Induced-fit Pathway Research Articles

Ligand-induced protein transition state stabilization switches the binding pathway from conformational selection to induced fit

Protein-ligand complex formation is fundamental to biological function. A central question is whether proteins spontaneously adopt binding-competent conformations to which ligands bind conformational selection (CS) or whether ligands induce the binding-competent conformation induced fit (IF). Here, we resolve the CS and IF binding pathways by characterizing protein conformational dynamics over a wide range of ligand concentrations using NMR relaxation dispersion. We determined the relative flux through the two pathways using a four-state binding model that includes both CS and IF. Experiments conducted without ligand show that galectin-3 exchanges between the ground-state conformation and a high-energy conformation similar to the ligand-bound conformation, demonstrating that CS is a plausible pathway. Near-identical crystal structures of the apo and ligand-bound states suggest that the high-energy conformation in solution corresponds to the apo crystal structure. Stepwise additions of the ligand lactose induce progressive changes in the relaxation dispersions that we fit collectively to the four-state model, yielding all microscopic rate constants and binding affinities. The ligand affinity is higher for the bound-like conformation than for the ground state, as expected for CS. Nonetheless, the IF pathway contributes greater than 70% of the total flux even at low ligand concentrations. The higher flux through the IF pathway is explained by considerably higher rates of exchange between the two protein conformations in the ligand-associated state. Thus, the ligand acts to decrease the activation barrier between protein conformations in a manner reciprocal to enzymatic transition-state stabilization of reactions involving ligand transformation.

From Selection to Instruction and Back: Competing Conformational Selection and Induced Fit Pathways in Abiotic Hosts.

Two limiting cases of molecular recognition, induced fit (IF) and conformational selection (CS), play a central role in allosteric regulation of natural systems. The IF paradigm states that a substrate "instructs" the host to change its shape after complexation, while CS asserts that a guest "selects" the optimal fit from an ensemble of preexisting host conformations. With no studies that quantitatively address the interplay of two limiting pathways in abiotic systems, we herein and for the first time describe the way by which twisted capsule M-1, encompassing two conformers M-1(+) and M-1(-), trap CX4 (X=Cl, Br) to give CX4 ⊂M-1(+) and CX4 ⊂M-1(-), with all four states being in thermal equilibrium. With the assistance of 2D EXSY, we found that CBr4 would, at its lower concentrations, bind M-1 via a M-1(+)→M-1(-)→CBr4 ⊂M-1(-) pathway corresponding to conformational selection. For M-1 complexing CCl4 though, data from 2D EXSY measurements and 1D NMR line-shape analysis suggested that lower CCl4 concentrations would favor CS while the IF pathway prevailed at higher proportions of the guest. Since CS and IF are not mutually exclusive, we reason that our work sets the stage for characterizing the dynamics of a wide range of already existing hosts to broaden our fundamental understanding of their action. The objective is to master the way in which encapsulation takes place for designing novel and allosteric sequestering agents, catalysts and chemosensors akin to those found in nature.

From Selection to Instruction and Back: Competing Conformational Selection and Induced Fit Pathways in Abiotic Hosts

AbstractTwo limiting cases of molecular recognition, induced fit (IF) and conformational selection (CS), play a central role in allosteric regulation of natural systems. The IF paradigm states that a substrate “instructs” the host to change its shape after complexation, while CS asserts that a guest “selects” the optimal fit from an ensemble of preexisting host conformations. With no studies that quantitatively address the interplay of two limiting pathways in abiotic systems, we herein and for the first time describe the way by which twisted capsule M‐1, encompassing two conformers M‐1(+) and M‐1(−), trap CX4 (X=Cl, Br) to give CX4⊂M‐1(+) and CX4⊂M‐1(−), with all four states being in thermal equilibrium. With the assistance of 2D EXSY, we found that CBr4 would, at its lower concentrations, bind M‐1 via a M‐1(+)→M‐1(−)→CBr4⊂M‐1(−) pathway corresponding to conformational selection. For M‐1 complexing CCl4 though, data from 2D EXSY measurements and 1D NMR line‐shape analysis suggested that lower CCl4 concentrations would favor CS while the IF pathway prevailed at higher proportions of the guest. Since CS and IF are not mutually exclusive, we reason that our work sets the stage for characterizing the dynamics of a wide range of already existing hosts to broaden our fundamental understanding of their action. The objective is to master the way in which encapsulation takes place for designing novel and allosteric sequestering agents, catalysts and chemosensors akin to those found in nature.

