The importance of G.A hydrogen bonding in the metal ion- and protein-induced folding of a kink turn RNA

The kink turn (K-turn) is a common motif in RNA structure, found in many RNA species important in translation, RNA modification and splicing, and the control of gene expression. In general the K-turn comprises a three nucleotide bulge followed by trans sugar-Hoogsteen G·A pairs. The RNA adopts a tightly kinked conformation, and is a common target for binding proteins, exemplified by the L7Ae family. We have measured the rates of association and dissociation for the binding of L7Ae to the Kt-7 kink turn, from which we calculate an affinity of K_D = 10 pM. This high affinity is consistent with the role of this binding as the first stage in the assembly of key functional nucleoproteins such as box C/D snoRNP. Kink-turn RNA undergoes a two-state transition between the kinked conformation, and a more extended structure, and folding into the kinked form is induced by divalent metal ions, or by binding of proteins of the L7Ae class. The K-turn provides an excellent, simple model for RNA folding, which can be dissected at the atomic level. We have analyzed the contributions of the hydrogen bonds that form the G·A pairs to the ion- and protein-induced folding of the K-turn. We find that all four hydrogen bonds are important to the stability of the kinked form of the RNA, and we can now define all the important hydrogen bonding interactions that stabilize the K-turn. The high affinity of L7Ae binding is coupled to the induced folding of the K-turn, allowing some sub-optimal variants to adopt the kinked geometry. However, in all such cases the affinity is lowered, and the results underline the importance of both G·A pairs to the stability of the K-turn.     

The Histone H4 Tail Regulates the Conformation of the ATP-Binding Pocket in the SNF2h Chromatin Remodeling Enzyme

The chromatin remodeling complex ACF helps establish the appropriate nucleosome spacing for generating repressed chromatin states. ACF activity is stimulated by two defining features of the nucleosomal substrate: a basic patch on the histone H4 N-terminal tail and the specific length of flanking DNA. However, the mechanisms by which these two substrate cues function in the ACF remodeling reaction is not well understood. Using electron paramagnetic resonance spectroscopy with spin-labeled ATP analogs to probe the structure of the ATP active site under physiological solution conditions, we identify a closed state of the ATP-binding pocket that correlates with ATPase activity. We find that the H4 tail promotes pocket closure. We further show that ATPase stimulation by the H4 tail does not require a specific structure connecting the H4 tail and the globular domain. In the case of many DNA helicases, closure of the ATP-binding pocket is regulated by specific DNA substrates. Pocket closure by the H4 tail may analogously provide a mechanism to directly couple substrate recognition to activity. Surprisingly, the flanking DNA, which also stimulates ATP hydrolysis, does not promote pocket closure, suggesting that the H4 tail and flanking DNA may be recognized in different reaction steps.     

Direct detection of the myosin super-relaxed state and interacting-heads motif in solution

The interacting-heads motif (IHM) is a structure of myosin that has been proposed to modulate cardiac output by occluding myosin molecules from undergoing the force-generating cycle. It is hypothesized to be the structural basis for the super-relaxed state (SRX), a low-ATPase kinetic state thought to be cardioprotective. The goal of the present study was to test this hypothesis by determining directly and quantitatively the fractions of myosin in the IHM and SRX under the same conditions in solution. To detect the structural IHM, we used time-resolved fluorescence resonance energy transfer to quantitate two distinct populations. One population was observed at a center distance of 2.0 nm, whereas the other was not detectable by fluorescence resonance energy transfer, implying a distance greater than 4 nm. We confirmed the IHM assignment to the 2.0-nm population by applying the same cross-linking protocol used previously to image the IHM by electron microscopy. Under the same conditions, we also measured the fraction of myosin in the SRX using stopped-flow kinetics. Our results show that the populations of SRX and IHM myosin were similar, unless treated with mavacamten, a drug that recently completed phase III clinical trials to treat hypertrophic cardiomyopathy and is proposed to act by stabilizing both the SRX and IHM. However, we found that mavacamten had a much greater effect on the SRX (55% increase) than on the IHM (4% increase). We conclude that the IHM structure is sufficient but not necessary to produce the SRX kinetic state.     

