Excited Conformational States Research Articles

Excited States of apo-Guanidine-III Riboswitch Contribute to Guanidinium Binding through Both Conformational and Induced-Fit Mechanisms.

Riboswitches are mRNA segments that regulate gene expression through conformational changes driven by their cognate ligand binding. The ykkC motif forms a riboswitch class that selectively senses a guanidinium ion (Gdm+) and regulates the downstream expression of proteins which aid in the efflux of excess Gdm+ from the cells. The aptamer domain (AD) of the guanidine-III riboswitch forms an H-type pseudoknot with a triple helical domain that binds a Gdm+. We studied the binding of Gdm+ to the AD of the guanidine (ykkC)-III riboswitch using computer simulations to probe the specificity of the riboswitch to Gdm+ binding. We show that Gdm+ binding is a fast process occurring on the nanosecond time scale, with minimal conformational changes to the AD. Using machine learning and Markov-state models, we identified the excited conformational states of the AD, which have a high Gdm+ binding propensity, making the Gdm+ binding landscape complex exhibiting both conformational selection and induced-fit mechanisms. The proposed apo-AD excited states and their role in the ligand-sensing mechanism are amenable to experimental verification. Further, targeting these excited-state conformations in discovering new antibiotics can be explored.

An RNA excited conformational state at atomic resolution

Sparse and short-lived excited RNA conformational states are essential players in cell physiology, disease, and therapeutic development, yet determining their 3D structures remains challenging. Combining mutagenesis, NMR spectroscopy, and computational modeling, we determined the 3D structural ensemble formed by a short-lived (lifetime ~2.1 ms) lowly-populated (~0.4%) conformational state in HIV-1 TAR RNA. Through a strand register shift, the excited conformational state completely remodels the 3D structure of the ground state (RMSD from the ground state = 7.2 ± 0.9 Å), forming a surprisingly more ordered conformational ensemble rich in non-canonical mismatches. The structure impedes the formation of the motifs recognized by Tat and the super elongation complex, explaining why this alternative TAR conformation cannot activate HIV-1 transcription. The ability to determine the 3D structures of fleeting RNA states using the presented methodology holds great promise for our understanding of RNA biology, disease mechanisms, and the development of RNA-targeting therapeutics.

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.

Pressure pushes tRNALys3 into excited conformational states

Conformational dynamics play essential roles in RNA function. However, detailed structural characterization of excited states of RNA remains challenging. Here, we apply high hydrostatic pressure (HP) to populate excited conformational states of tRNALys3, and structurally characterize them using a combination of HP 2D-NMR, HP-SAXS (HP-small-angle X-ray scattering), and computational modeling. HP-NMR revealed that pressure disrupts the interactions of the imino protons of the uridine and guanosine U-A and G-C base pairs of tRNALys3. HP-SAXS profiles showed a change in shape, but no change in overall extension of the transfer RNA (tRNA) at HP. Configurations extracted from computational ensemble modeling of HP-SAXS profiles were consistent with the NMR results, exhibiting significant disruptions to the acceptor stem, the anticodon stem, and the D-stem regions at HP. We propose that initiation of reverse transcription of HIV RNA could make use of one or more of these excited states.

High-pressure studies reveal the conformational landscape of the small GTPase Arf6.

Arf6, a small GTPase, functions as a protein “switch” by coupling the binding and subsequent hydrolysis of GTP to a variety of signaling cascades within the cell. Despite having significant degrees of sequence and structural homology in both the GDP and GTP-bound forms to the more well-characterized protein Arf1, the two proteins exhibit distinct spatial and functional segregation: Arf1 is responsible for membrane trafficking between the Endoplasmic Reticulum and Golgi Apparatus, while Arf6 is responsible for trafficking between the plasma membrane and endosome. Furthermore, the accessory proteins required for in vivo functionality of the switch are not interchangeable. Arf proteins are known to undergo massive structural rearrangements during the nucleotide switch transition, especially in the N-terminal and switch regions. Thus, it is unlikely that Arf protein binding partners’ specificity stems purely from the end state structures. We hypothesize that the excited conformations sampled during this transition are responsible for the observed specificity. To characterize these excited conformational states, our lab utilizes pressure-induced perturbation techniques which locally unfold the protein. We gain insights into the state of the protein using a two-pronged approach consisting of both high-pressure NMR and high-pressure SAXS; this has allowed us to develop residue-specific unfolding curves for Arf6 as well as pair-distance distribution curves to understand the global shape of the protein at various pressures. Using these results, we have begun to structurally map the conformational landscape of Arf6.

