Single VS ribozyme molecules reveal dynamic and hierarchical folding toward catalysis

Non-coding RNAs of complex tertiary structure are involved in numerous aspects of the replication and processing of genetic information in many organisms; however, an understanding of the complex relationship between their structural dynamics and function is only slowly emerging. The Neurospora Varkud Satellite (VS) ribozyme provides a model system to address this relationship. First, it adopts a tertiary structure assembled from common elements, a kissing loop and two three-way junctions. Second, catalytic activity of the ribozyme is essential for replication of VS RNA in vivo and can be readily assayed in vitro. Here we exploit single molecule FRET to show that the VS ribozyme exhibits previously unobserved dynamic and heterogeneous hierarchical folding into an active structure. Readily reversible kissing loop formation combined with slow cleavage of the upstream substrate helix suggests a model whereby the structural dynamics of the VS ribozyme favor cleavage of the substrate downstream of the ribozyme core instead. This preference is expected to facilitate processing of the multimeric RNA replication intermediate into circular VS RNA, which is the predominant form observed in vivo.     

Analysis of Four-Way Junctions in RNA Structures

RNA secondary structures can be divided into helical regions composed of canonical Watson-Crick and related base pairs, as well as single-stranded regions such as hairpin loops, internal loops, and junctions. These elements function as building blocks in the design of diverse RNA molecules with various fundamental functions in the cell. To better understand the intricate architecture of three-dimensional (3D) RNAs, we analyze existing RNA four-way junctions in terms of base-pair interactions and 3D configurations. Specifically, we identify nine broad junction families according to coaxial stacking patterns and helical configurations. We find that helices within junctions tend to arrange in roughly parallel and perpendicular patterns and stabilize their conformations using common tertiary motifs such as coaxial stacking, loop-helix interaction, and helix packing interaction. Our analysis also reveals a number of highly conserved base-pair interaction patterns and novel tertiary motifs such as A-minor-coaxial stacking combinations and sarcin/ricin motif variants. Such analyses of RNA building blocks can ultimately help in the difficult task of RNA 3D structure prediction.     

Thermodynamics and folding pathway of tetraloop receptor-mediated RNA helical packing

Little is known about the thermodynamic forces that drive the folding pathways of higher-order RNA structure. In this study, we employ calorimetric [isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC)] and spectroscopic (NMR and UV) methods to characterize the thermodynamics of the GAAA tetraloop-receptor interaction, utilizing a previously described bivalent construct. ITC studies indicate that the bivalent interaction is enthalpy driven and highly stable, with a binding constant (K(obs)) of 5.5x10(6) M(-1) and enthalpy (DeltaH(obs)(o)) of -33.8 kcal/mol at 45 degrees C in 20 mM KCl and 2 mM MgCl(2). Thus, we derive the DeltaH(obs)(o) for a single tetraloop-receptor interaction to be -16.9 kcal/mol at these conditions. UV absorbance data indicate that an increase in base stacking quality contributes to the enthalpy of complex formation. These highly favorable thermodynamics are consistent with the known critical role for the tetraloop-receptor motif in the folding of large RNAs. Additionally, a significant heat capacity change (DeltaC(p,obs)(o)) of -0.24 kcal mol(-1) K(-1) was determined by ITC. DSC and UV-monitored thermal denaturation experiments indicate that the bivalent tetraloop-receptor construct follows a minimally five-state unfolding pathway and suggest the observed DeltaC(p,obs)(o) for the interaction results from a temperature-dependent unbound receptor RNA structure.     