Secretory phospholipase A2 activity in blood serum: The challenge to sense

Excess levels of secretory phospholipase A2 (sPLA2) is known to contribute to several inflammatory diseases including vascular inflammation correlating with coronary events in coronary artery disease. Thus a method to monitor sPLA2 activity in blood serum is urgently needed. Such method is still a challenge since existing fluorescent probes do not allow to monitor sPLA2 activity directly in blood serum. Here we analyze and overcome barriers in sPLA2 sensing methodology and report a fluorescent probe and a kinetic model of its hydrolysis by sPLA2. New probe is designed with a fluorophore and a quencher not interfering binding to the enzyme. At the same time phospholipid matrix bearing the probe promotes efficient initial quenching of the fluorophore. Kinetic model of probe hydrolysis takes into account signal change due to the side processes. The probe and the kinetic model applied together prove the concept that the activity of sPLA can be measured directly in blood serum.     

Fast Subdomain Folding Prior to the Global Refolding Transition of E. coli Adenylate Kinase: A Double Kinetics Study

The rate of folding of globular proteins depends on specific local and nonlocal intramolecular interactions. What is the relative role of these two types of interaction at the initiation of refolding? We address this question by application of a “double kinetics” method based on fast initiation of refolding of site specifically labeled protein samples and detection of the transient distributions of selected intramolecular distances by means of fast measurements of time‐resolved fluorescence resonance energy transfer. We determined the distribution of the distance between the ends of a 44‐chain segment that includes the AMP_bind domain, by labeling residues 28 and 71, in Escherichia coli adenylate kinase (AK) and the distribution of the distance between residues 18 and 203, which depends on the overall order of the molecule. That distribution shows two-state transition to the native intramolecular distance at the same rate as that of the cooperative refolding transition of the AK molecule. In sharp contrast, the distance distribution between residues 28 and 71 is already native like at the end of the dead-time of the mixing device. This fast formation of native short distance between two widely separated chain sections can be either dependent on fast folding of the AMP_bind domain or a result of a very effective nonlocal interaction between specific short clusters of hydrophobic residues. Further experiments on studying the kinetics of folding of selected structural elements in the protein will help determination of the driving force of this early folding event.     

3D Cryo-Electron Reconstruction of BmrA,a Bacterial Multidrug ABC Transporter in an Inward-Facing Conformation and in a Lipidic Environment

ABC (ATP-binding cassette) membrane exporters are efflux transporters of a wide diversity of molecule across the membrane at the expense of ATP. A key issue regarding their catalytic cycle is whether or not their nucleotide-binding domains (NBDs) are physically disengaged in the resting state. To settle this controversy, we obtained structural data on BmrA, a bacterial multidrug homodimeric ABC transporter, in a membrane-embedded state. BmrA in the apostate was reconstituted in lipid bilayers forming a mixture of ring-shaped structures of 24 or 39 homodimers. Three-dimensional models of the ring-shaped structures of 24 or 39 homodimers were calculated at 2.3 nm and 2.5 nm resolution from cryo-electron microscopy, respectively. In these structures, BmrA adopts an inward-facing open conformation similar to that found in mouse P-glycoprotein structure with the NBDs separated by 3 nm. Both lipidic leaflets delimiting the transmembrane domains of BmrA were clearly resolved. In planar membrane sheets, the NBDs were even more separated. BmrA in an ATP-bound conformation was determined from two-dimensional crystals grown in the presence of ATP and vanadate. A projection map calculated at 1.6 nm resolution shows an open outward-facing conformation. Overall, the data are consistent with a mechanism of drug transport involving large conformational changes of BmrA and show that a bacterial ABC exporter can adopt at least two open inward conformations in lipid membrane.     

