Dynamic Monomer-dimer Equilibrium Research Articles

The Deinococcus protease PprI senses DNA damage by directly interacting with single-stranded DNA

Bacteria have evolved various response systems to adapt to environmental stress. A protease-based derepression mechanism in response to DNA damage was characterized in Deinococcus, which is controlled by the specific cleavage of repressor DdrO by metallopeptidase PprI (also called IrrE). Despite the efforts to document the biochemical, physiological, and downstream regulation of PprI-DdrO, the upstream regulatory signal activating this system remains unclear. Here, we show that single-stranded DNA physically interacts with PprI protease, which enhances the PprI-DdrO interactions as well as the DdrO cleavage in a length-dependent manner both in vivo and in vitro. Structures of PprI, in its apo and complexed forms with single-stranded DNA, reveal two DNA-binding interfaces shaping the cleavage site. Moreover, we show that the dynamic monomer-dimer equilibrium of PprI is also important for its cleavage activity. Our data provide evidence that single-stranded DNA could serve as the signal for DNA damage sensing in the metalloprotease/repressor system in bacteria. These results also shed light on the survival and acquired drug resistance of certain bacteria under antimicrobial stress through a SOS-independent pathway.

Dimerization mechanism of an inverted-topology ion channel in membranes.

Many ion channels are multisubunit complexes where oligomerization is an obligatory requirement for function as the binding axis forms the charged permeation pathway. However, the mechanisms of in-membrane assembly of thermodynamically stable channels are largely unknown. Here, we demonstrate a key advance by reporting the dimerization equilibrium reaction of an inverted-topology, homodimeric fluoride channel Fluc in lipid bilayers. While the wild-type channel is a long-lived dimer, we leverage a known mutation, N43S, that weakens Na+ binding in a buried site at the interface, thereby unlocking the complex for reversible association in lipid bilayers. Single-channel recordings show that Na+ binding is required for fluoride conduction while single-molecule microscopy experiments demonstrate that N43S Fluc exists in a dynamic monomer-dimer equilibrium in the membrane, even following removal of Na+. Quantifying the thermodynamic stability while titrating Na+ indicates that dimerization occurs first, providing a membrane-embedded binding site where Na+ binding weakly stabilizes the complex. To understand how these subunits form stable assemblies while presenting charged surfaces to the membrane, we carried out molecular dynamics simulations, which show the formation of a thinned membrane defect around the exposed dimerization interface. In simulations where subunits are permitted to encounter each other while preventing protein contacts, we observe spontaneous and selective association at the native interface, where stability is achieved by mitigation of the membrane defect. These results suggest a model wherein membrane-associated forces drive channel assembly in the native orientation while subsequent factors, such as Na+ binding, result in channel activation.

Conformational Heterogeneity and Cooperative Effects of Mammalian ALOX15.

Arachidonic acid lipoxygenases (ALOXs) have been suggested to function as monomeric enzymes, but more recent data on rabbit ALOX15 indicated that there is a dynamic monomer-dimer equilibrium in aqueous solution. In the presence of an active site ligand (the ALOX15 inhibitor RS7) rabbit ALOX15 was crystalized as heterodimer and the X-ray coordinates of the two monomers within the dimer exhibit subtle structural differences. Using native polyacrylamide electrophoresis, we here observed that highly purified and predominantly monomeric rabbit ALOX15 and human ALOX15B are present in two conformers with distinct electrophoretic mobilities. In silico docking studies, molecular dynamics simulations, site directed mutagenesis experiments and kinetic measurements suggested that in aqueous solutions the two enzymes exhibit motional flexibility, which may impact the enzymatic properties.

Glutamate binding triggers monomerization of unliganded mGluR2 dimers

The Metabotropic glutamate receptor 2 (mGluR2) is involved in several neurological and psychiatric disorders and is an attractive drug target. It is believed to form a strict dimer and the dimeric assembly is necessary for glutamate induced activation. Although many studies have focused on glutamate induced conformational changes, the dimerization propensity of mGluR2 with and without glutamate has never been investigated. Also, the role of the unstructured loop in dimerization of mGluR2 is not clear. Here, using Forster Resonance Energy Transfer (FRET) based assay in live cells we show that mGluR2 does not form a “strict dimer” rather it exists in a dynamic monomer-dimer equilibrium. The unstructured loop moderately destabilizes the dimers. Furthermore, binding of glutamate to mGluR2 induces conformational change that promotes monomerization of mGluR2. In the absence of an unstructured loop, mGluR2 neither undergoes conformational change nor monomerizes upon binding to glutamate.