Energetics and kinetics of substrate analog-coupled staphylococcal nuclease folding revealed by a statistical mechanical approach

Protein conformational changes associated with ligand binding, especially those involving intrinsically disordered proteins, are mediated by tightly coupled intra- and intermolecular events. Such reactions are often discussed in terms of two limiting kinetic mechanisms, conformational selection (CS), where folding precedes binding, and induced fit (IF), where binding precedes folding. It has been shown that coupled folding/binding reactions can proceed along both CS and IF pathways with the flux ratio depending on conditions such as ligand concentration. However, the structural and energetic basis of such complex reactions remains poorly understood. Therefore, we used experimental, theoretical, and computational approaches to explore structural and energetic aspects of the coupled-folding/binding reaction of staphylococcal nuclease in the presence of the substrate analog adenosine-3',5'-diphosphate. Optically monitored equilibrium and kinetic data, combined with a statistical mechanical model, gave deeper insight into the relative importance of specific and Coulombic protein-ligand interactions in governing the reaction mechanism. We also investigated structural aspects of the reaction at the residue level using NMR and all-atom replica-permutation molecular dynamics simulations. Both approaches yielded clear evidence for accumulation of a transient protein-ligand encounter complex early in the reaction under IF-dominant conditions. Quantitative analysis of the equilibrium/kinetic folding revealed that the ligand-dependent CS-to-IF shift resulted from stabilization of the compact transition state primarily by weakly ligand-dependent Coulombic interactions with smaller contributions from specific binding energies. At a more macroscopic level, the CS-to-IF shift was represented as a displacement of the reaction "route" on the free energy surface, which was consistent with a flux analysis.

Induced-Fit Pathway Accelerated Binding of Agitoxin-2 to A K+ Channel Imaged by HS-AFM

Agitoxin-2 (AgTx2) from scorpion venom is a potent blocker of K+ channels which consists of 38 amino acids. The docking model has been elucidated by using solution structure of AgTx2 and molecular dynamics simulation with K+ channel, thus electrostatic and hydrophobic interactions that contribute to binding have been revealed. However, it remains unclear whether binding dynamics are described by a two-state model (AgTx2-bound and AgTx2-unbound) or a more complicated mechanism, such as induced fit or conformational selection. Here, we observed the binding dynamics of AgTx2 to the KcsA channel using high-speed atomic force microscopy. From images of repeated binding and dissociation of AgTx2 to the channel, single-molecule kinetic analyses revealed that the affinity of the channel for AgTx2 increased during persistent binding and decreased during persistent dissociation. We propose a four-state model, including high- and low-affinity states of the channel, with relevant rate constants. An induced-fit pathway was dominant and accelerated binding by 400 times. This is the first analytical imaging of scorpion toxin binding in real time, which is applicable to various biological dynamics including channel ligands, DNA-modifier proteins, and antigen-antibody complexes.

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.

High-speed AFM reveals accelerated binding of agitoxin-2 to a K+ channel by induced fit.

Agitoxin-2 (AgTx2) from scorpion venom is a potent blocker of K+ channels. The docking model has been elucidated, but it remains unclear whether binding dynamics are described by a two-state model (AgTx2-bound and AgTx2-unbound) or a more complicated mechanism, such as induced fit or conformational selection. Here, we observed the binding dynamics of AgTx2 to the KcsA channel using high-speed atomic force microscopy. From images of repeated binding and dissociation of AgTx2 to the channel, single-molecule kinetic analyses revealed that the affinity of the channel for AgTx2 increased during persistent binding and decreased during persistent dissociation. We propose a four-state model, including high- and low-affinity states of the channel, with relevant rate constants. An induced-fit pathway was dominant and accelerated binding by 400 times. This is the first analytical imaging of scorpion toxin binding in real time, which is applicable to various biological dynamics including channel ligands, DNA-modifier proteins, and antigen-antibody complexes.

Exploration of Multistate Conformational Dynamics upon Ligand Binding of a Monomeric Enzyme Involved in Pyrophosphoryl Transfer.

HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase) is a monomeric protein with 158 residues, which undergoes large-scale conformational changes between apo, open, and holo states responding to ligand binding for its function. It has been explored widely as an excellent target for potential antibacterial drug development. However, little is known about how conformational dynamics between the native states influences the substrate recognition and the functionality of enzymatic catalysis. Here, we report a coarse-grained triple-basin structure-based model upon ligand binding to describe such multiple-state system by the molecular dynamics simulation. With our model, we have made theoretical predictions that are in good agreement with the experimental measurements. Our results revealed the intrinsic conformational fluctuations between apo and open states without ligand binding. We found that HPPK can switch to the activated holo state upon the ordered binding of the two ligands (ATP and HP). We uncovered the underlying mechanism by which major induced fit and minor population shift pathways coexist upon ligand binding by quantitative flux analysis. Additionally, we pointed out the structural origin for the conformational changes and identified the key residues as well as contact interactions. We further explored the temperature effect on the conformational distributions and pathway weights. It gave strong support that higher temperatures promote population shift, while the induced fit pathway is always the predominant activation route of the HPPK system. These findings will provide significant insights of the mechanisms of the multistate conformational dynamics of HPPK upon ligand binding.