Role of divalent metal cations in ATP hydrolysis catalyzed by the hepatitis C virus NS3 helicase: magnesium provides a bridge for ATP to fuel unwinding

This study investigates the role of magnesium ions in coupling ATP hydrolysis to the nucleic acid unwinding catalyzed by the NS3 protein encoded by the hepatitis C virus (HCV). Analyses of steady-state ATP hydrolysis rates at various RNA and magnesium concentrations were used to determine values for the 15 dissociation constants describing the formation of a productive enzyme-metal-ATP-RNA complex and the four rate constants describing hydrolysis of ATP by the possible enzyme-ATP complexes. These values coupled with direct binding studies, specificity studies and analyses of site-directed mutants reveal only one ATP binding site on HCV helicase centered on the catalytic base Glu291. An adjacent residue, Asp290, binds a magnesium ion that forms a bridge to ATP, reorienting the nucleotide in the active site. RNA stimulates hydrolysis while decreasing the affinity of the enzyme for ATP, magnesium, and MgATP. The binding scheme described here explains the unusual regulation of the enzyme by ATP that has been reported previously. Binding of either free magnesium or free ATP to HCV helicase competes with MgATP, the true fuel for helicase movements, and leads to slower hydrolysis and nucleic acid unwinding.     

Examining Polyglutamine Peptide Length: A Connection between Collapsed Conformations and Increased Aggregation

Abnormally expanded polyglutamine domains in proteins are associated with several neurodegenerative diseases, of which the best known is Huntington's. Expansion of the polyglutamine domain facilitates aggregation of the affected protein, and several studies directly link aggregation to neurotoxicity. The age of onset of disease is inversely correlated with the length of the polyglutamine domain; this correlation motivates an examination of the role of the length of the domain on aggregation. In this investigation, peptides containing 8 to 24 glutamines were synthesized, and their conformational and aggregation properties were examined. All peptides lacked secondary structure. Fluorescence resonance energy transfer studies revealed that the peptides became increasingly collapsed as the number of glutamine residues increased. The effective persistence length was estimated to decrease from ∼ 11 to ∼ 7 Å as the number of glutamines increased from 8 to 24. A comparison of our data with theoretical results suggests that phosphate-buffered saline is a good solvent for Q8 and Q12, a theta solvent for Q16, and a poor solvent for Q20 and Q24. By dynamic light scattering, we observed that Q16, Q20, and Q24, but not Q8 or Q12, immediately formed soluble aggregates upon dilution into phosphate-buffered saline at 37 °C. Thus, Q16 stands at the transition point between good and poor solvent and between stable and aggregation-prone peptide. Examination of aggregates by transmission electron microscopy, along with kinetic assays for sedimentation, provided evidence indicating that soluble aggregates mature into sedimentable aggregates. Together, the data support a mechanism of aggregation in which monomer collapse is accompanied by formation of soluble oligomers; these soluble species lack regular secondary structure but appear morphologically similar to the sedimentable aggregates into which they eventually mature.     

Determination of the Individual Roles of the Linker Residues in the Interdomain Motions of Calmodulin Using NMR Chemical Shifts

Many protein molecules are formed by two or more domains whose structures and dynamics are closely related to their biological functions. It is thus important to develop methods to determine the structural properties of these multidomain proteins. Here, we characterize the interdomain motions in the calcium-bound state of calmodulin (Ca^2 +-CaM) using NMR chemical shifts as replica-averaged structural restraints in molecular dynamics simulations. We find that the conformational fluctuations of the interdomain linker, which are largely responsible for the overall interdomain motions of CaM, can be well described by exploiting the information provided by chemical shifts. We thus identify 10 residues in the interdomain linker region that change their conformations upon substrate binding. Five of these residues (Met76, Lys77, Thr79, Asp80 and Ser81) are highly flexible and cover the range of conformations observed in the substrate-bound state, while the remaining five (Arg74, Lys75, Asp78, Glu82 and Glu83) are much more rigid and do not populate conformations typical of the substrate-bound form. The ensemble of conformations representing the Ca^2 +-CaM state obtained in this study is in good agreement with residual dipolar coupling, paramagnetic resonance enhancement, small-angle X-ray scattering and fluorescence resonance energy transfer measurements, which were not used as restraints in the calculations. These results provide initial evidence that chemical shifts can be used to characterize the conformational fluctuations of multidomain proteins.     

