A Small Helical Bundle Prepares Primer Synthesis by Binding Two Nucleotides that Enhance Sequence-Specific Recognition of the DNA Template.

Primases have a fundamental role in DNA replication. They synthesize a primer that is then extended by DNA polymerases. Archaeoeukaryotic primases require for synthesis a catalytic and an accessory domain, the exact contribution of the latter being unresolved. For the pRN1 archaeal primase, this domain is a 115-amino acid helix bundle domain (HBD). Our structural investigations of this small HBD by liquid- and solid-state nuclear magnetic resonance (NMR) revealed that only the HBD binds the DNA template. DNA binding becomes sequence-specific after a major allosteric change in the HBD, triggered by the binding of two nucleotide triphosphates. The spatial proximity of the two nucleotides and the DNA template in the quaternary structure of the HBD strongly suggests that this small domain brings together the substrates to prepare the first catalytic step of primer synthesis. This efficient mechanism is likely general for all archaeoeukaryotic primases.

Engineering a therapeutic lectin by uncoupling mitogenicity from antiviral activity.

A key effector route of the Sugar Code involves lectins that exert crucial regulatory controls by targeting distinct cellular glycans. We demonstrate that a single amino-acid substitution in a banana lectin, replacing histidine 84 with a threonine, significantly reduces its mitogenicity, while preserving its broad-spectrum antiviral potency. X-ray crystallography, NMR spectroscopy, and glycocluster assays reveal that loss of mitogenicity is strongly correlated with loss of pi-pi stacking between aromatic amino acids H84 and Y83, which removes a wall separating two carbohydrate binding sites, thus diminishing multivalent interactions. On the other hand, monovalent interactions and antiviral activity are preserved by retaining other wild-type conformational features and possibly through unique contacts involving the T84 side chain. Through such fine-tuning, target selection and downstream effects of a lectin can be modulated so as to knock down one activity, while preserving another, thus providing tools for therapeutics and for understanding the Sugar Code.

Initial Primer Synthesis of a DNA Primase Monitored by Real-Time NMR Spectroscopy.

Primases are crucial enzymes for DNA replication, as they synthesize a short primer required for initiating DNA replication. We herein present time-resolved nuclear magnetic resonance (NMR) spectroscopy in solution and in the solid state to study the initial dinucleotide formation reaction of archaeal pRN1 primase. Our findings show that the helix-bundle domain (HBD) of pRN1 primase prepares the two substrates and then hands them over to the catalytic domain to initiate the reaction. By using nucleotide triphosphate analogues, the reaction is substantially slowed down, allowing us to study the initial dinucleotide formation in real time. We show that the sedimented protein-DNA complex remains active in the solid-state NMR rotor and that time-resolved 31P-detected cross-polarization experiments allow monitoring the kinetics of dinucleotide formation. The kinetics in the sedimented protein sample are comparable to those determined by solution-state NMR. Protein conformational changes during primer synthesis are observed in time-resolved 1H-detected experiments at fast magic-angle spinning frequencies (100 kHz). A significant number of spectral changes cluster in the HBD pointing to the importance of the HBD for positioning the nucleotides and the dinucleotide.

Conformational Plasticity of Parallel G-Quadruplex─Implications on Duplex-Quadruplex Motifs.

DNA G-quadruplexes are essential motifs in molecular biology performing a wide range of functions enabled by their unique and diverse structures. In this study, we focus on the conformational plasticity of the most abundant and biologically relevant parallel G-quadruplex topology. A multipronged approach of structure survey, solution-state NMR spectroscopy, and molecular dynamics simulations unravels subtle yet essential features of the parallel G-quadruplex topology. Stark differences in flexibility are observed for the nucleotides depending upon their positioning in the tetrad planes that are intricately correlated with the conformational sampling of the propeller loop. Importantly, the terminal nucleotides in the 5'-end versus the 3'-end of the parallel quadruplex display differential dynamics that manifests their ability to accommodate a duplex on either end of the G-quadruplex. The conformational plasticity characterized in this study provides essential cues toward biomolecular processes such as small molecular binding, intermolecular quadruplex stacking, and implications on how a duplex influences the structure of a neighboring quadruplex.

Adenosine-Dependent Activation Mechanism of Prodrugs Targeting an Aminoacyl-tRNA Synthetase.