Systematic Mutational Analysis of Peptide Inhibition of the p53-MDM2/MDMX Interactions

Inhibition of the interaction between the tumor suppressor protein p53 and its negative regulators MDM2 and MDMX is of great interest in cancer biology and drug design. We previously reported a potent duodecimal peptide inhibitor, termed PMI (TSFAEYWNLLSP), of the p53-MDM2 and -MDMX interactions. PMI competes with p53 for MDM2 and MDMX binding at an affinity roughly 2 orders of magnitude higher than that of ^17-28p53 (ETFSDLWKLLPE) of the same length; both peptides adopt nearly identical α-helical conformations in the complexes, where the three highlighted hydrophobic residues Phe, Trp, and Leu dominate PMI or ^17-28p53 binding to MDM2 and MDMX. To elucidate the molecular determinants for PMI activity and specificity, we performed a systematic Ala scanning mutational analysis of PMI and ^17-28p53. The binding affinities for MDM2 and MDMX of a total of 35 peptides including 10 truncation analogs were quantified, affording a complete dissection of energetic contributions of individual residues of PMI and ^17-28p53 to MDM2 and MDMX association. Importantly, the N8A mutation turned PMI into the most potent dual-specific antagonist of MDM2 and MDMX reported to date, registering respective K_d values of 490 pM and 2.4 nM. The co-crystal structure of N8A-PMI-^25-109MDM2 was determined at 1.95 Å, affirming that high-affinity peptide binding to MDM2/MDMX necessitates, in addition to optimized intermolecular interactions, enhanced helix stability or propensity contributed by non-contact residues. The powerful empirical binding data and crystal structures present a unique opportunity for computational studies of peptide inhibition of the p53-MDM2/MDMX interactions.     

Exploring the Minimally Frustrated Energy Landscape of Unfolded ACBP

The unfolded state of globular proteins is not well described by a simple statistical coil due to residual structural features, such as secondary structure or transiently formed long-range contacts. The principle of minimal frustration predicts that the unfolded ensemble is biased toward productive regions in the conformational space determined by the native structure. Transient long-range contacts, both native-like and non-native-like, have previously been shown to be present in the unfolded state of the four-helix-bundle protein acyl co-enzyme binding protein (ACBP) as seen from both perturbations in nuclear magnetic resonance (NMR) chemical shifts and structural ensembles generated from NMR paramagnetic relaxation data. To study the nature of the contacts in detail, we used paramagnetic NMR relaxation enhancements, in combination with single-point mutations, to obtain distance constraints for the acid-unfolded ensemble of ACBP. We show that, even in the acid-unfolded state, long-range contacts are specific in nature and single-point mutations affect the free-energy landscape of the unfolded protein. Using this approach, we were able to map out concerted, interconnected, and productive long-range contacts. The correlation between the native-state stability and compactness of the denatured state provides further evidence for native-like contact formation in the denatured state. Overall, these results imply that, even in the earliest stages of folding, ACBP dynamics are governed by native-like contacts on a minimally frustrated energy landscape.     

Determination of the transition state ensemble for the folding of ubiquitin from a combination of Phi and Psi analyses

Protein engineering techniques have emerged as powerful tools for characterizing transition states (TSs) for protein folding. Recently, the Ψ analysis, in which double-histidine mutations create the possibility of reversible crosslinking in the native state, has been proposed as an additional approach to the well-established Φ analysis. We present here a combination of these two procedures for defining the structure of the TS of ubiquitin, a small α/β protein that has been used extensively as a model system for both experimental and computational studies of the protein-folding process. We performed a series of molecular dynamics simulations in which Φ and Ψ values were used as ensemble-averaged structural restraints to determine an ensemble of structures representing the TS of ubiquitin. Although the available Ψ values for ubiquitin did not, by themselves, generate well-defined TS ensembles, the inclusion of the restricted set of zero or unity values, but not fractional ones, provided useful complementary information to the Φ analysis. Our results show that the TS of ubiquitin is formed by a relatively narrow ensemble of structures exhibiting an overall native-like topology in which the N-terminal and C-terminal regions are in close proximity.     

Determinants of homodimerization specificity in histidine kinases

Two-component signal transduction pathways consisting of a histidine kinase and a response regulator are used by prokaryotes to respond to diverse environmental and intracellular stimuli. Most species encode numerous paralogous histidine kinases that exhibit significant structural similarity. Yet in almost all known examples, histidine kinases are thought to function as homodimers. We investigated the molecular basis of dimerization specificity, focusing on the model histidine kinase EnvZ and RstB, its closest paralog in Escherichia coli. Direct binding studies showed that the cytoplasmic domains of these proteins each form specific homodimers in vitro. Using a series of chimeric proteins, we identified specificity determinants at the base of the four-helix bundle in the dimerization and histidine phosphotransfer domain. Guided by molecular coevolution predictions and EnvZ structural information, we identified sets of residues in this region that are sufficient to establish homospecificity. Mutating these residues in EnvZ to the corresponding residues in RstB produced a functional kinase that preferentially homodimerized over interacting with EnvZ. EnvZ and RstB likely diverged following gene duplication to yield two homodimers that cannot heterodimerize, and the mutants we identified represent possible evolutionary intermediates in this process.     

