Conformational Selection Mechanism Research Articles

Conformational dynamics during high-fidelity DNA replication and translocation defined using a DNA polymerase with a fluorescent artificial amino acid

We address the role of enzyme conformational dynamics in specificity for a high-fidelity DNA polymerase responsible for genome replication. We present the complete characterization of the conformational dynamics during the correct nucleotide incorporation forward and reverse reactions using stopped-flow and rapid-quench methods with a T7 DNA polymerase variant containing a fluorescent unnatural amino acid, (7-hydroxy-4-coumarin-yl) ethylglycine, which provides a signal for enzyme conformational changes. We show that the forward conformational change (>6000 s−1) is much faster than chemistry (300 s−1) while the enzyme opening to allow release of bound nucleotide (1.7 s−1) is much slower than chemistry. These parameters show that the conformational change selects a correct nucleotide for incorporation through an induced-fit mechanism. We also measured conformational changes occurring after chemistry and during pyrophosphorolysis, providing new insights into processive polymerization. Pyrophosphorolysis occurs via a conformational selection mechanism as the pyrophosphate binds to a rare pretranslocation state of the enzyme–DNA complex. Global data fitting was achieved by including experiments in the forward and reverse directions to correlate conformational changes with chemical reaction steps. This analysis provided well-constrained values for nine rate constants to establish a complete free-energy profile including the rates of DNA translocation during processive synthesis. Translocation does not follow Brownian ratchet or power stroke models invoking nucleotide binding as the driving force. Rather, translocation is rapid and thermodynamically favorable after enzyme opening and pyrophosphate release, and it appears to limit the rate of processive synthesis at 4 °C.

Bringing the Ca2+ sensitivity of myristoylated recoverin into the physiological range.

The prototypical Ca2+-sensor protein recoverin (Rec) is thought to regulate the activity of rhodopsin kinase (GRK1) in photoreceptors by switching from a relaxed (R) disc membrane-bound conformation in the dark to a more compact, cytosol-diffusing tense (T) conformation upon cell illumination. However, the apparent affinity for Ca2+ of its physiologically relevant form (myristoylated recoverin) is almost two orders of magnitude too low to support this mechanism in vivo. In this work, we compared the individual and synergistic roles of the myristic moiety, the GRK1 target and the disc membrane in modulating the calcium sensitivity of Rec. We show that the sole presence of the target or the disc membrane alone are not sufficient to achieve a physiological response to changes in intracellular [Ca2+]. Instead, the simultaneous presence of GRK1 and membrane allows the T to R transition to occur in a physiological range of [Ca2+] with high cooperativity via a conformational selection mechanism that drives the structural transitions of Rec in the presence of multiple ligands. Our conclusions may apply to other sensory transduction systems involving protein complexes and biological membranes.

Phosphorylation regulates the binding of intrinsically disordered proteins via a flexible conformation selection mechanism

Phosphorylation is one of the most common post-translational modifications. The phosphorylation of the kinase-inducible domain (KID), which is an intrinsically disordered protein (IDP), promotes the folding of KID and binding with the KID-interacting domain (KIX). However, the regulation mechanism of the phosphorylation on KID is still elusive. In this study, the structural ensembles and binding process of pKID and KIX are studied by all-atom enhanced sampling technologies. The results show that more hydrophobic interactions are formed in pKID, which promote the formation of the special hydrophobic residue cluster (HRC). The pre-formed HRC promotes binding to the correct sites of KIX and further lead the folding of pKID. Consequently, a flexible conformational selection model is proposed to describe the binding and folding process of intrinsically disordered proteins. The binding mechanism revealed in this work provides new insights into the dynamic interactions and phosphorylation regulation of proteins.

Conformational Selection in Ligand Recognition by the First Tudor Domain of PHF20L1.