Demonstration that Small Molecules can Bind and Stabilize Low-abundance Short-lived RNA Excited Conformational States

Many promising RNA drug targets have functions that require the formation of RNA-protein complexes, but inhibiting RNA-protein interactions can prove difficult using small molecules. Regulatory RNAs have been shown to transiently form excited conformational states (ESs) that remodel local aspects of secondary structure. In some cases, the ES conformation has been shown to be inactive and to be poorly recognized by protein binding partners. In these cases, specifically targeting and stabilizing the RNA ES using a small molecule provides a rational structure-based strategy for inhibiting RNA activity. However, this requires that a small molecule discriminates between two conformations of the same RNA to preferentially bind and stabilize the short-lived low-abundance ES relative to the long-lived more abundant ground state (GS). Here, we tested the feasibility of this approach by designing a mutant that inverts the conformational equilibrium of the HIV-1 transactivation response element (TAR) RNA, such that the native GS conformation becomes a low-abundance ES. Using this mutant and NMR chemical shift mapping experiments, we show that argininamide, a ligand mimic of TAR's cognate protein binding partner Tat, is able to restore a native-like conformation by preferentially binding and stabilizing the transient and low-populated ES. A synthetic small molecule optimized to bind the TAR GS also partially stabilized the ES, whereas an aminoglycoside molecule that binds RNAs nonspecifically did not preferentially stabilize the ES to a similar extent. These results support the feasibility of inhibiting RNA activity using small molecules that preferentially bind and stabilize the ES.

Probing excited conformational states of nucleic acids by nitrogen CEST NMR spectroscopy.

Characterizing low-populated and short-lived excited conformational states has become increasingly important for understanding mechanisms of RNA function. Interconversion between RNA ground and excited conformational states often involves base pairing rearrangements that lead to changes in the hydrogen-bond network. Here, we present two 15N chemical exchange saturation transfer (CEST) NMR experiments that utilize protonated and non-protonated nitrogens, which are key hydrogen-bond donors and acceptors, for characterizing excited conformational states in RNA. We demonstrated these approaches on the B. Cereus fluoride riboswitch, where 15N CEST profiles complement 13C CEST profiles in depicting a potential pathway for ligand-dependent allosteric regulation of the excited conformational state of the fluoride riboswitch.

A Methyl‐TROSY‐Based 1H Relaxation Dispersion Experiment for Studies of Conformational Exchange in High Molecular Weight Proteins

AbstractMolecular complexes often sample conformational states that direct them to specific functions. These states can be difficult to observe through traditional biophysical approaches but they can be studied using a variety of different NMR spin relaxation experiments. However, these applications, when focused on moderate to high molecular weight proteins, are complicated by fast relaxing signals that negatively affect the sensitivity and resolution of spectra. Here a methyl 1H CPMG‐based experiment for studies of excited conformational states of protein machines is described that exploits a TROSY‐effect to increase signal‐to‐noise. Complexities from the multiplicity of methyl 1H transitions are addressed to generate a robust pulse scheme that is applied to a 320 kDa homeostasis protein, p97.

A Methyl-TROSY-Based 1 H Relaxation Dispersion Experiment for Studies of Conformational Exchange in High Molecular Weight Proteins.

Molecular complexes often sample conformational states that direct them to specific functions. These states can be difficult to observe through traditional biophysical approaches but they can be studied using a variety of different NMR spin relaxation experiments. However, these applications, when focused on moderate to high molecular weight proteins, are complicated by fast relaxing signals that negatively affect the sensitivity and resolution of spectra. Here a methyl 1 H CPMG-based experiment for studies of excited conformational states of protein machines is described that exploits a TROSY-effect to increase signal-to-noise. Complexities from the multiplicity of methyl 1 H transitions are addressed to generate a robust pulse scheme that is applied to a 320 kDa homeostasis protein, p97.

A spin-1 representation for dual-funnel energy landscapes.

The interconversion between the left- and right-handed helical folds of a polypeptide defines a dual-funneled free energy landscape. In this context, the funnel minima are connected through a continuum of unfolded conformations, evocative of the classical helix-coil transition. Physical intuition and recent conjectures suggest that this landscape can be mapped by assigning a left- or right-handed helical state to each residue. We explore this possibility using all-atom replica exchange molecular dynamics and an Ising-like model, demonstrating that the energy landscape architecture is at odds with a two-state picture. A three-state model-left, right, and unstructured-can account for most key intermediates during chiral interconversion. Competing folds and excited conformational states still impose limitations on the scope of this approach. However, the improvement is stark: Moving from a two-state to a three-state model decreases the fit error from 1.6 kBT to 0.3 kBT along the left-to-right interconversion pathway.

Retinal isomerization in bacteriorhodopsin captured by a femtosecond x-ray laser.

