Shift In Conformational Equilibrium Research Articles

Darobactin B Stabilises a Lateral-Closed Conformation of the BAM Complex in E. coli Cells.

The β-barrel assembly machinery (BAM complex) is essential for outer membrane protein (OMP) folding in Gram-negative bacteria, and represents a promising antimicrobial target. Several conformational states of BAM have been reported, but all have been obtained under conditions which lack the unique features and complexity of the outer membrane (OM). Here, we use Pulsed Electron-Electron Double Resonance (PELDOR, or DEER) spectroscopy distance measurements to interrogate the conformational ensemble of the BAM complex in E. coli cells. We show that BAM adopts a broad ensemble of conformations in the OM, while in the presence of the antibiotic darobactin B (DAR-B), BAM's conformational equilibrium shifts to a restricted ensemble consistent with the lateral closed state. Our in-cell PELDOR findings are supported by new cryoEM structures of BAM in the presence and absence of DAR-B. This work demonstrates the utility of PELDOR to map conformational changes in BAM within its native cellular environment.

Darobactin B Stabilises a Lateral-Closed Conformation of the BAM Complex in E. coli Cells.

The β-barrel assembly machinery (BAM complex) is essential for outer membrane protein (OMP) folding in Gram-negative bacteria, and represents a promising antimicrobial target. Several conformational states of BAM have been reported, but all have been obtained under conditions which lack the unique features and complexity of the outer membrane (OM). Here, we use Pulsed Electron-Electron Double Resonance (PELDOR, or DEER) spectroscopy distance measurements to interrogate the conformational ensemble of the BAM complex in E. coli cells. We show that BAM adopts a broad ensemble of conformations in the OM, while in the presence of the antibiotic darobactin B (DAR-B), BAM's conformational equilibrium shifts to a restricted ensemble consistent with the lateral closed state. Our in-cell PELDOR findings are supported by new cryoEM structures of BAM in the presence and absence of DAR-B. This work demonstrates the utility of PELDOR to map conformational changes in BAM within its native cellular environment.

Conformational changes in the human Cx43/GJA1 gap junction channel visualized using cryo-EM

Connexin family proteins assemble into hexameric hemichannels in the cell membrane. The hemichannels dock together between two adjacent membranes to form gap junction intercellular channels (GJIChs). We report the cryo-electron microscopy structures of Cx43 GJICh, revealing the dynamic equilibrium state of various channel conformations in detergents and lipid nanodiscs. We identify three different N-terminal helix conformations of Cx43—gate-covering (GCN), pore-lining (PLN), and flexible intermediate (FIN)—that are randomly distributed in purified GJICh particles. The conformational equilibrium shifts to GCN by cholesteryl hemisuccinates and to PLN by C-terminal truncations and at varying pH. While GJIChs that mainly comprise GCN protomers are occluded by lipids, those containing conformationally heterogeneous protomers show markedly different pore sizes. We observe an α-to-π-helix transition in the first transmembrane helix, which creates a side opening to the membrane in the FIN and PLN conformations. This study provides basic structural information to understand the mechanisms of action and regulation of Cx43 GJICh.

Pressure resolved DEER to define the conformational landscape governing ligand-mediated β2-adrenergic receptor signal transduction.