Prodrugs have little or no pharmacological activity and are converted to active drugs in the body by enzymes, metabolic reactions, or through human-controlled actions. However, prodrugs promoting their chemical bioconversion without any of these processes have not been reported before. Here, we present an enzyme-independent prodrug activation mechanism by boron-based compounds (benzoxaboroles) targeting leucyl-tRNA synthetase (LeuRS), including an antibiotic that recently has completed phase II clinical trials to cure tuberculosis. We combine nuclear magnetic resonance spectroscopy and X-ray crystallography with isothermal titration calorimetry to show that these benzoxaboroles do not bind directly to their drug target LeuRS, instead they are prodrugs that activate their bioconversion by forming a highly specific and reversible LeuRS inhibition adduct with ATP, AMP, or the terminal adenosine of the tRNALeu. We demonstrate how the oxaborole group of the prodrugs cyclizes with the adenosine ribose at physiological concentrations to form the active molecule. This bioconversion mechanism explains the remarkably good druglike properties of benzoxaboroles showing efficacy against radically different human pathogens and fully explains the mechanism of action of these compounds. Thus, this adenosine-dependent activation mechanism represents a novel concept in prodrug chemistry that can be applied to improve the solubility, permeability and metabolic stability of challenging drugs.

Kinetic Resolution of the Atomic 3D Structures Formed by Ground and Excited Conformational States in an RNA Dynamic Ensemble.

Knowing the 3D structures formed by the various conformations populating the RNA free-energy landscape, their relative abundance, and kinetic interconversion rates is required to obtain a quantitative and predictive understanding of how RNAs fold and function at the atomic level. While methods integrating ensemble-averaged experimental data with computational modeling are helping define the most abundant conformations in RNA ensembles, elucidating their kinetic rates of interconversion and determining the 3D structures of sparsely populated short-lived RNA excited conformational states (ESs) remains challenging. Here, we developed an approach integrating Rosetta-FARFAR RNA structure prediction with NMR residual dipolar couplings and relaxation dispersion that simultaneously determines the 3D structures formed by the ground-state (GS) and ES subensembles, their relative abundance, and kinetic rates of interconversion. The approach is demonstrated on HIV-1 TAR, whose six-nucleotide apical loop was previously shown to form a sparsely populated (∼13%) short-lived (lifetime ∼ 45 µs) ES. In the GS, the apical loop forms a broad distribution of open conformations interconverting on the pico-to-nanosecond time scale. Most residues are unpaired and preorganized to bind the Tat-superelongation protein complex. The apical loop zips up in the ES, forming a narrow distribution of closed conformations, which sequester critical residues required for protein recognition. Our work introduces an approach for determining the 3D ensemble models formed by sparsely populated RNA conformational states, provides a rare atomic view of an RNA ES, and kinetically resolves the atomic 3D structures of RNA conformational substates, interchanging on time scales spanning 6 orders of magnitude, from picoseconds to microseconds.

Elongated Bacterial Pili as a Versatile Alignment Medium for NMR Spectroscopy.

In NMR spectroscopy, residual dipolar couplings (RDCs) have emerged as one of the most exquisite probes of biological structure and dynamics. The measurement of RDCs relies on the partial alignment of the molecule of interest, for example by using a liquid crystal as a solvent. Here, we establish bacterial type 1 pili as an alternative liquid-crystalline alignment medium for the measurement of RDCs. To achieve alignment at pilus concentrations that allow for efficient NMR sample preparation, we elongated wild-type pili by recombinant overproduction of the main structural pilus subunit. Building on the extraordinary stability of type 1 pili against spontaneous dissociation and unfolding, we show that the medium is compatible with challenging experimental conditions such as high temperature, the presence of detergents, organic solvents or very acidic pH, setting it apart from most established alignment media. Using human ubiquitin, HIV-1 TAR RNA and camphor as spectroscopic probes, we demonstrate the applicability of the medium for the determination of RDCs of proteins, nucleic acids and small molecules. Our results show that type 1 pili represent a very useful alternative to existing alignment media and may readily assist the characterization of molecular structure and dynamics by NMR.

Multimodal Response to Copper Binding in Superoxide Dismutase Dynamics.

Copper/zinc superoxide dismutase (SOD) is a homodimeric metalloenzyme that has been extensively studied as a benchmark for structure-function relationships in proteins, in particular because of its implication in the familial form of the neurodegenerative disease amyotrophic lateral sclerosis. Here, we investigate microcrystalline preparations of two differently metalated forms of SOD, namely, the fully mature functional Cu,Zn state and the E,Zn-SOD state in which the Cu site is empty. By using solid-state NMR with fast magic-angle spinning (MAS) at high magnetic fields (1H Larmor frequency of 800-1000 MHz), we quantify motions spanning a dynamic range from ns to ms. We determine that metal ion uptake does not act as a rigidification element but as a switch redistributing motional processes on different time scales, with coupling of the dynamics of histidine side chains and those of remote key backbone elements of the protein.