Modulation of Guanylate Cyclase Activating Protein 1 (GCAP1) Dimeric Assembly by Ca2+ or Mg2+: Hints to Understand Protein Activity.

The guanylyl cyclase-activating protein 1, GCAP1, activates or inhibits retinal guanylyl cyclase (retGC) depending on cellular Ca2+ concentrations. Several point mutations of GCAP1 have been associated with impaired calcium sensitivity that eventually triggers progressive retinal degeneration. In this work, we demonstrate that the recombinant human protein presents a highly dynamic monomer-dimer equilibrium, whose dissociation constant is influenced by salt concentration and, more importantly, by protein binding to Ca2+ or Mg2+. Based on small-angle X-ray scattering data, protein-protein docking, and molecular dynamics simulations we propose two novel three-dimensional models of Ca2+-bound GCAP1 dimer. The different propensity of human GCAP1 to dimerize suggests structural differences induced by cation binding potentially involved in the regulation of retGC activity.

Single-molecule analysis reveals agonist-specific dimer formation of µ-opioid receptors

G-protein-coupled receptors (GPCRs) are key signaling proteins that mostly function as monomers, but for several receptors constitutive dimer formation has been described and in some cases is essential for function. Using single-molecule microscopy combined with super-resolution techniques on intact cells, we describe here a dynamic monomer-dimer equilibrium of µ-opioid receptors (µORs), where dimer formation is driven by specific agonists. The agonist DAMGO, but not morphine, induces dimer formation in a process that correlates both temporally and in its agonist- and phosphorylation-dependence with β-arrestin2 binding to the receptors. This dimerization is independent from, but may precede, µOR internalization. These data suggest a new level of GPCR regulation that links dimer formation to specific agonists and their downstream signals.

Solution NMR structure of yeast Rcf1, a protein involved in respiratory supercomplex formation

The Saccharomyces cerevisiae respiratory supercomplex factor 1 (Rcf1) protein is located in the mitochondrial inner membrane where it is involved in formation of supercomplexes composed of respiratory complexes III and IV. We report the solution structure of Rcf1, which forms a dimer in dodecylphosphocholine (DPC) micelles, where each monomer consists of a bundle of five transmembrane (TM) helices and a short flexible soluble helix (SH). Three TM helices are unusually charged and provide the dimerization interface consisting of 10 putative salt bridges, defining a "charge zipper" motif. The dimer structure is supported by molecular dynamics (MD) simulations in DPC, although the simulations show a more dynamic dimer interface than the NMR data. Furthermore, CD and NMR data indicate that Rcf1 undergoes a structural change when reconstituted in liposomes, which is supported by MD data, suggesting that the dimer structure is unstable in a planar membrane environment. Collectively, these data indicate a dynamic monomer-dimer equilibrium. Furthermore, the Rcf1 dimer interacts with cytochrome c, suggesting a role as an electron-transfer bridge between complexes III and IV. The Rcf1 structure will help in understanding its functional roles at a molecular level.

Insights into CC Chemokine Ligand 2/Chemokine Receptor 2 Molecular Recognition: A Step Forward toward Antichemotactic Agents.

Chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein 1 (MCP-1), is a chemokine that recruits immune cells to inflammatory sites by interacting with G protein-coupled receptor CCR2. The CCL2/CCR2 axis is also involved in pathological processes such as tumor growth and metastasis and hence is currently considered as an important drug target. CCL2 exists in a dynamic monomer-dimer equilibrium that is modulated by CCR2 binding. We used solution nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulations to study the interactions between CCL2 and a sulfopeptide corresponding to the N-terminal sequence of CCR2 (CCR218-31). Peptide binding induced the dissociation of CCL2 into monomers, forming stable CCL2/CCR218-31 complexes. NMR relaxation measurements indicated that residues around the CCR218-31 binding site, which are located at the dimer interface, undergo a complex regime of motions. NMR data were used to construct a three-dimensional structural model of the CCL2/CCR218-31 complex, revealing that CCR218-31 occupies a binding site juxtaposed to the dimer interface, partially replacing monomer-monomer contacts, explaining why CCR218-31 binding weakens the dimer interface and induces dissociation. We found that the main interactions governing receptor binding are highly stable salt bridges with conserved chemokine residues as well as hydrophobic interactions. These data provide new insights into the structure-function relationship of the CCL2-CCR2 interaction and may be helpful for the design of novel antichemotactic agents.

SecA functions in vivo as a discrete anti-parallel dimer to promote protein transport.