The Three-Dimensional Structure of [NiFeSe] Hydrogenase from Desulfovibrio vulgaris Hildenborough: A Hydrogenase without a Bridging Ligand in the Active Site in Its Oxidised, “as-Isolated” State

Hydrogen is a good energy vector, and its production from renewable sources is a requirement for its widespread use. [NiFeSe] hydrogenases (Hases) are attractive candidates for the biological production of hydrogen because they are capable of high production rates even in the presence of moderate amounts of O₂, lessening the requirements for anaerobic conditions. The three-dimensional structure of the [NiFeSe] Hase from Desulfovibrio vulgaris Hildenborough has been determined in its oxidised “as-isolated” form at 2.04-Å resolution. Remarkably, this is the first structure of an oxidised Hase of the [NiFe] family that does not contain an oxide bridging ligand at the active site. Instead, an extra sulfur atom is observed binding Ni and Se, leading to a SeCys conformation that shields the NiFe site from contact with oxygen. This structure provides several insights that may explain the fast activation and O₂ tolerance of these enzymes.     

Probing conformational changes in lipoxygenases upon membrane binding: Fine-tuning by the active site inhibitor ETYA

Lipoxygenases (LOXs) are lipid-peroxidizing enzymes that are involved in the metabolism of polyunsaturated fatty acids. Their biological activity includes a membrane binding process whose molecular details are not completely understood. The mechanism of enzyme–membrane interactions is thought to involve conformational changes at the level of the protein tertiary structure, and the extent of such alterations depends on the degree of structural flexibility of the different LOX isoforms. In this study, we have tested the resilience properties of a plant and a mammalian LOX, by using high pressure fluorescence measurements at different temperatures. The binding of LOXs to the lipid bilayer has been characterized using both large and giant unilamellar vesicles and electron transfer particles (inner mitochondrial membranes) as model membranes. The data indicate that the degree of LOXs' flexibility is strictly dependent on the two distinct N- and C-terminal domains that characterize the 3D structure of these enzymes. Furthermore, they demonstrate that increasing the rigidity of protein scaffolding by the presence of an active site ligand impairs the membrane binding ability of LOXs. These findings provide evidence that the amphitropic nature of LOXs is finely tuned by the interaction of the substrate with the residues of the active site, suggesting new strategies for the design of enzyme inhibitors.     

Sensory rhodopsin II/transducer complex formation in detergent and in lipid bilayers studied with FRET

The photophobic receptor from Natronomonas pharaonis (NpSRII) forms a photo-signalling complex with its cognate transducer (NpHtrII). In order to elucidate the complex formation in more detail, we have studied the intermolecular binding of both constituents (NpSRII and NpHtrII₁₅₇; truncated at residue 157) in detergent buffers, and in lipid bilayers using FRET. The data for hetero-dimer formation of NpSRII/NpHtrII in detergent agrees well with K_D values (∼ 200 nM) described in the literature. In lipid bilayers, the binding affinity between proteins in the NpSRII/NpHtrII complex is at least one order of magnitude stronger. In detergent the strength of binding is similar for both homo-dimers (NpSRII/NpSRII and NpHtrII/NpHtrII) but significantly weaker (K_D  ∼ 16 μM) when compared to the hetero-dimer. The intermolecular binding is again considerably stronger in lipid bilayers; however, it is not as strong as that observed for the hetero-dimer. At a molar transducer/lipid ratio of 1:2000, which is still well above physiological concentrations, only 40% homo-dimers are formed. Apparently, in cell membranes the formation of the assumed functionally active oligomeric 2:2 complex depends on the full-length transducer including the helical cytoplasmic part, which is thought to tighten the transducer-dimer association.     