Activity of Tumor Necrosis Factor α Is Modulated by Dynamic Conformational Rearrangements.

The homotrimeric ligand tumor necrosis factor α (TNFα) is a key cytokine and immune regulator; however, when deregulated, it leads to several major chronic inflammatory diseases. Perturbation of the protein-protein interface has proven to be an efficient strategy to inactivate TNFα, but the atomic-resolution mechanism of its inactivation remains poorly understood. Here, we probe the solution structure and dynamics of active and inactive TNFα using NMR spectroscopy. The data reveal that TNFα undergoes motions on different time scales. Furthermore, by site-directed mutagenesis of residues at the trimerization interface and by targeting the interface with a low molecular weight inhibitor, we show that TNFα retains its overall structure and trimeric state. However, upon perturbation, TNFα exhibits increased conformational dynamics spanning from the trimerization interface to the regions mediating receptor binding. These findings provide novel insights into the inactivation mechanism of TNFα and the basis for strategies to target TNFα activity.

Dynamic Descriptions of Highly Flexible Molecules from NMR Dipolar Couplings: Physical Basis and Limitations.

Biomolecules that control physiological function by changing their conformation play key roles in biology and remain poorly characterized. NMR dipolar couplings (DCs) depend intrinsically on both molecular shape and structural fluctuations, thereby providing the enticing prospect of tracking these conformational changes at atomic detail. Although this dual dependence has until now severely complicated analysis of DCs from highly dynamic systems, general approaches have recently been proposed that simplify interpretation of experimental DCs, by entirely eliminating molecular alignment from the analysis. Using simple and intuitive simulation of target ensembles, we investigate the impact of such approaches on the resulting descriptions of the conformational energy landscape. We find that ensemble descriptions of highly flexible systems derived from DCs without explicit consideration of the alignment properties of the constituent conformations can be compromised and inaccurate, despite exhibiting high correlation with experimental measurement.

Measuring similarity between dynamic ensembles of biomolecules.

We present a simple and general approach termed REsemble for quantifying population overlap and structural similarity between ensembles. This approach captures improvements in the quality of ensembles determined using increasing input experimental data--improvements that go undetected when conventional methods for comparing ensembles are used--and reveals unexpected similarities between RNA ensembles determined using NMR and molecular dynamics simulations.

HdeB functions as an acid-protective chaperone in bacteria.

Enteric bacteria such as Escherichia coli utilize various acid response systems to counteract the acidic environment of the mammalian stomach. To protect their periplasmic proteome against rapid acid-mediated damage, bacteria contain the acid-activated periplasmic chaperones HdeA and HdeB. Activation of HdeA at pH 2 was shown to correlate with its acid-induced dissociation into partially unfolded monomers. In contrast, HdeB, which has high structural similarities to HdeA, shows negligible chaperone activity at pH 2 and only modest chaperone activity at pH 3. These results raised intriguing questions concerning the physiological role of HdeB in bacteria, its activation mechanism, and the structural requirements for its function as a molecular chaperone. In this study, we conducted structural and biochemical studies that revealed that HdeB indeed works as an effective molecular chaperone. However, in contrast to HdeA, whose chaperone function is optimal at pH 2, the chaperone function of HdeB is optimal at pH 4, at which HdeB is still fully dimeric and largely folded. NMR, analytical ultracentrifugation, and fluorescence studies suggest that the highly dynamic nature of HdeB at pH 4 alleviates the need for monomerization and partial unfolding. Once activated, HdeB binds various unfolding client proteins, prevents their aggregation, and supports their refolding upon subsequent neutralization. Overexpression of HdeA promotes bacterial survival at pH 2 and 3, whereas overexpression of HdeB positively affects bacterial growth at pH 4. These studies demonstrate how two structurally homologous proteins with seemingly identical in vivo functions have evolved to provide bacteria with the means for surviving a range of acidic protein-unfolding conditions.

Capturing a Dynamic Chaperone-Substrate Interaction Using NMR-Informed Molecular Modeling.