SecA ATPase motor protein plays a central role in bacterial protein transport by binding substrate proteins and the SecY channel complex and utilizing its ATPase activity to drive protein translocation across the plasma membrane. SecA has been shown to exist in a dynamic monomer-dimer equilibrium modulated by translocation ligands, and multiple structural forms of the dimer have been crystallized. Since the structural form of the dimer remains a controversial and unresolved question, we addressed this matter by engineering ρ-benzoylphenylalanine along dimer interfaces corresponding to the five different SecA X-ray structures and assessing their in vivo photo-crosslinking pattern. A discrete anti-parallel 1M6N-like dimer was the dominant if not exclusive dimer found in vivo, whether SecA was cytosolic or in lipid or SecYEG-bound states. SecA bound to a stable translocation intermediate was crosslinked in vivo to a second SecA protomer at its 1M6N interface, suggesting that this specific dimer likely promotes active protein translocation. Taken together, our studies strengthen models that posit, at least in part, a SecA dimer-driven translocation mechanism.

Defining the Escherichia coli SecA Dimer Interface Residues through In Vivo Site-Specific Photo-Cross-Linking

The motor protein SecA is a core component of the bacterial general secretory (Sec) pathway and is essential for cell viability. Despite evidence showing that SecA exists in a dynamic monomer-dimer equilibrium favoring the dimeric form in solution and in the cytoplasm, there is considerable debate as to the quaternary structural organization of the SecA dimer. Here, a site-directed photo-cross-linking technique was utilized to identify residues on the Escherichia coli SecA (ecSecA) dimer interface in the cytosol of intact cells. The feasibility of this method was demonstrated with residue Leu6, which is essential for ecSecA dimerization based on our analytical ultracentrifugation studies of SecA L6A and shown to form the cross-linked SecA dimer in vivo with p-benzoyl-phenylalanine (pBpa) substituted at position 6. Subsequently, the amino terminus (residues 2 to 11) in the nucleotide binding domain (NBD), Phe263 in the preprotein binding domain (PBD), and Tyr794 and Arg805 in the intramolecular regulator of the ATPase 1 domain (IRA1) were identified to be involved in ecSecA dimerization. Furthermore, the incorporation of pBpa at position 805 did not form a cross-linked dimer in the SecA Δ2-11 context, indicating the possibility that the amino terminus may directly contact Arg805 or that the deletion of residues 2 to 11 alters the topology of the naturally occurring ecSecA dimer.

Insights into the regulation of the human COP9 signalosome catalytic subunit, CSN5/Jab1

The COP9 (Constitutive photomorphogenesis 9) signalosome (CSN), a large multiprotein complex that resembles the 19S lid of the 26S proteasome, plays a central role in the regulation of the E3-cullin RING ubiquitin ligases (CRLs). The catalytic activity of the CSN complex, carried by subunit 5 (CSN5/Jab1), resides in the deneddylation of the CRLs that is the hydrolysis of the cullin-neural precursor cell expressed developmentally downregulated gene 8 (Nedd8)isopeptide bond. Whereas CSN-dependent CSN5 displays isopeptidase activity, it is intrinsically inactive in other physiologically relevant forms. Here we analyze the crystal structure of CSN5 in its catalytically inactive form to illuminate the molecular basis for its activation state. We show that CSN5 presents a catalytic domain that brings essential elements to understand its activity control. Although the CSN5 active site is catalytically competent and compatible with di-isopeptide binding, the Ins-1 segment obstructs access to its substrate-binding site, and structural rearrangements are necessary for the Nedd8-binding pocket formation. Detailed study of CSN5 by molecular dynamics unveils signs of flexibility and plasticity of the Ins-1 segment. These analyses led to the identification of a molecular trigger implicated in the active/inactive switch that is sufficient to impose on CSN5 an active isopeptidase state. We show that a single mutation in the Ins-1 segment restores biologically relevant deneddylase activity. This study presents detailed insights into CSN5 regulation. Additionally, a dynamic monomer-dimer equilibrium exists both in vitro and in vivo and may be functionally relevant.