The β-adrenergic receptors (βARs) are a subfamily of G protein-coupled receptors (GPCRs) that are expressed by most cell types in humans; as such, signaling through βARs regulates a wide variety of physiological processes. Upon binding extracellular ligands, these receptors couple to one of several subtypes of G-protein which reside at the intracellular side of the plasma membrane to trigger signaling events. During receptor signaling, ligand binding is converted into an intracellular signal via conformational changes in the GPCR. The ligand shifts the conformational equilibrium of the receptor toward active or inactive states, dependent on whether it is an agonist, inverse agonist, or antagonist. The prevailing conformational selection model suggests that receptors exist as an ensemble of distinct conformations in dynamic equilibrium where each conformation translates functionally by possessing distinct affinities and catalytic activities for the different intracellular transducer proteins. Experimental techniques that characterize structural differences of functionally distinct receptor conformations and their equilibrium populations are paramount in furthering our understanding of ligand-mediated GPCR signal transduction. Pressure induces a reversible shift in conformational equilibria to populate excited states, allowing for the characterization of sparsely-populated states and the identification of their functional relevance. Pressure perturbation grants the ability to systematically populate states that may otherwise be invisible. With pressure-resolved double electron-electron resonance (DEER), we are able to capture the relative populations of each conformation under different hydrostatic pressures. I have demonstrated a pressure dependence for the β2-adrenergic receptor. These data indicate we can populate the active conformation of the β2AR without a ligand, but with sufficient pressure of which we have the autonomy to adjust, which allows for us to populate otherwise spectroscopically “invisible members” of the β2AR conformational ensemble.

Shift in Conformational Equilibrium Underlies the Oscillatory Phosphoryl Transfer Reaction in the Circadian Clock.

Oscillatory phosphorylation/dephosphorylation can be commonly found in a biological system as a means of signal transduction though its pivotal presence in the workings of circadian clocks has drawn significant interest: for example in a significant portion of the physiology of Synechococcus elongatus PCC 7942. The biological oscillatory reaction in the cyanobacterial circadian clock can be visualized through its reconstitution in a test tube by mixing three proteins—KaiA, KaiB and KaiC—with adenosine triphosphate and magnesium ions. Surprisingly, the oscillatory phosphorylation/dephosphorylation of the hexameric KaiC takes place spontaneously and almost indefinitely in a test tube as long as ATP is present. This autonomous post-translational modification is tightly regulated by the conformational change of the C-terminal peptide of KaiC called the “A-loop” between the exposed and the buried states, a process induced by the time-course binding events of KaiA and KaiB to KaiC. There are three putative hydrogen-bond forming residues of the A-loop that are important for stabilizing its buried conformation. Substituting the residues with alanine enabled us to observe KaiB’s role in dephosphorylating hyperphosphorylated KaiC, independent of KaiA’s effect. We found a novel role of KaiB that its binding to KaiC induces the A-loop toward its buried conformation, which in turn activates the autodephosphorylation of KaiC. In addition to its traditional role of sequestering KaiA, KaiB’s binding contributes to the robustness of cyclic KaiC phosphorylation by inhibiting it during the dephosphorylation phase, effectively shifting the equilibrium toward the correct phase of the clock.

Ammonia Borane in Nanotubes: The Preference of Eclipsed Conformation

The DFT PBE/3ζ modeling of conformational transformations of an ammonia borane molecule BH3 ← NH3 in model single-layer carbon nanotubes indicates an unusual shift of conformational equilibrium to the eclipsed form. In cluster, the В ← N coordination bond demonstrates a noticeable decrease in length and an increase in its order. The increasing diameter of nanoobject weakens the observed effects and shifts conformation equilibrium to the staggered conformer.

Shift in Conformational Equilibrium Induces Constitutive Activity of G-Protein-Coupled Receptor, Rhodopsin.

Constitutively active mutants (CAMs) of G-protein-coupled receptors (GPCRs) cause various kinds of diseases. Rhodopsin, a light-absorbing GPCR in animal retinas, has retinal as an endogenous ligand; only very low levels of activation of G-protein can be obtained with the ligand-free opsin. However, the CAM of opsin activates G-protein much more efficiently than the wild type, but the mechanism underlying this remains unclear. The present work revisits the constitutive activity of rhodopsin from the standpoint of conformational dynamics. Single-molecule observation of the M257Y mutant of bovine rhodopsin demonstrated that the switch between active and inactive conformations frequently occurred in M257Y opsin, and frequent generation of the active state results in the population shift toward the active state, which accounts for the constitutive activity of M257Y opsin. Our findings demonstrate that the protein function has a direct connection with the structural dynamics.