The first Tudor domain (Tudor1) of PHF20L1 recognizes (non)histone methylation to play versatile roles. However, the underlying ligand-recognition mechanism remains unknown as a closed state revealed in the free-form structure. NMR relaxation dispersion and molecular dynamics simulations suggest a pre-existing low-population conformation with a remarkable rearrangement of aromatic cage residues of PHF20L1 Tudor1. Such an open-form conformation is utilized to recognize lysine 142 methylated DNMT1, a cosolvent, and an NMR fragment screening hit, as revealed by the complex crystal structures. Intriguingly, the ligand binding capacity was enhanced by mutation that tunes up the open-state population only. The recognition of DNMT1 by PHF20L1 was further validated in cancer cells. This conformational selection mechanism will enable the discovery of small molecule inhibitors against the seemingly "undruggable" PHF20L1 Tudor1.

Schistosomal Sulfotransferase Interaction with Oxamniquine Involves Hybrid Mechanism of Induced-fit and Conformational Selection.

Sulfotransferase family comprises key enzymes involved in drug metabolism. Oxamniquine is a pro-drug converted into its active form by schistosomal sulfotransferase. The conformational dynamics of side-chain amino acid residues at the binding site of schistosomal sulfotransferase towards activation of oxamniquine has not received attention. The study investigated the conformational dynamics of binding site residues in free and oxamniquine bound schistosomal sulfotransferase systems and their contribution to the mechanism of oxamniquine activation by schistosomal sulfotransferase using molecular dynamics simulations and binding energy calculations. Schistosomal sulfotransferase was obtained from Protein Data Bank and both the free and oxamniquine bound forms were subjected to molecular dynamics simulations using GROMACS-4.5.5 after modeling it's missing amino acid residues with SWISS-MODEL. Amino acid residues at its binding site for oxamniquine was determined and used for Principal Component Analysis and calculations of side-chain dihedrals. In addition, binding energy of the oxamniquine bound system was calculated using g_MMPBSA. The results showed that binding site amino acid residues in free and oxamniquine bound sulfotransferase sampled different conformational space involving several rotameric states. Importantly, Phe45, Ile145 and Leu241 generated newly induced conformations, whereas Phe41 exhibited shift in equilibrium of its conformational distribution. In addition, the result showed binding energy of -130.091 ± 8.800 KJ/mol and Phe45 contributed -9.8576 KJ/mol. The results showed that schistosomal sulfotransferase binds oxamniquine by relying on hybrid mechanism of induced fit and conformational selection models. The findings offer new insight into sulfotransferase engineering and design of new drugs that target sulfotransferase.

Sequential Folding of the Nickel/Cobalt Riboswitch Is Facilitated by a Conformational Intermediate: Insights from Single-Molecule Kinetics and Thermodynamics.

The present work presents the first single-molecule fluorescence resonant energy transfer (smFRET) studies of the nickel/cobalt (NiCo) riboswitch, with temperature-dependent, single-molecule confocal microscopy to provide comprehensive kinetic and thermodynamic information on folding into a biochemically competent structure. The results indicate that the NiCo riboswitch first folds into a more compact "prefolded" conformation, with a preorganized binding pocket partially stabilized under physiological conditions by noncognate monovalent/divalent cations. Such a prefolded intermediate then has opportunity to fold further into a tightly ligand-bound structure, in response to the cognate ligands, Ni2+ or Co2+, with submicromolar affinities. Such stepwise ligand-induced folding represents a particularly clean example of a conformational selection ("fold-then-bind") mechanism, whereby a configuration dynamically accessible by thermal fluctuation is stabilized into the final folded state by ligand association. In addition, we observe a strong positive cooperativity in the ligand-induced folding kinetics with respect to both Ni2+ and Co2+ ligands. This provides maximal sensitivity in the riboswitch conformational response near [Ni2+] or [Co2+] ≈ Kd, which facilitates more accurate biochemical probing of the cell environment and therefore bioregulation of gene expression. Temperature-dependent kinetics at the single-molecule level has also been explored, which permits free energies to be deconstructed into enthalpic and entropic components along the folding coordinate. In the absence of the cognate ligand, a predominantly enthalpic barrier between the unfolded riboswitch (U) and the prefolded intermediate (I) suggests a rearrangement of the hydrogen bonding network, whereas in the presence of the cognate ligand, a large entropic penalty (-TΔS0 > 0) in forming the folded riboswitch conformation (F) is almost perfectly counterbalanced by an equivalent enthalpic gain (ΔH0 < 0) to yield ΔG0 ≈ 0. The thermodynamic results are therefore consistent with a simple physical picture of riboswitch folding, whereby association of the cognate ligand is strongly stabilized by Coulombic attraction while forming an entropically more ordered structure around the binding site.