Chaperones maintain a healthy proteome by preventing aggregation and by aiding in protein folding. Precisely how chaperones influence the conformational properties of their substrates, however, remains unclear. To achieve a detailed description of dynamic chaperone-substrate interactions, we fused site-specific NMR information with coarse-grained simulations. Our model system is the binding and folding of a chaperone substrate, immunity protein 7 (Im7), with the chaperone Spy. We first used an automated procedure in which NMR chemical shifts inform the construction of system-specific force fields that describe each partner individually. The models of the two binding partners are then combined to perform simulations on the chaperone-substrate complex. The binding simulations show excellent agreement with experimental data from multiple biophysical measurements. Upon binding, Im7 interacts with a mixture of hydrophobic and hydrophilic residues on Spy's surface, causing conformational exchange within Im7 to slow down as Im7 folds. Meanwhile, the motion of Spy's flexible loop region increases, allowing for better interaction with different substrate conformations, and helping offset losses in Im7 conformational dynamics that occur upon binding and folding. Spy then preferentially releases Im7 into a well-folded state. Our strategy has enabled a residue-level description of a dynamic chaperone-substrate interaction, improving our understanding of how chaperones facilitate substrate folding. More broadly, we validate our approach using two other binding partners, showing that this approach provides a general platform from which to investigate other flexible biomolecular complexes through the integration of NMR data with efficient computational models.

Inter-helical conformational preferences of HIV-1 TAR-RNA from maximum occurrence analysis of NMR data and molecular dynamics simulations.

Detecting conformational heterogeneity in biological macromolecules is a key for the understanding of their biological function. We here provide a comparison between two independent approaches to assess conformational heterogeneity: molecular dynamics simulations, performed without inclusion of any experimental data, and maximum occurrence (MaxOcc) distribution over the topologically available conformational space. The latter only reflects the extent of the averaging and identifies regions which are most compliant with the experimentally measured NMR Residual Dipolar Couplings (RDCs). The analysis was performed for the HIV-1 TAR RNA, consisting of two helical domains connected by a flexible bulge junction, for which four sets of RDCs were available as well as an 8.2 µs all-atom molecular dynamics simulation. A sample and select approach was previously applied to extract from the molecular dynamics trajectory conformational ensembles in agreement with the four sets of RDCs. The MaxOcc analysis performed here identifies the most likely sampled region in the conformational space of the system which, strikingly, overlaps well with the structures independently sampled in the molecular dynamics calculations and even better with the RDC selected ensemble.

Efficient in silico exploration of RNA interhelical conformations using Euler angles and WExplore.

HIV-1 TAR RNA is a two-helix bulge motif that plays a critical role in HIV viral replication and is an important drug target. However, efforts at designing TAR inhibitors have been challenged by its high degree of structural flexibility, which includes slow large-amplitude reorientations of its helices with respect to one another. Here, we use the recently introduced algorithm WExplore in combination with Euler angles to achieve unprecedented sampling of the TAR conformational ensemble. Our ensemble achieves similar agreement with experimental NMR data when compared with previous TAR computational studies, and is generated at a fraction of the computational cost. It clearly emerges from configuration space network analysis that the intermittent formation of the A22-U40 base pair acts as a reversible switch that enables sampling of interhelical conformations that would otherwise be topologically disallowed. We find that most previously determined ligand-bound structures are found in similar location in the network, and we use a sample-and-select approach to guide the construction of a set of novel conformations which can serve as the basis for future drug development efforts. Collectively, our findings demonstrate the utility of WExplore in combination with suitable order parameters as a method for exploring RNA conformational space.

Modulating RNA Alignment Using Directional Dynamic Kinks: Application in Determining an Atomic-Resolution Ensemble for a Hairpin using NMR Residual Dipolar Couplings.

Approaches that combine experimental data and computational molecular dynamics (MD) to determine atomic resolution ensembles of biomolecules require the measurement of abundant experimental data. NMR residual dipolar couplings (RDCs) carry rich dynamics information, however, difficulties in modulating overall alignment of nucleic acids have limited the ability to fully extract this information. We present a strategy for modulating RNA alignment that is based on introducing variable dynamic kinks in terminal helices. With this strategy, we measured seven sets of RDCs in a cUUCGg apical loop and used this rich data set to test the accuracy of an 0.8 µs MD simulation computed using the Amber ff10 force field as well as to determine an atomic resolution ensemble. The MD-generated ensemble quantitatively reproduces the measured RDCs, but selection of a sub-ensemble was required to satisfy the RDCs within error. The largest discrepancies between the RDC-selected and MD-generated ensembles are observed for the most flexible loop residues and backbone angles connecting the loop to the helix, with the RDC-selected ensemble resulting in more uniform dynamics. Comparison of the RDC-selected ensemble with NMR spin relaxation data suggests that the dynamics occurs on the ps-ns time scales as verified by measurements of R(1ρ) relaxation-dispersion data. The RDC-satisfying ensemble samples many conformations adopted by the hairpin in crystal structures indicating that intrinsic plasticity may play important roles in conformational adaptation. The approach presented here can be applied to test nucleic acid force fields and to characterize dynamics in diverse RNA motifs at atomic resolution.