Water-mediated conformational preselection mechanism in substrate binding cooperativity to protein kinase A

Substrate binding cooperativity in protein kinase A (PKA) seems to involve allosteric coupling between the two binding sites. It received significant attention, but its molecular basis still remains not entirely clear. Based on long molecular dynamics of PKA and its complexes, we characterized an allosteric pathway that links ATP binding to the redistribution of states adopted by a protein substrate positioning segment in favor of those that warrant correct binding. We demonstrate that the cooperativity mechanism critically depends on the presence of water in two distinct, buried hydration sites. One holds just a single water molecule, which acts as a switchable hydrogen bond bridge along the allosteric pathway. The second, filled with partially disordered solvent, is essential for providing a smooth free energy landscape underlying conformational transitions of the peptide binding region. Our findings remain in agreement with experimental data, also concerning the cooperativity abolishing effect of the Y204A mutation, and indicate a plausible molecular mechanism contributing to experimentally observed binding cooperativity of the two substrates.

Selective exclusion and selective binding both contribute to ion selectivity in KcsA, a model potassium channel

The selectivity filter in potassium channels, a main component of the ion permeation pathway, configures a stack of binding sites (sites S1-S4) to which K+ and other cations may bind. Specific ion binding to such sites induces changes in the filter conformation, which play a key role in defining both selectivity and permeation. Here, using the potassium channel KcsA as a model, we contribute new evidence to reinforce this assertion. First, ion binding to KcsA blocked by tetrabutylammonium at the most cytoplasmic site in the selectivity filter (S4) suggests that such a site, when in the nonconductive filter conformation, has a higher affinity for cation binding than the most extracellular S1 site. This filter asymmetry, along with differences in intracellular and extracellular concentrations of K+versus Na+ under physiological conditions, should strengthen selection of the permeant K+ by the channel. Second, we used different K+ concentrations to shift the equilibrium between nonconductive and conductive states of the selectivity filter in which to test competitive binding of Na+ These experiments disclosed a marked decrease in the affinity of Na+ to bind the channel when the conformational equilibrium shifts toward the conductive state. This finding suggested that in addition to the selective binding of K+ and other permeant species over Na+, there is a selective exclusion of nonpermeant species from binding the channel filter, once it reaches a fully conductive conformation. We conclude that selective binding and selective exclusion of permeant and nonpermeant cations, respectively, are important determinants of ion channel selectivity.

Kinetic modulation of a disordered protein domain by phosphorylation

Phosphorylation is a major post-translational mechanism of regulation that frequently targets disordered protein domains, but it remains unclear how phosphorylation modulates disordered states of proteins. Here we determine the kinetics and energetics of a disordered protein domain the kinase-inducible domain (KID) of the transcription factor CREB and that of its phosphorylated form pKID, using high-throughput molecular dynamic simulations. We identify the presence of a metastable, partially ordered state with a 60-fold slowdown in conformational kinetics that arises due to phosphorylation, kinetically stabilizing residues known to participate in an early binding intermediate. We show that this effect is only partially reconstituted by mutation to glutamate, indicating that the phosphate is uniquely required for the long-lived state to arise. This mechanism of kinetic modulation could be important for regulation beyond conformational equilibrium shifts.

Allosteric Regulation of the Hsp90 Dynamics and Stability by Client Recruiter Cochaperones: Protein Structure Network Modeling