Effector role of cytochrome P450 reductase for androstenedione binding to human aromatase

Cytochromes P450 constitute a large superfamily of monooxygenases involved in many metabolic pathways. Most of them are not self-sufficient and need a reductase protein to provide the electrons necessary for catalysis. It was shown that the redox partner plays a role in the modulation of the structure and function of some bacterial P450 enzymes.Here, the effect of NADPH-cytochrome reductase (CPR) on human aromatase (Aro) is studied for what concerns its role in substrate binding. Pre-steady-state kinetic experiments indicate that both the substrate binding rates and the percentage of spin shift detected for aromatase are increased when CPR is present. Moreover, aromatase binds the substrate through a conformational selection mechanism, suggesting a possible effector role of CPR. The thermodynamic parameters for the formation of the CPR-Aro complex were studied by isothermal titration calorimetry. The dissociation constant of the complex formation is 4.5 folds lower for substrate-free compared to the substrate-bound enzyme. The enthalpy change observed when the CPR-Aro complex forms in the absence of the substrate are higher than in its presence, indicating that more interactions are formed/broken in the former case.Taken together, our data confirm that CPR has a role in promoting aromatase conformation optimal for substrate binding.

Homodimerization of a glycoside hydrolase family GH1 β-glucosidase suggests distinct activity of enzyme different states.

In this work, we investigated how activity and oligomeric state are related in a purified GH1 β-glucosidase from Spodoptera frugiperda (Sfβgly). Gel filtration chromatography coupled to a multiple angle light scattering detector allowed separation of the homodimer and monomer states and determination of the dimer dissociation constant (KD ), which was in the micromolar range. Enzyme kinetic parameters showed that the dimer is on average 2.5-fold more active. Later, we evaluated the kinetics of homodimerization, scanning the changes in the Sfβgly intrinsic fluorescence over time when the dimer dissociates into the monomer after a large dilution. We described how the rate constant of monomerization (koff ) is affected by temperature, revealing the enthalpic and entropic contributions to the process. We also evaluated how the rate constant (kobs ) by which equilibrium is reached after dimer dilution behaves when varying the initial Sfβgly concentration. These data indicated that Sfβgly dimerizes through the conformational selection mechanism, in which the monomer undergoes a conformational exchange and then binds to a similar monomer, forming a more active homodimer. Finally, we noted that conformational selection reports and experiments usually rely on a ligand whose concentration is in excess, but for homodimerization, this approach does not hold. Hence, since our approach overcomes this limitation, this study not only is a new contribution to the comprehension of GH1 β-glucosidases, but it can also help to elucidate protein interaction pathways.

Dynamical Behavior and Conformational Selection Mechanism of the Intrinsically Disordered Sic1 Kinase-Inhibitor Domain.

Intrinsically Disordered Peptides and Proteins (IDPs) in solution can span a broad range of conformations that often are hard to characterize by both experimental and computational methods. However, obtaining a significant representation of the conformational space is important to understand mechanisms underlying protein functions such as partner recognition. In this work, we investigated the behavior of the Sic1 Kinase-Inhibitor Domain (KID) in solution by Molecular Dynamics (MD) simulations. Our results point out that application of common descriptors of molecular shape such as Solvent Accessible Surface (SAS) area can lead to misleading outcomes. Instead, more appropriate molecular descriptors can be used to define 3D structures. In particular, we exploited Weighted Holistic Invariant Molecular (WHIM) descriptors to get a coarse-grained but accurate definition of the variegated Sic1 KID conformational ensemble. We found that Sic1 is able to form a variable amount of folded structures even in absence of partners. Among them, there were some conformations very close to the structure that Sic1 is supposed to assume in the binding with its physiological complexes. Therefore, our results support the hypothesis that this protein relies on the conformational selection mechanism to recognize the correct molecular partners.