Ultrafast isomerization of retinal is the primary step in photoresponsive biological functions including vision in humans and ion transport across bacterial membranes. We used an x-ray laser to study the subpicosecond structural dynamics of retinal isomerization in the light-driven proton pump bacteriorhodopsin. A series of structural snapshots with near-atomic spatial resolution and temporal resolution in the femtosecond regime show how the excited all-trans retinal samples conformational states within the protein binding pocket before passing through a twisted geometry and emerging in the 13-cis conformation. Our findings suggest ultrafast collective motions of aspartic acid residues and functional water molecules in the proximity of the retinal Schiff base as a key facet of this stereoselective and efficient photochemical reaction.

Overview of Relaxation Dispersion NMR Spectroscopy to Study Protein Dynamics and Protein-Ligand Interactions.

Proteins and nucleic acids are central to all biological processes. NMR spectroscopy has proven to be excellent for studying the dynamics of these macromolecules over various timescales. Relaxation rates and heteronuclear nuclear Overhauser-effect values can resolve motion on pico- to nanosecond timescales, residual dipolar couplings provide information on submicro- to millisecond timescales, and even slower dynamics over seconds to hours can be resolved by hydrogen-exchange experiments. Relaxation dispersion experiments are especially valuable because they resolve motion on micro- to millisecond timescales, encompassing biomolecular motions associated with ligand binding, enzymatic catalysis, and domain-domain opening. These experiments provide structural, kinetic, and thermodynamic information on "invisible" excited conformational states. Relaxation dispersion can be applied not only to single biomolecules but also to protein-ligand complexes to study the kinetics and thermodynamics of association and dissociation. We review recent developments in relaxation dispersion methodology, outline the R1ρ relaxation dispersion experiment, and discuss application to biomolecular interactions. © 2018 by John Wiley & Sons, Inc.

Shortening the HIV-1 TAR RNA Bulge by a Single Nucleotide Preserves Motional Modes over a Broad Range of Time Scales.

Helix-junction-helix (HJH) motifs are flexible building blocks of RNA architecture that help define the orientation and dynamics of helical domains. They are also frequently involved in adaptive recognition of proteins and small molecules and in the formation of tertiary contacts. Here, we use a battery of nuclear magnetic resonance techniques to examine how deleting a single bulge residue (C24) from the human immunodeficiency virus type 1 (HIV-1) transactivation response element (TAR) trinucleotide bulge (U23-C24-U25) affects dynamics over a broad range of time scales. Shortening the bulge has an effect on picosecond-to-nanosecond interhelical and local bulge dynamics similar to that casued by increasing the Mg(2+) and Na(+) concentration, whereby a preexisting two-state equilibrium in TAR is shifted away from a bent flexible conformation toward a coaxial conformation, in which all three bulge residues are flipped out and flexible. Surprisingly, the point deletion minimally affects microsecond-to-millisecond conformational exchange directed toward two low-populated and short-lived excited conformational states that form through reshuffling of bases pairs throughout TAR. The mutant does, however, adopt a slightly different excited conformational state on the millisecond time scale, in which U23 is intrahelical, mimicking the expected conformation of residue C24 in the excited conformational state of wild-type TAR. Thus, minor changes in HJH topology preserve motional modes in RNA occurring over the picosecond-to-millisecond time scales but alter the relative populations of the sampled states or cause subtle changes in their conformational features.

Measuring Residual Dipolar Couplings in Excited Conformational States of Nucleic Acids by CEST NMR Spectroscopy.

Nucleic acids undergo structural transitions to access sparsely populated and transiently lived conformational states--or excited conformational states--that play important roles in diverse biological processes. Despite ever-increasing detection of these functionally essential states, 3D structure determination of excited states (ESs) of RNA remains elusive. This is largely due to challenges in obtaining high-resolution structural constraints in these ESs by conventional structural biology approaches. Here, we present nucleic-acid-optimized chemical exchange saturation transfer (CEST) NMR spectroscopy for measuring residual dipolar couplings (RDCs), which provide unique long-range angular constraints in ESs of nucleic acids. We demonstrate these approaches on a fluoride riboswitch, where one-bond (13)C-(1)H RDCs from both base and sugar moieties provide direct structural probes into an ES of the ligand-free riboswitch.

Characterizing excited conformational states of RNA by NMR spectroscopy

Characterizing excited conformational states of RNA by NMR spectroscopy

Measuring Residual Dipolar Couplings in Excited Conformational States of Nucleic Acids by CEST NMR Spectroscopy

Measuring Residual Dipolar Couplings in Excited Conformational States of Nucleic Acids by CEST NMR Spectroscopy

Measurement of Proton Chemical Shifts in Invisible States of Slowly Exchanging Protein Systems by Chemical Exchange Saturation Transfer

Chemical exchange saturation transfer (CEST) NMR spectroscopy has emerged as a powerful technique for studies of transiently formed, sparsely populated (excited) conformational states of protein molecules in slow exchange with a dominant structure. The most popular form of the experiment, and the version originally developed, uses a weak (1)H radio frequency field to perturb longitudinal magnetization of one state with the effect transferred to magnetization in the second conformation via chemical exchange. A significant limitation of the method for protein applications emerges from (1)H magnetization transfer via dipolar relaxation (NOE effect) that can severely complicate analysis of the resulting CEST profile. This is particularly an issue since the (1)H chemical shifts of the excited state, critical for structural studies of these elusive conformers, become difficult to extract. Here we present a method for measurement of these shifts via CEST experiments in which the NOE effect is not an issue. The methodology is illustrated through applications to a pair of exchanging systems where the results are cross-validated.