The fundamental role of the Hsp90 chaperone in supporting functional activity of diverse protein clients is anchored by specific cochaperones. A family of immune sensing client proteins is delivered to the Hsp90 system with the aid of cochaperones Sgt1 and Rar1 that act cooperatively with Hsp90 to form allosterically regulated dynamic complexes. In this work, functional dynamics and protein structure network modeling are combined to dissect molecular mechanisms of Hsp90 regulation by the client recruiter cochaperones. Dynamic signatures of the Hsp90-cochaperone complexes are manifested in differential modulation of the conformational mobility in the Hsp90 lid motif. Consistent with the experiments, we have determined that targeted reorganization of the lid dynamics is a unifying characteristic of the client recruiter cochaperones. Protein network analysis of the essential conformational space of the Hsp90-cochaperone motions has identified structurally stable interaction communities, interfacial hubs and key mediating residues of allosteric communication pathways that act concertedly with the shifts in conformational equilibrium. The results have shown that client recruiter cochaperones can orchestrate global changes in the dynamics and stability of the interaction networks that could enhance the ATPase activity and assist in the client recruitment. The network analysis has recapitulated a broad range of structural and mutagenesis experiments, particularly clarifying the elusive role of Rar1 as a regulator of the Hsp90 interactions and a stability enhancer of the Hsp90-cochaperone complexes. Small-world organization of the interaction networks in the Hsp90 regulatory complexes gives rise to a strong correspondence between highly connected local interfacial hubs, global mediator residues of allosteric interactions and key functional hot spots of the Hsp90 activity. We have found that cochaperone-induced conformational changes in Hsp90 may be determined by specific interaction networks that can inhibit or promote progression of the ATPase cycle and thus control the recruitment of client proteins.

Visualizing Structures, Dynamics and Function of Enzymes

In order to understand how enzymes work it is required to know about its conformational landscape, its enzymatic state or interaction with the substrate or product, and the exchange times between conformations and enzymatic states. Experimentally this is extremely challenging because it requires to sample over large time domains, as well as resolving low transiently populated states. Using a hybrid approach involving multiparameter fluorescence spectroscopy, multiscale computer simulations and mutagenesis we are able to map the conformational landscape involving three conformational states (C1, C2, and C3) that are sampled from ns to ms. Furthermore, we present a state matrix that links the enzyme state of the bacteriophage T4 (T4L) as it follows a Michaelis-Menten like formalism. In all we identify T4L as a three state system linking each of the fundamental steps in enzyme function: Substrate binding in C1, catalysis in C2, and product release in C3. We validate the conformational state C1 against the crystallographic structure with identification code in the protein data bank (PDBID) 172L with an accuracy of 1.8 A RMSD and 2σ precision of 0.6 A. The C2 agrees very well with the PDBID 148L with an accuracy of 2.1 A RMSD and 2σ precision of 0.9 A. The conformational state C3 has not been identified before and has a 2σ precision of 2.6 A. Our findings for T4L demonstrate that a fine-tuned shift of conformational equilibrium leads to motions of active cleaning where the energies of substrate binding and product formation drive the conformational equilibrium. The directionality of the overall process is manifested by the free reaction enthalpy of the glycosidic bond cleavage. In all, our work highlights the potential of our hybrid approach to observe thermally accessible, low-populated intermediates with high spatial and temporal resolution.

Chirped-Pulse and Cavity-Based Fourier Transform Microwave Spectroscopy of a Chiral Epoxy Ester: Methyl Glycidate

Rotational spectra of a chiral epoxy ester, methyl glycidate, were measured using a chirped-pulse and a cavity-based Fourier transform microwave spectrometer. The two lowest energy conformers where the epoxy oxygen and the ester oxygen atoms are in the syn and anti relative orientation with respect to each other were identified experimentally. Spectra of four (13)C isotopologues of the lowest energy conformer of methyl glycidate were also measured and assigned. All of the observed rotational transitions are split into doublets due to the presence of the ester methyl internal rotor. The rotational constants and the internal rotation barrier height for the ester methyl group were determined for both conformers of methyl glycidate and for the four (13)C isotopologues of the most stable conformer. A value for the interconversion barrier between the two most stable conformers was estimated. Furthermore, comparison to strawberry aldehyde, a larger derivative of methyl glycidate, shows how the syn-anti conformational equilibrium shifts as a result of the additional bulky substituents at the epoxy ring and at the ester oxygen atom.