Rotations of the 2B sub-domain of E. coli UvrD helicase/translocase coupled to nucleotide and DNA binding

Escherichia coli UvrD is a superfamily 1 DNA helicase and single-stranded DNA (ssDNA) translocase that functions in DNA repair and plasmid replication and as an anti-recombinase by removing RecA protein from ssDNA. UvrD couples ATP binding and hydrolysis to unwind double-stranded DNA and translocate along ssDNA with 3′-to-5′ directionality. Although a UvrD monomer is able to translocate along ssDNA rapidly and processively, DNA helicase activity in vitro requires a minimum of a UvrD dimer. Previous crystal structures of UvrD bound to a ssDNA/duplex DNA junction show that its 2B sub-domain exists in a “closed” state and interacts with the duplex DNA. Here, we report a crystal structure of an apo form of UvrD in which the 2B sub-domain is in an “open” state that differs by an ∼ 160° rotation of the 2B sub-domain. To study the rotational conformational states of the 2B sub-domain in various ligation states, we constructed a series of double-cysteine UvrD mutants and labeled them with fluorophores such that rotation of the 2B sub-domain results in changes in fluorescence resonance energy transfer. These studies show that the open and closed forms can interconvert in solution, with low salt favoring the closed conformation and high salt favoring the open conformation in the absence of DNA. Binding of UvrD to DNA and ATP binding and hydrolysis also affect the rotational conformational state of the 2B sub-domain, suggesting that 2B sub-domain rotation is coupled to the function of this nucleic acid motor enzyme.     

Dissecting the non-specific and specific components of the initial folding reaction of barstar by multi-site FRET measurements

Initial polypeptide chain collapse plays a major role in the development of subsequent structure during protein folding, but it has been difficult to elucidate the coupling between its cooperativity and specificity. To better understand this important aspect of protein folding, nine different intramolecular distances in the protein have been measured by fluorescence resonance energy transfer (FRET) in the product(s) of the initial, sub-millisecond collapse reaction during the folding of barstar, under different folding conditions. All nine distances contract in these initial folding products, when the denaturant concentration is reduced. Two of these distances were also measured in peptides corresponding to sequence segments 38-55 and 51-69 of the protein. Surprisingly, both distances do not contract in the peptides which remain fully unfolded when the denaturant concentration is reduced. This suggests that the contraction of at least some segments of the polypeptide chain may be facilitated only by contraction of other segments. In the case of the initial product of folding of the protein, the dependence on denaturant concentration of the relative change in each distance suggests that there are two components to the initial folding reaction. One is a nonspecific component, which appears to be driven by the change in denaturant concentration that is used to initiate refolding. This component corresponds to the collapse of completely unfolded protein (U) to unfolded protein in refolding conditions (U(C)). The extent of nonspecific collapse can be predicted by the response of completely unfolded protein to a change in denaturant concentration. All distances undergo such solvent-induced contraction, but each distance contracts to a different extent. There is also a specific component to initial sub-millisecond folding, in which some distances (but not all) contract more than that predicted by solvent-induced contraction. The observation that only some of the distances undergo contraction over and above solvent-induced contraction, suggest that this specific component is associated with the formation of a specific intermediate (I(E)). FRET efficiency and distance change differently for the different donor-acceptor pairs, with a change in denaturant concentration, indicating that the formation or dissolution of structure in U(C) and I(E) does not happen in a synchronized manner across different regions of the protein molecule. Also, all nine FRET efficiencies and intramolecular distances in the product(s) of sub-ms folding, change continuously with a change in denaturant concentration. Hence, it appears that the transitions from U to U(C) and to I(E) are gradual transformations, and not all-or-none structural transitions. Nevertheless, the product of these gradual transitions, I(E), possesses specific structure.     