Protein-protein and protein-lipid interactions in domain-assembly: Lessons from giant unilamellar vesicles

Giant Unilamellar Vesicles (GUVs) provide a key model membrane system to study lipid-lipid and lipid-protein interactions, which are relevant to vital cellular processes, by (single-molecule) optical microscopy. Here, we review the work on reconstitution techniques for membrane proteins and other preparation methods for developing GUVs towards most suitable close-to-native membrane systems. Next, we present a few applications of protein-containing GUVs to study domain assembly and protein partitioning into raft-like domains.     

Early Closure of a Long Loop in the Refolding of Adenylate Kinase: A Possible Key Role of Non-Local Interactions in the Initial Folding Steps

Most globular protein chains, when transferred from high to low denaturant concentrations, collapse instantly before they refold to their native state. The initial compaction of the protein molecule is assumed to have a key effect on the folding pathway, but it is not known whether the earliest structures formed during or instantly after collapse are defined by local or by non-local interactions—that is, by secondary structural elements or by loop closure of long segments of the protein chain. Stable closure of one or several long loops can reduce the chain entropy at a very early stage and can prevent the protein from following non-productive pathways whose number grows exponentially with the length of the protein chain. In Escherichia coli adenylate kinase (AK), about seven long loops define the topology of the native structure. We selected four loop-forming sections of the chain and probed the time course of loop formation during refolding of AK. We labeled the termini of the loop segments with tryptophan and cysteine-5-amidosalicylic acid. This donor-acceptor pair of probes used with fluorescence resonance excitation energy transfer spectroscopy (FRET) is suitable for detecting very short distances and thus is able to distinguish between random and specific compactions. Refolding of AK was initiated by stopped-flow mixing, followed simultaneously by donor and acceptor fluorescence, and analyzed in terms of energy transfer efficiency and distance. In the collapsed state of AK, observed after the 5-ms dead time of the instrument, one of the selected segments shows a native-like separation of its termini; it forms a loop already in the collapsed state. A second segment that includes the first but is longer by 15 residues shows an almost native-like separation of its termini. In contrast, a segment that is shorter but part of the second segment shows a distance separation of its termini as high as a segment that spans almost the whole protein chain. We conclude that a specific network of non-local interactions, the closure of one or several loops, can play an important role in determining the protein folding pathway at its early phases.     

The Role of L1 Stalk-tRNA Interaction in the Ribosome Elongation Cycle

The ribosomal L1 stalk is a mobile structure implicated in directing tRNA movement during translocation through the ribosome. This article investigates three aspects of L1 stalk-tRNA interaction. First, by combining data from cryo electron microscopy, X-ray crystallography, and molecular dynamics simulations through the molecular dynamics flexible fitting method, we obtained atomic models of different tRNAs occupying the hybrid P/E state interacting with the L1 stalk. These models confirm the assignment of fluorescence resonance energy transfer states from previous single-molecule investigations of L1 stalk dynamics. Second, the models reconcile how initiator tRNA^fMet interacts less strongly with the L1 stalk compared to elongator tRNA^Phe, as seen in previous single-molecule experiments. Third, results from a simulation of the entire ribosome in which the L1 stalk is moved from a half-closed conformation to its open conformation are found to support the hypothesis that L1 stalk opening is involved in tRNA release from the ribosome.     

EPR properties of a g=2 broad signal trapped in an S1 state in Ca-depleted Photosystem II

We investigated a new EPR signal that gives a broad line shape around g=2 in Ca²⁺-depleted Photosystem (PS) II. The signal was trapped by illumination at 243 K in parallel with the formation of Y_Z. The ratio of the intensities between the g=2 broad signal and the Y_Z signal was 1:3, assuming a Gaussian line shape for the former. The g=2 broad signal and the Y_Z signal decayed together in parallel with the appearance of the S₂ state multiline at 243 K. The g=2 broad signal was assigned to be an intermediate S₁X state in the transition from the S₁ to the S₂ state, where X represents an amino acid radical nearby manganese cluster, such as D1-His337. The signal is in thermal equilibrium with Y_Z. Possible reactions in the S state transitions in Ca²⁺-depleted PS II were discussed.     