Monitoring Shifts in the Conformation Equilibrium of the Membrane Protein Cytochrome P450 Reductase (POR) in Nanodiscs

Nanodiscs are self-assembled ∼50-nm(2) patches of lipid bilayers stabilized by amphipathic belt proteins. We demonstrate that a well ordered dense film of nanodiscs serves for non-destructive, label-free studies of isolated membrane proteins in a native like environment using neutron reflectometry (NR). This method exceeds studies of membrane proteins in vesicle or supported lipid bilayer because membrane proteins can be selectively adsorbed with controlled orientation. As a proof of concept, the mechanism of action of the membrane-anchored cytochrome P450 reductase (POR) is studied here. This enzyme is responsible for catalyzing the transfer of electrons from NADPH to cytochrome P450s and thus is a key enzyme in the biosynthesis of numerous primary and secondary metabolites in plants. Neutron reflectometry shows a coexistence of two different POR conformations, a compact and an extended form with a thickness of 44 and 79 Å, respectively. Upon complete reduction by NADPH, the conformational equilibrium shifts toward the compact form protecting the reduced FMN cofactor from engaging in unspecific electron transfer reaction.

Crystal structure of Onconase at 1.1 Å resolution – insights into substrate binding and collective motion

Onconase® (ONC) is an amphibian member of the pancreatic ribonuclease superfamily that is selectively toxic to tumor cells. It is a much less efficient enzyme than the archetypal ribonuclease A and, in an attempt to gain further insight, we report the first atomic resolution crystal structure of ONC, determined in complex with sulfate ions at 100 K. The electron density map is of a quality sufficient to reveal significant nonplanarity in several peptide bonds. The majority of active site residues are very well defined, with the exceptions being Lys31 from the catalytic triad and Lys33 from the B1 subsite, which are relatively mobile but rigidify upon nucleotide binding. Cryocooling causes a compaction of the unit cell and the protein contained within. This is principally the result of an inward movement of one of the lobes of the enzyme (lobe 2), which also narrows the active site cleft. Binding a nucleotide in place of sulfate is associated with an approximately perpendicular movement of lobe 2 and has little further effect on the cleft width. Aspects of this deformation are present in the principal axes of anisotropy extracted from Cα atomic displacement parameters, indicating its intrinsic nature. The three lowest-frequency modes of ONC motion predicted by an anisotropic network model are compaction/expansion variations in which lobe 2 is the prime mover. Two of these have high similarity to the cryocooling response and imply that the essential ‘breathing’ motion of ribonuclease A is conserved in ONC. Instead, shifts in conformational equilibria may contribute to the reduced ribonucleolytic activity of ONC.DatabaseStructural data have been submitted to the Protein Data Bank under accession number 3SNF.

Pressure-Induced Spectral Changes of Room-Temperature Ionic Liquid, N,N-Diethyl-N-methyl-N-(2-methoxyethyl)ammonium Bis(trifluoromethylsulfonyl)imide, [DEME][TFSI

We have investigated pressure-induced Raman spectral changes of ionic liquid, N,N-diethyl-N-methyl-N-(2-methoxyethyl)ammonium bis(trifluoromethylsulfonyl)imide ([DEME][TFSI]) up to 5.5 GPa. We found that the CH stretching spectrum of [DEME] cation changes significantly above 4 GPa together with the large shift of conformational equilibrium between C1 and C2 conformers of [TFSI] anion toward C2 one. The results along with the visual observations show that no crystallization occurs even at 5.5 GPa. We suppose that structural organization in [DEME][TFSI] might significantly change at 4 to 5 GPa region.

Conformational Partitioning in pH-Induced Fluorescence of the Kindling Fluorescent Protein (KFP)

Kindling fluorescent protein (KFP) is considered as a prospective fluorophore for high-resolution nanoscopy. Analysis of pH dependence of the absorption and fluorescence spectra of KFP in aqueous solutions prompted us to assume that a shift in conformational equilibrium is responsible for substantial enhancement of red fluorescence in KFP at alkaline pH. Variations in pH also resulted in noticeable shifts in band maxima for absorption, fluorescence excitation, and fluorescence emission. These observations can be interpreted as an appearance of pH-induced fluorescent conformational states of the protein. On the basis of the available crystal structures of the protein and the results of molecular modeling, we suggest that appearance of these pH-induced fluorescent states is due to changes in the hydrogen bond network around the chromophore moiety (but not the cromophore itself), especially those associated with the side chains of Cys62 and Ser158. We hypothesize that conformational partitioning and fluctuations in protein ionization at alkali pH play an essential role in the appearance of fluorescent properties of KFP.