A supramolecular system that strictly follows the binding mechanism of conformational selection

Induced fit and conformational selection are two dominant binding mechanisms in biology. Although induced fit has been widely accepted by supramolecular chemists, conformational selection is rarely studied with synthetic systems. In the present research, we report a macrocyclic host whose binding mechanism is unambiguously assigned to conformational selection. The kinetic and thermodynamic aspects of this system are studied in great detail. It reveals that the kinetic equation commonly used for conformational selection is strictly followed here. In addition, two mathematical models are developed to determine the association constants of the same guest to the two host conformations. A “conformational selectivity factor” is defined to quantify the fidelity of conformational selection. Many details about the kinetic and thermodynamic aspects of conformational selection are revealed by this synthetic system. The conclusion and the mathematical models reported here should be helpful in understanding complex molecular recognition in both biological and synthetic systems.

Probing the correlation between ligand efficacy and conformational diversity at the α1A-adrenoreceptor reveals allosteric coupling of its microswitches

G protein-coupled receptors (GPCRs) use a series of conserved microswitches to transmit signals across the cell membrane via an allosteric network encompassing the ligand-binding site and the G protein-binding site. Crystal structures of GPCRs provide snapshots of their inactive and active states, but poorly describe the conformational dynamics of the allosteric network that underlies GPCR activation. Here, we analyzed the correlation between ligand binding and receptor conformation of the α1A-adrenoreceptor, a GPCR that stimulates smooth muscle contraction in response to binding noradrenaline. NMR of [13CϵH3]methionine-labeled α1A-adrenoreceptor variants, each exhibiting differing signaling capacities, revealed how different classes of ligands modulate the conformational equilibria of this receptor. [13CϵH3]Methionine residues near the microswitches exhibited distinct states that correlated with ligand efficacies, supporting a conformational selection mechanism. We propose that allosteric coupling among the microswitches controls the conformation of the α1A-adrenoreceptor and underlies the mechanism of ligand modulation of GPCR signaling in cells.

NMR-Derived Conformational Ensemble of State 1 of Activated Ras Reveals Insights into a Druggable Pocket.

The lack of apparent pockets in the ground conformation of Ras has long challenged the rational design of inhibitors against this oncogenic protein. The sparsely populated, transiently formed state 1 of activated Ras, on the other hand, shows appreciable surface roughness and is increasingly recognized as a potential target for drug discovery. State 1, however, is extremely flexible, and a static structure cannot fully unveil its conformational space that can be exploited for drug design. Here, we present a conformational ensemble of state 1 that was derived using chemical shift-based modeling. The ensemble reveals the intrinsic plasticity of a druggable pocket in state 1 and demonstrates the mechanism of conformational selection for inhibitor recognition. The large set of structural templates in the ensemble, providing a comprehensive description of thermally accessible pocket conformations, is expected to significantly aid the rational design of anti-Ras drugs.

Human POT1 unfolds G-quadruplexes by conformational selection

The reaction mechanism by which the shelterin protein POT1 (Protection of Telomeres 1) unfolds human telomeric G-quadruplex structures is not fully understood. We report here kinetic, thermodynamic, hydrodynamic and computational studies that show that a conformational selection mechanism, in which POT1 binding is coupled to an obligatory unfolding reaction, is the most plausible mechanism. Stopped-flow kinetic and spectroscopic titration studies, along with isothermal calorimetry, were used to show that binding of the single-strand oligonucleotide d[TTAGGGTTAG] to POT1 is both fast (80 ms) and strong (−10.1 ± 0.3 kcal mol−1). In sharp contrast, kinetic studies showed the binding of POT1 to an initially folded 24 nt G-quadruplex structure is four orders of magnitude slower. Fluorescence, circular dichroism and analytical ultracentrifugation studies showed that POT1 binding is coupled to quadruplex unfolding, with a final complex with a stoichiometry of 2 POT1 per 24 nt DNA. The binding isotherm for the POT1-quadruplex interaction was sigmoidal, indicative of a complex reaction. A conformational selection model that includes equilibrium constants for both G-quadruplex unfolding and POT1 binding to the resultant single-strand provided an excellent quantitative fit to the experimental binding data. POT1 unfolded and bound to any conformational form of human telomeric G-quadruplex (antiparallel, hybrid, parallel monomers or a 48 nt sequence with two contiguous quadruplexes), but did not avidly interact with duplex DNA or with other G-quadruplex structures. Finally, molecular dynamics simulations provided a detailed structural model of a 2:1 POT1:DNA complex that is fully consistent with experimental biophysical results.

