Ligand binding to a high‐energy partially unfolded protein

The conformational energy landscape of a protein determines populations of all possible conformations of the protein and also determines the kinetics of the conversion between the conformations. Interaction with ligands influences the conformational energy landscapes of proteins and shifts populations of proteins in different conformational states. To investigate the effect of ligand binding on partial unfolding of a protein, we use Escherichia coli dihydrofolate reductase (DHFR) and its functional ligand NADP⁺ as a model system. We previously identified a partially unfolded form of DHFR that is populated under native conditions. In this report, we determined the free energy for partial unfolding of DHFR at varying concentrations of NADP⁺ and found that NADP⁺ binds to the partially unfolded form as well as the native form. DHFR unfolds partially without releasing the ligand, though the binding affinity for NADP⁺ is diminished upon partial unfolding. Based on known crystallographic structures of NADP⁺‐bound DHFR and the model of the partially unfolded protein we previously determined, we propose that the adenosine‐binding domain of DHFR remains folded in the partially unfolded form and interacts with the adenosine moiety of NADP⁺. Our result demonstrates that ligand binding may affect the conformational free energy of not only native forms but also high‐energy non‐native forms.     

Mg2+-induced conformational changes in the btuB riboswitch from E. coli

Mg²⁺ has been shown to modulate the function of riboswitches by facilitating the ligand-riboswitch interactions. The btuB riboswitch from Escherichia coli undergoes a conformational change upon binding to its ligand, coenzyme B₁₂ (adenosyl-cobalamine, AdoCbl), and down-regulates the expression of the B₁₂ transporter protein BtuB in order to control the cellular levels of AdoCbl. Here, we discuss the structural folding attained by the btuB riboswitch from E. coli in response to Mg²⁺ and how it affects the ligand binding competent conformation of the RNA. The btuB riboswitch notably adopts different conformational states depending upon the concentration of Mg²⁺. With the help of in-line probing, we show the existence of at least two specific conformations, one being achieved in the complete absence of Mg²⁺ (or low Mg²⁺ concentration) and the other appearing above ∼0.5 mM Mg²⁺. Distinct regions of the riboswitch exhibit different dissociation constants toward Mg²⁺, indicating a stepwise folding of the btuB RNA. Increasing the Mg²⁺ concentration drives the transition from one conformation toward the other. The conformational state existing above 0.5 mM Mg²⁺ defines the binding competent conformation of the btuB riboswitch which can productively interact with the ligand, coenzyme B₁₂, and switch the RNA conformation. Moreover, raising the Mg²⁺ concentration enhances the ratio of switched RNA in the presence of AdoCbl. The lack of a AdoCbl-induced conformational switch experienced by the btuB riboswitch in the absence of Mg²⁺ indicates a crucial role played by Mg²⁺ for defining an active conformation of the riboswitch.     

Atomic-level insights into metabolite recognition and specificity of the SAM-II riboswitch

Although S-adenosylhomocysteine (SAH), a metabolic by-product of S-adenosylmethionine (SAM), differs from SAM only by a single methyl group and an overall positive charge, SAH binds the SAM-II riboswitch with more than 1000-fold less affinity than SAM. Using atomistic molecular dynamics simulations, we investigated the molecular basis of such high selectivity in ligand recognition by SAM-II riboswitch. The biosynthesis of SAM exclusively generates the (S,S) stereoisomer, and (S,S)-SAM can spontaneously convert to the (R,S) form. We, therefore, also examined the effects of (R,S)-SAM binding to SAM-II and its potential biological function. We find that the unfavorable loss in entropy in SAM-II binding is greater for (S,S)- and (R,S)-SAM than SAH, which is compensated by stabilizing electrostatic interactions with the riboswitch. The positively charged sulfonium moiety on SAM acts as the crucial anchor point responsible for the formation of key ionic interactions as it fits favorably in the negatively charged binding pocket. In contrast, SAH, with its lone pair of electrons on the sulfur, experiences repulsion in the binding pocket of SAM-II and is enthalpically destabilized. In the presence of SAH, similar to the unbound riboswitch, the pseudoknot structure of SAM-II is not completely formed, thus exposing the Shine-Dalgarno sequence. Unlike SAM, this may further facilitate ribosomal assembly and translation initiation. Our analysis of the conformational ensemble sampled by SAM-II in the absence of ligands and when bound to SAM or SAH reveals that ligand binding follows a combination of conformational selection and induced-fit mechanisms.     