Structure, solvation, and acid–base property in ionic liquids

Ionic liquids (ILs) are expected to have specific properties as solvents for chemical reactions in view of solution chemistry. Among physicochemical properties, liquid structure, acid–base, and electron-pair donating and accepting abilities of solvent play a crucial role in ion-solvation and acid–base, metal-ion complexation, and electrochemical reactions. Various types of ILs have been developed, and among others, the bis(trifluoromethanesulfonyl)amide (TFSA–)-based ILs are extensively used. TFSA–is a flexible molecule to give two stable conformers,cis(C1) andtrans(C2), which are present in equilibrium in the liquid state. The conformational equilibrium shifts upon solvation to the metal ion. This is quantitatively studied to obtain thermodynamic parameters of conformational change from C2 to C1 in the bulk and in the solvation sphere of the lithium ion. On the other hand, with ethylammonium nitrate (EAN), a typical protic IL, it is revealed that the ammonium group is hydrogen-bonded with three nitrate ions to form a heterogeneous liquid structure. The solvent acid–base property of EAN and acid dissociation reaction in EAN have been quantitatively revealed, and the results will be discussed in comparison with those in normal molecular solvents.

From structural to functional glycomics: core substitutions as molecular switches for shape and lectin affinity of N-glycans

Glycan epitopes of cellular glycoconjugates act as versatile biochemical signals (sugar coding). Here, we test the hypothesis that the common N-glycan modifications by core fucosylation and introduction of the bisecting N-acetylglucosamine moiety have long-range effects with functional consequences. Molecular dynamics simulations indicate a shift in conformational equilibria between linear extension or backfolding of the glycan antennae upon substitution. We also present a new fingerprint-like mode of presentation for this multi-parameter system. In order to delineate definite structure-function relationships, we strategically combined chemoenzymatic synthesis with bioassaying cell binding and the distribution of radioiodinated neoglycoproteins in vivo. Of clinical relevance, tailoring the core region affects serum clearance markedly, e.g., prolonging circulation time for the neoglycoprotein presenting the N-glycan with both substitutions. alpha2,3-Sialylation is another means toward this end, similarly seen for type II branching in triantennary N-glycans. This discovery signifies that rational glycoengineering along the given lines is an attractive perspective to optimize pharmacokinetic behavior of glycosylated pharmaproteins. Of general importance for the concept of the sugar code, the presented results teach the fundamental lesson that N-glycan core substitutions convey distinct characteristics to the concerned oligosaccharide relevant for cis and trans biorecognition processes. These modifications are thus molecular switches.

KIT kinase mutants show unique mechanisms of drug resistance to imatinib and sunitinib in gastrointestinal stromal tumor patients

Most gastrointestinal stromal tumors (GISTs) exhibit aberrant activation of the receptor tyrosine kinase (RTK) KIT. The efficacy of the inhibitors imatinib mesylate and sunitinib malate in GIST patients has been linked to their inhibition of these mutant KIT proteins. However, patients on imatinib can acquire secondary KIT mutations that render the protein insensitive to the inhibitor. Sunitinib has shown efficacy against certain imatinib-resistant mutants, although a subset that resides in the activation loop, including D816H/V, remains resistant. Biochemical and structural studies were undertaken to determine the molecular basis of sunitinib resistance. Our results show that sunitinib targets the autoinhibited conformation of WT KIT and that the D816H mutant undergoes a shift in conformational equilibrium toward the active state. These findings provide a structural and enzymologic explanation for the resistance profile observed with the KIT inhibitors. Prospectively, they have implications for understanding oncogenic kinase mutants and for circumventing drug resistance.