Dynamic Monomer-Dimer Equilibrium of a Prototypical GPCR, Beta2 Adrenergic Receptor: A Single Molecule Imaging Study

Recently, by developing special single-molecule techniques and analysis theory, we first time ever succeeded in obtaining the two-dimensional association equilibrium constant in the membrane and fully characterizing the equilibrium by also obtaining both dissociation and association rate constants (Kasai et al., 2011). As an important paradigm for this work, we examined the monomer-dimer equilibrium of a GPCR, formyl-peptide receptor (FPR). Here, we applied this method to a prototypical class-A GPCR, beta2-adrenergic receptor (B-AR), to clarify whether it forms dimers. Although B-AR has been studied extensively, whether it (and other class-A GPCRs in addition to FPR) exists as dimers has been controversial. We found that B-AR does form dimers with rate and equilibrium constants similar to those for FPR (FPR values in parentheses): the monomer-dimer equilibrium constant = 1.6 (3.6) copies/square microns; the dimer dissociation rate constant of 12.6 (11.0) /s [dimer lifetime of 80 (90) ms]; and the monomer association rate constant of 7.9 (3.1)/[copies/square microns]/s. However, under physiological conditions, the behaviors of B-AR and FPR were quite different due to the difference in the expression levels, ∼260 and ∼2.1 copies/square microns [740,000 (6,000) copies/cell] in dog heart cells and neutrophils, respectively. The majority (95%) of B-AR would exist as dimers, whereas about half (42%) of FPR would do so. Interestingly, their dimer lifetimes are similar, ∼80 and ∼90 ms, respectively, but their monomer lifetimes would be vastly different, i.e., 0.49 ms and 154 ms, respectively. These results suggest that the dynamic monomer-dimer equilibrium might play a role in signal transduction and/or its regulation.

Molecular interactions between multihaem cytochromes: probing the protein–protein interactions between pentahaem cytochromes of a nitrite reductase complex

The cytochrome c nitrite reductase NrfA is a 53kDa pentahaem enzyme that crystallizes as a decahaem homodimer. NrfA catalyses the reduction of NO2- to NH4+ through a six electron reduction pathway that is of major physiological significance to the anaerobic metabolism of enteric and sulfate reducing bacteria. NrfA receives electrons from the 21kDa pentahaem NrfB donor protein. This requires that redox complexes form between the NrfA and NrfB pentahaem cytochromes. The formation of these complexes can be monitored using a range of methodologies for studying protein-protein interactions, including dynamic light scattering, gel filtration, analytical ultracentrifugation and visible spectroscopy. These methods have been used to show that oxidized NrfA exists in dynamic monomer-dimer equilibrium with a Kd (dissociation constant) of 4 μM. Significantly, the monomeric and dimeric forms of NrfA are equally active for either the six electron reduction of NO2- or HSO3-. When mixed together, NrfA and NrfB exist in equilibrium with NrfAB, which is described by a Kd of 50nM. Thus, since NrfA and NrfB are present in micromolar concentrations in the periplasmic compartment, it is likely that NrfB remains tightly associated with its NrfA redox partner under physiological conditions.

2K1436 GPCR(β2アドレナリン受容体)のダイマー・モノマー平衡 : 1分子イメジング法による研究(細胞生物的課題2,第49回日本生物物理学会年会)

2K1436 GPCR(β2アドレナリン受容体)のダイマー・モノマー平衡 : 1分子イメジング法による研究(細胞生物的課題2,第49回日本生物物理学会年会)

Characterization of the monomer–dimer equilibrium of recombinant histo-aspartic protease from Plasmodium falciparum

Histo-aspartic protease (HAP) from Plasmodium falciparum is an intriguing aspartic protease due to its unique structure. Our previous study reported the first recombinant expression of soluble HAP, in its truncated form (lys77p-Leu328) (p denotes prosegment), as a thioredoxin (Trx) fusion protein Trx–tHAP. The present study found that the recombinant Trx–tHAP fusion protein aggregated during purification which could be prevented through the addition of 0.2% CHAPS. Trx–tHAP fusion protein was processed into a mature form of tHAP (mtHAP) by both autoactivation, and activation with either enterokinase or plasmepsin II. Using gel filtration chromatography as well as sedimentation velocity and equilibrium ultracentrifugation, it was shown that the recombinant mtHAP exists in a dynamic monomer–dimer equilibrium with an increasing dissociation constant in the presence of CHAPS. Enzymatic activity data indicated that HAP was most active as a monomer. The dominant monomeric form showed a K m of 2.0 μM and a turnover number, k cat, of 0.036 s −1 using the internally quenched fluorescent synthetic peptide substrate EDANS–CO–CH 2–CH 2–CO–Ala-Leu-Glu-Arg-Met-Phe-Leu-Ser-Phe-Pro–Dap–(DABCYL)–OH (2837b) at pH 5.2.