Molecular simulation uncovers the conformational space of the λ Cro dimer in solution

The significant variation among solved structures of the λ Cro dimer suggests its flexibility. However, contacts in the crystal lattice could have stabilized a conformation which is unrepresentative of its dominant solution form. Here we report on the conformational space of the Cro dimer in solution using replica exchange molecular dynamics in explicit solvent. The simulated ensemble shows remarkable correlation with available x-ray structures. Network analysis and a free energy surface reveal the predominance of closed and semi-open dimers, with a modest barrier separating these two states. The fully open conformation lies higher in free energy, indicating that it requires stabilization by DNA or crystal contacts. Most NMR models are found to be unstable conformations in solution. Intersubunit salt bridging between Arg⁴ and Glu⁵³ during simulation stabilizes closed conformations. Because a semi-open state is among the low-energy conformations sampled in simulation, we propose that Cro-DNA binding may not entail a large conformational change relative to the dominant dimer forms in solution.     

Investigation of the influence of external factors on the conformational dynamics of rhodopsin-like receptors by means of molecular dynamics simulation

Abstract

The study reports about the influence of binding of orthosteric ligands on the conformational dynamics of β-2-adrenoreceptor. Using molecular dynamics (MD) simulation, we found that there was a little fraction of active states of the receptor in its apo (ligand-free) ensemble. Analysis of MD trajectories indicated that such spontaneous activation of the receptor is accompanied by the motion in intracellular part of its alpha-helices. Thus, receptor’s constitutive activity directly results from its conformational dynamics. On the other hand, the binding of a full agonist resulted in a significant shift of the initial equilibrium towards its active state. Finally, the binding of the inverse agonist stabilized the receptor in its inactive state. It is likely that the binding of inverse agonists might be a universal way of constitutive activity inhibition in vivo. Our results indicate that ligand binding redistribute pre-existing conformational degrees of freedom (in accordance to the Monod–Wyman–Changeux Model) of the receptor rather than cause induced fit in it. Therefore, the ensemble of biologically relevant receptor conformations is encoded in its spatial structure, and individual conformations from that ensemble might be used by the cell in conformity with the physiological behavior.     

Single-Molecule Folding Mechanisms of the apo- and Mg2+-Bound States of Human Neuronal Calcium Sensor-1

Neuronal calcium sensor-1 (NCS-1) is the primordial member of a family of proteins responsible primarily for sensing changes in neuronal Ca²⁺ concentration. NCS-1 is a multispecific protein interacting with a number of binding partners in both calcium-dependent and independent manners, and acting in a variety of cellular processes in which it has been linked to a number of disorders such as schizophrenia and autism. Despite extensive studies on the Ca²⁺-activated state of NCS proteins, little is known about the conformational dynamics of the Mg²⁺-bound and apo states, both of which are populated, at least transiently, at resting Ca²⁺ conditions. Here, we used optical tweezers to study the folding behavior of individual NCS-1 molecules in the presence of Mg²⁺ and in the absence of divalent ions. Under tension, the Mg²⁺-bound state of NCS-1 unfolds and refolds in a three-state process by populating one intermediate state consisting of a folded C-domain and an unfolded N-domain. The interconversion at equilibrium between the different molecular states populated by NCS-1 was monitored in real time through constant-force measurements and the energy landscapes underlying the observed transitions were reconstructed through hidden Markov model analysis. Unlike what has been observed with the Ca²⁺-bound state, the presence of Mg²⁺ allows both the N- and C-domain to fold through all-or-none transitions with similar refolding rates. In the absence of divalent ions, NCS-1 unfolds and refolds reversibly in a two-state reaction involving only the C-domain, whereas the N-domain has no detectable transitions. Overall, the results allowed us to trace the progression of NCS-1 folding along its energy landscapes and provided a solid platform for understanding the conformational dynamics of similar EF-hand proteins.     