Acylation and cholesterol binding are not required for targeting of influenza A virus M2 protein to the hemagglutinin-defined budozone

Influenza virus assembles in the budozone, a cholesterol-/sphingolipid-enriched (“raft”) domain at the apical plasma membrane, organized by hemagglutinin (HA). The viral protein M2 localizes to the budozone edge for virus particle scission. This was proposed to depend on acylation and cholesterol binding. We show that M2–GFP without these motifs is still transported apically in polarized cells. Employing FRET, we determined that clustering between HA and M2 is reduced upon disruption of HA’s raft-association features (acylation, transmembranous VIL motif), but remains unchanged with M2 lacking acylation and/or cholesterol-binding sites. The motifs are thus irrelevant for M2 targeting in cells.     

A Structural Model for the DEAD Box Helicase YxiN in Solution: Localization of the RNA Binding Domain

DEAD box proteins consist of a common helicase core formed by two globular RecA domains that are separated by a cleft. The helicase core acts as a nucleotide-dependent switch that alternates between open and closed conformations during the catalytic cycle of duplex separation, thereby providing basic helicase activity. Flanking domains can direct the helicase core to a specific RNA substrate by mediating high-affinity or high-specificity RNA binding. In addition, they may position RNA for the helicase core or may directly contribute to unwinding. While structures of different helicase cores have been determined previously, little is known about the orientation of flanking domains relative to the helicase core.YxiN is a DEAD box protein that consists of a helicase core and a C-terminal RNA binding domain (RBD) that mediates specific binding to hairpin 92 in 23S rRNA. To provide a framework for understanding the functional cooperation of the YxiN helicase core and the RBD, we mapped the orientation of the RBD in single-molecule fluorescence resonance energy transfer experiments. We present a model for the global conformation of YxiN in which the RBD lies above a slightly concave patch that is formed by flexible loops on the surface of the C-terminal RecA domain. The orientation of the RBD is different from the orientations of flanking domains in the Thermus thermophilus DEAD box protein Hera and in Saccharomyces cerevisiae Mss116p, in line with the different functions of these DEAD box proteins and of their RBDs. Interestingly, the corresponding patch on the C-terminal RecA domain that is covered by the YxiN RBD is also part of the interface between the translation factors eIF4A and eIF4G. Possibly, this region constitutes an adaptable interface that generally allows for the interaction of the helicase core with additional domains or interacting factors.     

A Link between Hinge-Bending Domain Motions and the Temperature Dependence of Catalysis in 3-Isopropylmalate Dehydrogenase

Enzyme function depends on specific conformational motions. We show that the temperature dependence of enzyme kinetic parameters can provide insight into these functionally relevant motions. While investigating the catalytic properties of IPMDH from Escherichia coli, we found that its catalytic efficiency (k_cat/K_M,IPM) for the substrate IPM has an unusual temperature dependence, showing a local minimum at ∼35°C. In search of an explanation, we measured the individual constants k_cat and K_M,IPM as a function of temperature, and found that the van 't Hoff plot of K_M,IPM shows sigmoid-like transition in the 20-40°C temperature range. By means of various measurements including hydrogen-deuterium exchange and fluorescence resonance energy transfer, we showed that the conformational fluctuations, including hinge-bending domain motions increase more steeply with temperatures >30°C. The thermodynamic parameters of ligand binding determined by isothermal titration calorimetry as a function of temperature were found to be strongly correlated to the conformational fluctuations of the enzyme. Because the binding of IPM is associated with a hinge-bending domain closure, the more intense hinge-bending fluctuations at higher temperatures increasingly interfere with IPM binding, thereby abruptly increasing its dissociation constant and leading to the observed unusual temperature dependence of the catalytic efficiency.