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.     

Mechanistic Insights into Ferredoxin-NADP(H) Reductase Catalysis Involving the Conserved Glutamate in the Active Site

Plant-type ferredoxin-NADP(H) reductases (FNRs) are flavoenzymes harboring one molecule of noncovalently bound flavin adenine dinucleotide that catalyze reversible reactions between obligatory one-electron carriers and obligatory two-electron carriers. A glutamate next to the C-terminus is strictly conserved in FNR and has been proposed to function as proton donor/acceptor during catalysis. However, experimental studies of this proposed function led to contradicting conclusions about the role of this glutamate in the catalytic mechanism. In the present work, we study the titration behavior of the glutamate in the active site of FNR using theoretical methods. Protonation probabilities for maize FNR were computed for the reaction intermediates of the catalytic cycle by Poisson-Boltzmann electrostatic calculations and Metropolis Monte Carlo titration. The titration behavior of the highly conserved glutamate was found to vary depending on the bound substrates NADP(H) and ferredoxin and also on the redox states of these substrates and the flavin adenine dinucleotide. Our results support the involvement of the glutamate in the FNR catalytic mechanism not only as a proton donor but also as a key residue for stabilizing and destabilizing reaction intermediates. On the basis of our findings, we propose a model rationalizing the function of the glutamate in the reaction cycle, which allows reinterpretation of previous experimental results.     

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.     

Minimalist representations and the importance of nearest neighbor effects in protein folding simulations

In order to investigate the level of representation required to simulate folding and predict structure, we test the ability of a variety of reduced representations to identify native states in decoy libraries and to recover the native structure given the advanced knowledge of the very broad native Ramachandran basin assignments. Simplifications include the removal of the entire side-chain or the retention of only the Cbeta atoms. Scoring functions are derived from an all-atom statistical potential that distinguishes between atoms and different residue types. Structures are obtained by minimizing the scoring function with a computationally rapid simulated annealing algorithm. Results are compared for simulations in which backbone conformations are sampled from a Protein Data Bank-based backbone rotamer library generated by either ignoring or including a dependence on the identity and conformation of the neighboring residues. Only when the Cbeta atoms and nearest neighbor effects are included do the lowest energy structures generally fall within 4 A of the native backbone root-mean square deviation (RMSD), despite the initial configuration being highly expanded with an average RMSD > or = 10 A. The side-chains are reinserted into the Cbeta models with minimal steric clash. Therefore, the detailed, all-atom information lost in descending to a Cbeta-level representation is recaptured to a large measure using backbone dihedral angle sampling that includes nearest neighbor effects and an appropriate scoring function.     

In vivo interaction between the tobacco lectin and the core histone proteins

Nictaba, a lectin accumulating in tobacco (Nicotiana tabacum) leaves treated with jasmonate, is considered to act as a signaling protein in the stress physiology of the plant. Immunolocalization studies revealed that Nictaba has a nucleocytoplasmic localization. In previous research, histones were identified as primary interaction partners for Nictaba. Here, the interaction between Nictaba and tobacco histones was scrutinized in vivo. Localization studies, performed in stably transformed Nicotiana benthamiana plants, confirmed the nucleocytoplasmic localization of the lectin and colocalization with the presumed binding partners in the nucleus. Furthermore, bimolecular fluorescence complementation (BiFC) assays confirmed the interaction in vivo. Since BiFC signals were also observed for a Nictaba mutant incapable of binding sugar moieties, this interaction may be mediated by alternative binding sites. The interaction of Nictaba with core histones possibly reflects a role of this stress inducible lectin in gene regulation or chromatin remodeling.     