Combining LE4PD Normal Modes and Markov State Modeling to Elucidate the Fluctuation Dynamics of Ubiquitin

Local fluctuations are important for protein binding and molecular recognition because they provide conformational states that can be trapped through a conformational selection mechanism of binding. Thus, an accurate characterization of local fluctuations can be important for modeling the mechanisms leading to the biological activity of a protein. In this presentation we quantify the fluctuation dynamics of the regulatory protein ubiquitin and propose a novel theoretical approach to modelling its fluctuations. A coarse-grained, diffusive, mode-dependent description of the protein's fluctuation dynamics is accomplished using the Langevin Equation for Protein Dynamics (LE4PD); this equation decomposes the dynamics of a protein, modeled through a molecular dynamics (MD) simulation, into dynamical pathways that explore mode-dependent free energy surfaces. Using as input to the theory a one-microsecond, atomistic MD simulation, we calculate the time scales of the slow, high-amplitude fluctuations by modeling the kinetics of barrier crossing in the two-dimensional free-energy surfaces using a Markov state model (MSM) approach. We find that the LE4PD predicts slow fluctuations in three biologically significant binding regions in ubiquitin: the C-terminal tail, the Lys11 loop, and the 50 s loop. The combination of the LE4PD modes with the MSM analysis can provide useful information on the role of fluctuations in the process of molecular recognition regulating the biological activity of ubiquitin, with the LE4PD modes describing the location and magnitude of high-amplitude conformational changes and the MSM giving the associated timescales.

Macromolecular crowding induces compaction and DNA binding in the disordered N-terminal domain of hUNG2

Many human DNA repair proteins have disordered domains at their N- or C-termini with poorly defined biological functions. We recently reported that the partially structured N-terminal domain (NTD) of human uracil DNA glycosylase 2 (hUNG2), functions to enhance DNA translocation in crowded environments and also targets the enzyme to single-stranded/double-stranded DNA junctions. To understand the structural basis for these effects we now report high-resolution heteronuclear NMR studies of the isolated NTD in the presence and absence of an inert macromolecular crowding agent (PEG8K). Compared to dilute buffer, we find that crowding reduces the degrees of freedom for the structural ensemble, increases the order of a PCNA binding motif and dramatically promotes binding of the NTD for DNA through a conformational selection mechanism. These findings shed new light on the function of this disordered domain in the context of the crowded nuclear environment.

Binding-induced folding under unfolding conditions: Switching between induced fit and conformational selection mechanisms

The chemistry of protein-ligand binding is the basis of virtually every biological process. Ligand binding can be essential for a protein to function in the cell by stabilizing or altering the conformation of a protein, particularly for partially or completely unstructured proteins. However, the mechanisms by which ligand binding impacts disordered proteins or influences the role of disorder in protein folding is not clear. To gain insight into this question, the mechanism of folding induced by the binding of a Pro-rich peptide ligand to the SH3 domain of phosphatidylinositol 3-kinase unfolded in the presence of urea has been studied using kinetic methods. Under strongly denaturing conditions, folding was found to follow a conformational selection (CS) mechanism. However, under mildly denaturing conditions, a ligand concentration-dependent switch in the mechanism was observed. The folding mechanism switched from being predominantly a CS mechanism at low ligand concentrations to being predominantly an induced fit (IF) mechanism at high ligand concentrations. The switch in the mechanism manifests itself as an increase in the reaction flux along the IF pathway at high ligand concentrations. The results indicate that, in the case of intrinsically disordered proteins too, the folding mechanism is determined by the concentration of the ligand that induces structure formation.

Branch site bulge conformations in domain 6 determine functional sugar puckers in group II intron splicing.

