation is not understood. The proteins most closely involved in motor rotation are MotA, MotB, and FliG. MotA and MotB are transmembrane proteins that function in transmembrane proton conduction and that are believed to form the stator. FliG is a soluble protein located on the cytoplasmic face of the rotor. Two other proteins, FliM and FliN, are known to bind to FliG and have also been suggested to be involved to some extent in torque generation. Proton (or sodium)-binding sites in the motor are likely to be important to its function and might be formed from the side chains of acidic residues. To investigate the role of acidic residues in the function of the flagellar motor, we mutated each of the conserved acidic residues in the five proteins that have been suggested to be involved in torque generation and measured the effects on motility. None of the conserved acidic residues of MotA, FliG, FliM, or FliN proved essential for torque generation. An acidic residue at position 32 of MotB did prove essential. Of 15 different substitutions studied at this position, only the conservative-replacement D32E mutant retained any function. Previous studies, together with additional data presented here, indicate that the proteins involved in motor rotation do not contain any conserved basic residues that are critical for motor rotation per se. We propose that Asp 32 of MotB functions as a proton-binding site in the bacterial flagellar motor and that no other conserved, protonatable residues function in this capacity.

Identification and purification of a novel G protein from neutrophils

A novel G protein which appears to couple chemotactic peptide receptors to a polyphosphoinositide phospholipase C has been purified from rabbit neutrophils. Neutrophil membranes were solubilized with sodium cholate and fractionated by successive anion exchange, gel filtration and hydrophobic chromatography. Guanosine-5′-(3- O-thio)triphosphate binding activity was purified 170-fold from the soluble extract. The α-subunit of the purified G protein was identified by pertussis toxin-catalyzed ADP-ribosylation, and found to have an M r of 40 000. The β-subunit ( M r 36 000) comigrated on SDS-polyacrylamide gel electrophoresis with the β-subunits of bovine brain Gi and Go. The neutrophil pertussis toxin substrate is highly unstable in cholate solution unless 30% ethylene glycol is added. Structural and functional analysis of this novel G protein will advance our understanding of the molecular mechanisms of coupling of receptors to phospholipase C.