The energetic contribution of induced electrostatic asymmetry to DNA bending by a site-specific protein

DNA bending can be promoted by reducing the net negative electrostatic potential around phosphates on one face of the DNA, such that electrostatic repulsion among phosphates on the opposite face drives bending toward the less negative surface. To provide the first assessment of energetic contribution to DNA bending when electrostatic asymmetry is induced by a site-specific DNA binding protein, we manipulated the electrostatics in the EcoRV endonuclease-DNA complex by mutation of cationic side chains that contact DNA phosphates and/or by replacement of a selected phosphate in each strand with uncharged methylphosphonate. Reducing the net negative charge at two symmetrically located phosphates on the concave DNA face contributes − 2.3 kcal mol^− 1 to − 0.9 kcal mol^− 1 (depending on position) to complex formation. In contrast, reducing negative charge on the opposing convex face produces a penalty of + 1.3 kcal mol^− 1. Förster resonance energy transfer experiments show that the extent of axial DNA bending (about 50°) is little affected in modified complexes, implying that modification affects the energetic cost but not the extent of DNA bending. Kinetic studies show that the favorable effects of induced electrostatic asymmetry on equilibrium binding derive primarily from a reduced rate of complex dissociation, suggesting stabilization of the specific complex between protein and markedly bent DNA. A smaller increase in the association rate may suggest that the DNA in the initial encounter complex is mildly bent. The data imply that protein-induced electrostatic asymmetry makes a significant contribution to DNA bending but is not itself sufficient to drive full bending in the specific EcoRV-DNA complex.     

Crystal Structure of Zebrafish Hatching Enzyme 1 from the Zebrafish Danio rerio

Fish hatching enzymes are zinc metalloproteases that digest the egg envelope (chorion) at the time of hatching. The crystal structure of zebrafish hatching enzyme 1 (ZHE1) has been solved at 1.10 Å resolution. ZHE1 is monomeric, is mitten shaped, and has a cleft at the center of the molecule. ZHE1 consists of three 3₁₀-helices, three α-helices, and two β-sheets. The central cleft represents the active site of the enzyme that is crucial for substrate recognition and catalysis. Alanine-scanning mutagenesis of the two substrate peptides has shown that AspP1′ contributes the most and that the residues at P4-P2′ also contribute to the recognition of the major substrate peptide by ZHE1, whereas GluP3′ and the hydrophobic residues at P4-P2, P2′, and P5′ contribute significantly to the recognition of the minor substrate peptide by ZHE1. Molecular models of these two substrate peptides bound to ZHE1 have been built based on the crystal structure of a transition-state analog inhibitor bound to astacin. In substrate-recognition models, the AspP1′ in the major substrate peptide forms a salt bridge with Arg182 of ZHE1, while the GluP3′ in the minor substrate peptide instead forms a salt bridge with Arg182. Thus, these two substrate peptides would be differently recognized by ZHE1. The shapes and electrostatic potentials of the substrate-binding clefts of ZHE1 and the structurally similar proteins astacin and bone morphogenetic protein 1 are significantly dissimilar due to different side chains, which would confer their distinctive substrate preferences.     

The influence of proline isomerization and off-pathway intermediates on the folding mechanism of eukaryotic UMP/CMP Kinase

The globular 22-kDa protein UMP/CMP from Dictyostelium discoideum (UmpK) belongs to the family of nucleoside monophosphate (NMP) kinases. These enzymes not only show high sequence and structure similarities but also share the α/β-fold, a very common protein topology. We investigated the protein folding mechanism of UmpK as a representative for this ubiquitous enzyme class. Equilibrium stability towards urea and the unfolding and refolding kinetics were studied by means of fluorescence and far-UV CD spectroscopy. Although the unfolding can be described by a two-state process, folding kinetics are rather complex with four refolding phases that can be resolved and an additional burst phase. Moreover, two of these phases exhibit a pronounced rollover in the refolding limb that cannot be explained by aggregation. Whilst secondary structure formation is not observed in the burst phase reaction, folding to the native structure is strongly influenced by the slowest phase, since 30% of the α-helical CD signal is restored therein. This process can be assigned to proline isomerization and is strongly accelerated by the Escherichia coli peptidyl-prolyl isomerase trigger factor. The analysis of our single-mixing and double-mixing experiments suggests the occurrence of an off-pathway intermediate and an unproductive collapsed structure, which appear to be rate limiting for the folding of UmpK.