Characterization of the Monomer-Dimer Equilibrium of Recombinant Histo-aspartic Protease from Plasmodium falciparum

Malaria is a devastating disease that infects and kills 1-2 million people annually. Histo-aspartic protease (HAP) from Plasmodium falciparum, the most lethal of all Plasmodium parasites, is an intriguing aspartic protease due to its unique structure and its potential as an antimalarial target. Substantial effort has been devoted to investigate the structure function of this protease. The present study investigated the molecular state of HAP as related to enzymatic activity. Gel filtration chromatography indicated that recombinant Trx-tHAP fusion protein aggregated during purification and that aggregation could be prevented through the addition of 0.2% CHAPS. Using this latter technique as well as sedimentation velocity and sedimentation equilibrium ultracentrifugation, it was shown that the recombinant mature HAP (mtHAP), in which the His-tag, thioredoxin and prosegement were removed, exists in a dynamic monomer-dimer equilibrium in solution and the dissociation constant is 20-30 μM. Enzymatic activity data also indicated that HAP was most active as a monomer. The monomeric form of mtHAP showed a Km of 9.7 μM and a turnover number, kcat, of 0.044s-1 on the internally quenched fluorescent synthetic peptide substrate EDANS-CO-CH2-CH2-CO-Ala-Leu-Glu-Arg-Met-Phe-Leu-Ser-Phe-Pro-Dap-(DABCYL)-OH (2837b) at pH 6.5. Inhibition studies showed that the activity of mtHAP was completely inhibited by 1 mM PMSF and to a lesser degree by 10 μM ALLN, 10 mM EDTA and 10 mM 1,10-phenanthroline, and was inhibited strongly by ZnCl2 and to a lesser extent by NaCl and KBr. The effects of temperature and salts on the monomer-dimer equilibrium of mtHAP were also investigated by using sedimentation equilibrium ultracentrifugation.

Interactions of succinate dehydrogenase with cyanide

Succinate dehydrogenase (succinate:(acceptor) oxidoreductase, EC 1.3.99.1) solubilized by KCN, differs from other soluble preparations of this enzyme for the following properties. 1. 1. It has 16–20 nmoles of tightly bound cyanide per mg protein. 2. 2. It contains 3.6–4.1 nmoles of peptide-bound flavin, 24–32 natoms of iron and 16–17 nmoles of acid-labile sulphide per mg protein. An Alteration of the iron-sulphur prosthetic group is thus indicated. 3. 3. Density gradient centrifugation shows two active species of mol. wt 180 000 and 140 000. By gel filtration in chaotropic agents, two components of mol. wt 180 000 and 100 000 can be isolated. The molecular weights, by gradient ultra-centrifugation, of the isolated components correspond to the original values obtained for the unfractionated enzyme. It is proposed that the heavier species is a dimer stabilized by cyanide; the ligher one represents the monomeric form of the flavo-protein, which exists in a dynamic monomer dimer equilibrium. The mechanism of action of KCN in the dissociation of succinate dehydrogenase from the membrane was also investigated. 1. 1. CN is shown to be the species acting in the process. The reaction is found to be first order with respect to CN −. 2. 2. KCN induces spectral modifications on soluble succinate dehydrogenase and an anomalous stability to iron: two moles of CN − per gatom iron remain bound to protein after mild acid treatment. 3. 3. It is concluded taht CN − binds to the iron of the membrane-bound succinate dehydrogenase. As a result of a probable conformation change, the resolution of the linkages to the membrane occurs.

The resolution of some discrepancies concerning the molecular weight and subunit structure of succinate dehydrogenase

Values in the literature for the molecular weight of mammalian succinate dehydrogenase, based on a variety of physical and chemical measurements, range from 100,000 to 200,000 and the enzyme has been generally assumed to exist as a monomer in solution. Recent reports indicate the presence of two subunits of 70,000 and 30,000 molecular weight, which have been assumed (but not proven) to be present in a 1:1 ratio. It has been shown in the present study for two types of soluble preparations that analysis for histidyl flavin indicates a minimum molecular weight of 10 5 ± 5,000, but a variety of physical measurements (chromatography on Agarose and Sephadex, density gradient centrifugation, and sedimentation-diffusion) indicate a molecular weight of 175,000 ± 10,000. The reason for this discrepancy appears to be that the enzyme exists in solution in a dynamic monomer-dimer equilibrium. This interpretation is supported by the concentration dependence of the apparent molecular weight and the broadness of the peaks in gel elution and in density gradient profiles. The subunit molecular weights have been confirmed but the ratio of the 30,000 to 70,000 subunits appears to be considerably greater than 1. This is because even the purest preparations available appear to contain a 30,000 molecular weight impurity, which is not separated from the subunit of the dehydrogenase on Sephadex or on acrylamide gels.