Role of Detergents in Conformational Exchange of a G Protein-coupled Receptor

The G protein-coupled β₂-adrenoreceptor (β₂AR) signals through the heterotrimeric G proteins G_s and G_i and β-arrestin. As such, the energy landscape of β₂AR-excited state conformers is expected to be complex. Upon tagging Cys-265 of β₂AR with a trifluoromethyl probe, ¹⁹F NMR was used to assess conformations and possible equilibria between states. Here, we report key differences in β₂AR conformational dynamics associated with the detergents used to stabilize the receptor. In dodecyl maltoside (DDM) micelles, the spectra are well represented by a single Lorentzian line that shifts progressively downfield with activation by appropriate ligand. The results are consistent with interconversion between two or more states on a time scale faster than the greatest difference in ligand-dependent chemical shift (i.e. >100 Hz). Given that high detergent off-rates of DDM monomers may facilitate conformational exchange between functional states of β₂AR, we utilized the recently developed maltose-neopentyl glycol (MNG-3) diacyl detergent. In MNG-3 micelles, spectra indicated at least three distinct states, the relative populations of which depended on ligand, whereas no ligand-dependent shifts were observed, consistent with the slow exchange limit. Thus, detergent has a profound effect on the equilibrium kinetics between functional states. MNG-3, which has a critical micelle concentration in the nanomolar regime, exhibits an off-rate that is 4 orders of magnitude lower than that of DDM. High detergent off-rates are more likely to facilitate conformational exchange between distinct functional states associated with the G protein-coupled receptor.     

Folding of the SAM-I riboswitch: A tale with a twist

Riboswitches are ligand-dependent RNA genetic regulators that control gene expression by altering their structures. The elucidation of riboswitch conformational changes before and after ligand recognition is crucial to understand how riboswitches can achieve high ligand binding affinity and discrimination against cellular analogs. The detailed characterization of riboswitch folding pathways suggest that they may use their intrinsic conformational dynamics to sample a large array of structures, some of which being nearly identical to ligand-bound molecules. Some of these structural conformers can be "captured" upon ligand binding, which is crucial for the outcome of gene regulation. Recent studies about the SAM-I riboswitch have revealed unexpected and previously unknown RNA folding mechanisms. For instance, the observed helical twist of the P1 stem upon ligand binding to the SAM-I aptamer adds a new element in the repertoire of RNA strategies for recognition of small metabolites. From an RNA folding perspective, these findings also strongly indicate that the SAM-I riboswitch could achieve ligand recognition by using an optimized combination of conformational capture and induced-fit approaches, a feature that may be shared by other RNA regulatory sequences.     

Mapping the dynamics of ligand reorganization via 13CH3 and 13CH2 relaxation dispersion at natural abundance

Flexible ligands pose challenges to standard structure-activity studies since they frequently reorganize their conformations upon protein binding and catalysis. Here, we demonstrate the utility of side chain ¹³C relaxation dispersion measurements to identify and quantify the conformational dynamics that drive this reorganization. The dispersion measurements probe methylene ¹³CH₂ and methyl ¹³CH₃ groups; the latter are highly prevalent side chain moieties in known drugs. Combining these side chain studies with existing backbone dispersion studies enables a comprehensive investigation of μs–ms conformational dynamics related to binding and catalysis. We perform these measurements at natural ¹³C abundance, in congruence with common pharmaceutical research settings. We illustrate these methods through a study of the interaction of a phosphopeptide ligand with the peptidyl-prolyl isomerase, Pin1. The results illuminate the side-chain moieties that undergo conformational readjustments upon complex formation. In particular, we find evidence that multiple exchange processes influence the side chain dispersion profiles. Collectively, our studies illustrate how side-chain relaxation dispersion can shed light on ligand conformational transitions required for activity, and thereby suggest strategies for its optimization.     

Enhanced meta-analysis of acetylcholine binding protein structures reveals conformational signatures of agonism in nicotinic receptors

The soluble acetylcholine binding protein (AChBP) is the default structural proxy for pentameric ligand‐gated ion channels (LGICs). Unfortunately, it is difficult to recognize conformational signatures of LGIC agonism and antagonism within the large set of AChBP crystal structures in both apo and ligand‐bound states, primarily because AChBP conformations in this set are nearly superimposable (root mean square deviation < 1.5 Å). We have undertaken a systematic, alignment‐free approach to elucidate conformational differences displayed by AChBP that cleanly differentiate apo/antagonist‐bound from agonist‐bound states. Our approach uses statistical inference based on both crystallographic states and conformations sampled during long molecular dynamics simulations to select important inter‐C_α distances and map their collective values onto functional states. We observe that binding of (nAChR) agonists to AChBP elicits clockwise rotation of the inner β‐sheet with respect to the outer β‐sheet, causing tilting of the cys‐loop away from the five‐fold axis, in a manner quite similar to that speculated for α‐subunits of the heteromeric nAChR structure (Unwin, J Mol Biol 2005;346:967), making this motion potentially important in transmission of the gating signal to the transmembrane domain of a LGIC. The method is also successful at discriminating partial from full agonists and supports the hypothesis that a particularly controversial ligand, lobeline, is in fact an LGIC antagonist.     