Lipid-Mediated Folding/Unfolding of Phospholamban as a Regulatory Mechanism for the Sarcoplasmic Reticulum Ca2+-ATPase

The integral membrane protein complex between phospholamban (PLN) and sarcoplasmic reticulum Ca2+-ATPase (SERCA) regulates cardiac contractility. In the unphosphorylated form, PLN binds SERCA and inhibits Ca2+ flux. Upon phosphorylation of PLN at Ser16, the inhibitory effect is reversed. Although structural details on both proteins are emerging from X-ray crystallography, cryo-electron microscopy, and NMR studies, the molecular mechanisms of their interactions and regulatory process are still lacking. It has been speculated that SERCA regulation depends on PLN structural transitions (order to disorder, i.e., folding/unfolding). Here, we investigated PLN conformational changes upon chemical unfolding by a combination of electron paramagnetic resonance and NMR spectroscopies, revealing that the conformational transitions involve mostly the cytoplasmic regions, with two concomitant phenomena: (1) membrane binding and folding of the amphipathic domain Ia and (2) folding/unfolding of the juxtamembrane domain Ib of PLN. Analysis of phosphorylated and unphosphorylated PLN with two phosphomimetic mutants of PLN (S16E and S16D) shows that the population of an unfolded state in domains Ia and Ib (T′ state) is linearly correlated to the extent of SERCA inhibition measured by activity assays. Inhibition of SERCA is carried out by the folded ground state (T state) of the protein (PLN), while the relief of inhibition involves promotion of PLN to excited conformational states (Ser16 phosphorylated PLN). We propose that PLN population shifts (folding/unfolding) are a key regulatory mechanism for SERCA.

An Energetic Representation of Protein Architecture that is Independent of Primary and Secondary Structure

Protein fold classification often assumes that similarity in primary, secondary, or tertiary structure signifies a common evolutionary origin. However, when similarity is not obvious, it is sometimes difficult to conclude that particular proteins are completely unrelated. Clearly, a set of organizing principles that is independent of traditional classification could be valuable in linking different structural motifs and identifying common ancestry from seemingly disparate folds. Here, a four-dimensional ensemble-based energetic space spanned by a diverse set of proteins was defined and its characteristics were contrasted with those of Cartesian coordinate space. Eigenvector decomposition of this energetic space revealed the dominant physical processes contributing to the more or less stable regions of a protein. Unexpectedly, those processes were identical for proteins with different secondary structure content and were also identical among different amino acid types. The implications of these results are two-fold. First, it indicates that excited conformational states comprising the protein native state ensemble, largely invisible upon inspection of the high-resolution structure, are the major determinant of the energetic space. Second, it suggests that folds dissimilar in sequence or structure could nonetheless be energetically similar if their respective excited conformational states are considered, one example of which was observed in the N-terminal region of the Arc repressor switch mutant. Taken together, these results provide a surface area-based framework for understanding folds in energetic terms, a framework that may eventually yield a means of identifying common ancestry among structurally dissimilar proteins.

An Energetic Representation of Protein Architecture that Is Independent of Primary and Secondary Structure

Protein fold classification often assumes that similarity in primary, secondary, or tertiary structure signifies a common evolutionary origin. However, when similarity is not obvious, it is sometimes difficult to conclude that particular proteins are completely unrelated. Clearly, a set of organizing principles that is independent of traditional classification could be valuable in linking different structural motifs and identifying common ancestry from seemingly disparate folds. Here, a four-dimensional ensemble-based energetic space spanned by a diverse set of proteins was defined and its characteristics were contrasted with those of Cartesian coordinate space. Eigenvector decomposition of this energetic space revealed the dominant physical processes contributing to the more or less stable regions of a protein. Unexpectedly, those processes were identical for proteins with different secondary structure content and were also identical among different amino-acid types. The implications of these results are twofold. First, it indicates that excited conformational states comprising the protein native state ensemble, largely invisible upon inspection of the high-resolution structure, are the major determinant of the energetic space. Second, it suggests that folds dissimilar in sequence or structure could nonetheless be energetically similar if their respective excited conformational states are considered, one example of which was observed in the N-terminal region of the Arc repressor switch mutant. Taken together, these results provide a surface area-based framework for understanding folds in energetic terms, a framework that may eventually yield a means of identifying common ancestry among structurally dissimilar proteins.