Investigating protein conformational energy landscapes and atomic resolution dynamics from NMR dipolar couplings: a review.

Nuclear magnetic resonance spectroscopy is exquisitely sensitive to protein dynamics. In particular inter-nuclear dipolar couplings, that become measurable in solution when the protein is dissolved in a dilute liquid crystalline solution, report on all conformations sampled up to millisecond timescales. As such they provide the opportunity to describe the Boltzmann distribution present in solution at atomic resolution, and thereby to map the conformational energy landscape in unprecedented detail. The development of analytical methods and approaches based on numerical simulation and their application to numerous biologically important systems is presented.

Insights into domain-domain motions in proteins and RNA from solution NMR.

Many multidomain proteins and ribonucleic acids consist of domains that autonomously fold and that are linked together by flexible junctions. This architectural design allows domains to sample a wide range of positions with respect to one another, yet do so in a way that retains structural specificity, since the number of sampled conformations remains extremely small compared to the total conformations that would be sampled if the domains were connected by an infinitely long linker. This "tuned" flexibility in interdomain conformation is in turn used in many biochemical processes. There is great interest in characterizing the dynamic properties of multidomain systems, and moving beyond conventional descriptions in terms of static structures, toward the characterization of population-weighted ensembles describing a distribution of many conformations sampled in solution. There is also great interest in understanding the design principles and underlying physical and chemical interactions that specify the nature of interdomain flexibility. NMR spectroscopy is one of the most powerful techniques for characterizing motions in complex biomolecules and has contributed greatly toward our basic understanding of dynamics in proteins and nucleic acids and its role in folding, recognition, and signaling. Here, we review methods that have been developed in our laboratories to address these challenges. Our approaches are based on the ability of one domain of the molecule to self-align in a magnetic field, or to dominate the overall orientation of the molecule, so that the conformational freedom of other domains can be assessed by their degree of alignment induced by the aligned part. In turn, this self-alignment ability can be intrinsic or can be caused by tagging appropriate constructs to the molecule of interest. In general, self-alignment is due to magnetic susceptibility anisotropy. Nucleic acids with elongated helices have this feature, as well as several paramagnetic metal centers that can be found in, or attached to, a protein domain.

Advances in the determination of nucleic acid conformational ensembles.

Conformational changes in nucleic acids play a key role in the way genetic information is stored, transferred, and processed in living cells. Here, we describe new approaches that employ a broad range of experimental data, including NMR-derived chemical shifts and residual dipolar couplings, small-angle X-ray scattering, and computational approaches such as molecular dynamics simulations to determine ensembles of DNA and RNA at atomic resolution. We review the complementary information that can be obtained from diverse sets of data and the various methods that have been developed to combine these data with computational methods to construct ensembles and assess their uncertainty. We conclude by surveying RNA and DNA ensembles determined using these methods, highlighting the unique physical and functional insights obtained so far.

Intrinsic disorder in measles virus nucleocapsids.

The genome of measles virus is encapsidated by multiple copies of the nucleoprotein (N), forming helical nucleocapsids of molecular mass approaching 150 Megadalton. The intrinsically disordered C-terminal domain of N (N(TAIL)) is essential for transcription and replication of the virus via interaction with the phosphoprotein P of the viral polymerase complex. The molecular recognition element (MoRE) of N(TAIL) that binds P is situated 90 amino acids from the folded RNA-binding domain (N(CORE)) of N, raising questions about the functional role of this disordered chain. Here we report the first in situ structural characterization of N(TAIL) in the context of the entire N-RNA capsid. Using nuclear magnetic resonance spectroscopy, small angle scattering, and electron microscopy, we demonstrate that N(TAIL) is highly flexible in intact nucleocapsids and that the MoRE is in transient interaction with N(CORE). We present a model in which the first 50 disordered amino acids of N(TAIL) are conformationally restricted as the chain escapes to the outside of the nucleocapsid via the interstitial space between successive N(CORE) helical turns. The model provides a structural framework for understanding the role of N(TAIL) in the initiation of viral transcription and replication, placing the flexible MoRE close to the viral RNA and, thus, positioning the polymerase complex in its functional environment.