Fine structure of conformational ensembles in adenylate kinase

Adenylate kinase (ADK) catalyzes the reversible Mg²⁺‐dependent phosphoryl transfer reaction Mg²⁺+2ADP ?Mg²⁺+ATP + AMP in essential cellular systems. This reaction is a major player in cellular energy homeostasis and the isoform network of ADK plays an important role in AMP metabolic signaling circuits. ADK has 3 domains, the LID, NMP, and CORE domains, that undergo large conformational rearrangements during ADK's catalytic cycle. In spite of extensive experimental and computational studies, details of the conformational pathway from open to closed forms remain uncertain. In this paper we explore this pathway using coarse‐grained molecular dynamics (MD) trajectories of ADK calculated by GROMACS using a SMOG model and classify the conformations within the resultant trajectories by K‐means clustering. ADK conformations segregate naturally into open; intermediate; and closed forms with long‐term residence in the intermediate state. Structural clustering divides the intermediate conformation into 3 sub‐states that are distinguished from one another on the basis of differences in both structure and dynamics. These distinctions are defined on the basis of a number of different metrics including radius of gyration, dihedral angle fluctuation, and fluctuations of interatomic pair distances. Furthermore, differences in the sub‐states appear to correspond to the distinct ways each sub‐state contributes to the molecular mechanism of catalysis: One sub‐state acts as a gate‐way to the open conformation; one sub‐state a gate‐way to the closed conformation. A third intermediate sub‐state appears to represent a metastable off‐pathway structure that is nevertheless frequently visited during the passage from open to closed state.     

Dissecting the influence of Mg2+ on 3D architecture and ligand-binding of the guanine-sensing riboswitch aptamer domain

Long-range tertiary interactions determine the three-dimensional structure of a number of metabolite-binding riboswitch RNA elements and were found to be important for their regulatory function. For the guanine-sensing riboswitch of the Bacillus subtilis xpt-pbuX operon, our previous NMR-spectroscopic studies indicated pre-formation of long-range tertiary contacts in the ligand-free state of its aptamer domain. Loss of the structural pre-organization in a mutant of this RNA (G37A/C61U) resulted in the requirement of Mg²⁺ for ligand binding. Here, we investigate structural and stability aspects of the wild-type aptamer domain (Gsw) and the G37A/C61U-mutant (Gsw^loop) of the guanine-sensing riboswitch and their Mg²⁺-induced folding characteristics to dissect the role of long-range tertiary interactions, the link between pre-formation of structural elements and ligand-binding properties and the functional stability. Destabilization of the long-range interactions as a result of the introduced mutations for Gsw^loop or the increase in temperature for both Gsw and Gsw^loop involves pronounced alterations of the conformational ensemble characteristics of the ligand-free state of the riboswitch. The increased flexibility of the conformational ensemble can, however, be compensated by Mg²⁺. We propose that reduction of conformational dynamics in remote regions of the riboswitch aptamer domain is the minimal pre-requisite to pre-organize the core region for specific ligand binding.     

Threshold Occupancy and Specific Cation Binding Modes in the Hammerhead Ribozyme Active Site are Required for Active Conformation

The relationship between formation of active in-line attack conformations and monovalent (Na⁺) and divalent (Mg²⁺) metal ion binding in hammerhead ribozyme (HHR) has been explored with molecular dynamics simulations. To stabilize repulsions between negatively charged groups, different requirements of the threshold occupancy of metal ions were observed in the reactant and activated precursor states both in the presence and in the absence of a Mg²⁺ in the active site. Specific bridging coordination patterns of the ions are correlated with the formation of active in-line attack conformations and can be accommodated in both cases. Furthermore, simulation results suggest that the HHR folds to form an electronegative recruiting pocket that attracts high local concentrations of positive charge. The present simulations help to reconcile experiments that probe the metal ion sensitivity of HHR catalysis and support the supposition that Mg²⁺, in addition to stabilizing active conformations, plays a specific chemical role in catalysis.