Although group II intron ribozymes are intensively studied the question how structural dynamics affects splicing catalysis has remained elusive. We report for the first time that the group II intron domain 6 exists in a secondary structure equilibrium between a single- and a two-nucleotide bulge conformation, which is directly linked to a switch between sugar puckers of the branch site adenosine. Our study determined a functional sugar pucker equilibrium between the transesterification active C2′-endo conformation of the branch site adenosine in the 1nt bulge and an inactive C3′-endo state in the 2nt bulge fold, allowing the group II intron to switch its activity from the branching to the exon ligation step. Our detailed NMR spectroscopic investigation identified magnesium (II) ions and the branching reaction as regulators of the equilibrium populations. The tuneable secondary structure/sugar pucker equilibrium supports a conformational selection mechanism to up- and downregulate catalytically active and inactive states of the branch site adenosine to orchestrate the multi-step splicing process. The conformational dynamics of group II intron domain 6 is also proposed to be a key aspect for the directionality selection in reversible splicing.

How to distinguish ligand-binding mechanisms: an example of conformational selection disguised as an induced fit

ABSTRACT This report describes the implementation of a laboratory exercise for an advanced biochemistry or enzyme kinetics class at the undergraduate or graduate level, designed to improve understanding of protein conformational changes associated with the binding of a ligand. Students measure the fluorescence changes induced by the conformational transition of a glycoprotein (the Na,K-ATPase) upon addition of different ligands (Pi and BeF3 −) and analyse the results in order to determine the mechanism of the process. The results show that Pi and BeF3 − present opposite effects on the observed rate constants (kobs ) with ligand concentration: kobs decreases with [Pi] and increases with [BeF3 −]. This observation, together with the frequently used assumption that binding occurs under rapid equilibrium, led to propose different models for ligand-induced conformational transitions: a conformational selection for Pi and an induced fit for BeF3 −. In this paper, we show that if the rapid-equilibrium approximation for ligand binding is not assumed, a conformational selection mechanism can account for the effects of both ligands. This active-learning exercise serves as the basis for discussing the consequences of not being extremely cautious when invoking approximations about not-very-well-known systems and the importance of a correct understanding of models assigned to chemical processes.

Binding site plasticity in viral PPxY Late domain recognition by the third WW domain of human NEDD4

The recognition of PPxY viral Late domains by the third WW domain of the HECT-E3 ubiquitin ligase NEDD4 (hNEDD4-WW3) is essential for the completion of the budding process of numerous enveloped viruses, including Ebola, Marburg, HTLV1 or Rabies. hNEDD4-WW3 has been validated as a promising target for the development of novel host-oriented broad spectrum antivirals. Nonetheless, finding inhibitors with good properties as therapeutic agents remains a challenge since the key determinants of binding affinity and specificity are still poorly understood. We present here a detailed structural and thermodynamic study of the interactions of hNEDD4-WW3 with viral Late domains combining isothermal titration calorimetry, NMR structural determination and molecular dynamics simulations. Structural and energetic differences in Late domain recognition reveal a highly plastic hNEDD4-WW3 binding site that can accommodate PPxY-containing ligands with varying orientations. These orientations are mostly determined by specific conformations adopted by residues I859 and T866. Our results suggest a conformational selection mechanism, extensive to other WW domains, and highlight the functional relevance of hNEDD4-WW3 domain conformational flexibility at the binding interface, which emerges as a key element to consider in the search for potent and selective inhibitors of therapeutic interest.

Residues W215, E217 and E192 control the allosteric E*-E equilibrium of thrombin

A pre-existing, allosteric equilibrium between closed (E*) and open (E) conformations of the active site influences the level of activity in the trypsin fold and defines ligand binding according to the mechanism of conformational selection. Using the clotting protease thrombin as a model system, we investigate the molecular determinants of the E*-E equilibrium through rapid kinetics and X-ray structural biology. The equilibrium is controlled by three residues positioned around the active site. W215 on the 215–217 segment defining the west wall of the active site controls the rate of transition from E to E* through hydrophobic interaction with F227. E192 on the opposite 190–193 segment defining the east wall of the active site controls the rate of transition from E* to E through electrostatic repulsion of E217. The side chain of E217 acts as a lever that moves the entire 215–217 segment in the E*-E equilibrium. Removal of this side chain converts binding to the active site to a simple lock-and-key mechanism and freezes the conformation in a state intermediate between E* and E. These findings reveal a simple framework to understand the molecular basis of a key allosteric property of the trypsin fold.