Bridging Microscopic and Macroscopic Mechanisms of p53-MDM2 Binding with Kinetic Network Models

Under normal cellular conditions, the tumor suppressor protein p53 is kept at low levels in part due to ubiquitination by MDM2, a process initiated by binding of MDM2 to the intrinsically disordered transactivation domain (TAD) of p53. Many experimental and simulation studies suggest that disordered domains such as p53 TAD bind their targets nonspecifically before folding to a tightly associated conformation, but the microscopic details are unclear. Toward a detailed prediction of binding mechanisms, pathways, and rates, we have performed large-scale unbiased all-atom simulations of p53-MDM2 binding. Markov state models (MSMs) constructed from the trajectory data predict p53 TAD binding pathways and on-rates in good agreement with experiment. The MSM reveals that two key bound intermediates, each with a nonnative arrangement of hydrophobic residues in the MDM2 binding cleft, control the overall on-rate. Using microscopic rate information from the MSM, we parameterize a simple four-state kinetic model to 1) determine that induced-fit pathways dominate the binding flux over a large range of concentrations, and 2) predict how modulation of residual p53 helicity affects binding, in good agreement with experiment. These results suggest new ways in which microscopic models of peptide binding, coupled with simple few-state binding flux models, can be used to understand biological function in physiological contexts.

A Kinetic Signature for Parallel Pathways: Conformational Selection and Induced Fit. Links and Disconnects between Observed Relaxation Rates and Fractional Equilibrium Flux under Pseudo-First-Order Conditions.

Molecular association plays a ubiquitous role in biochemistry and is often accompanied by conformational exchange in one or both binding partners. Traditionally, two limiting mechanisms are considered for the association of two molecules. In a conformational selection (CS) mechanism, a ligand preferentially binds to a subset of conformations in its binding partner. In contrast, an induced fit (IF) mechanism describes the ligand-dependent isomerization of the binding partner in which binding occurs prior to conformational exchange. Measurements of the ligand concentration dependence of observed rates of relaxation are commonly used to probe whether CS or IF is taking place. Here we consider a four-state thermodynamic cycle subject to detailed balance and demonstrate the existence of a relatively unexplored class of kinetic signatures where an initial decrease in the observed rate is followed by a subsequent increase under pseudo-first-order conditions. We elucidate regions of rate space necessary to generate a nonmonotonic observed rate and show that, under certain conditions, the position of the minimum of the observed rate correlates with a transition in equilibrium flux between CS and IF pathways. Furthermore, we demonstrate that monotonic trends in the observed rate can occur when both CS and IF mechanisms are taking place, suggesting that caution must be taken not to overinterpret monotonic trends as evidence of the absence of either CS or IF. Lastly, we conclude that a nonmonotonic kinetic signature is uniquely unambiguous in the sense that when this trend is observed, one may conclude that both CS and IF mechanistic paths are utilized.

RNA Structural Modules Control the Rate and Pathway of RNA Folding and Assembly

Structured RNAs fold through multiple pathways, but we have little understanding of the molecular features that dictate folding pathways and determine rates along a given pathway. Here, we asked whether folding of a complex RNA can be understood from its structural modules. In a two-piece version of the Tetrahymena group I ribozyme, the separated P5abc subdomain folds to local native secondary and tertiary structure in a linked transition and assembles with the ribozyme core via three tertiary contacts: a kissing loop (P14), a metal core–receptor interaction, and a tetraloop–receptor interaction, the first two of which are expected to depend on native P5abc structure from the local transition. Native gel, NMR, and chemical footprinting experiments showed that mutations that destabilize the native P5abc structure slowed assembly up to 100-fold, indicating that P5abc folds first and then assembles with the core by conformational selection. However, rate decreases beyond 100-fold were not observed because an alternative pathway becomes dominant, with nonnative P5abc binding the core and then undergoing an induced-fit rearrangement. P14 is formed in the rate-limiting step along the conformational selection pathway but after the rate-limiting step along the induced-fit pathway. Strikingly, the assembly rate along the conformational selection pathway resembles that of an isolated kissing loop similar to P14, and the rate along the induced-fit pathway resembles that of an isolated tetraloop–receptor interaction. Our results indicate substantial modularity in RNA folding and assembly and suggest that these processes can be understood in terms of underlying structural modules.

Enzymes with lid-gated active sites must operate by an induced fit mechanism instead of conformational selection

The induced fit and conformational selection/population shift models are two extreme cases of a continuum aimed at understanding the mechanism by which the final key-lock or active enzyme conformation is achieved upon formation of the correctly ligated enzyme. Structures of complexes representing the Michaelis and enolate intermediate complexes of the reaction catalyzed by phosphoenolpyruvate carboxykinase provide direct structural evidence for the encounter complex that is intrinsic to the induced fit model and not required by the conformational selection model. In addition, the structural data demonstrate that the conformational selection model is not sufficient to explain the correlation between dynamics and catalysis in phosphoenolpyruvate carboxykinase and other enzymes in which the transition between the uninduced and the induced conformations occludes the active site from the solvent. The structural data are consistent with a model in that the energy input from substrate association results in changes in the free energy landscape for the protein, allowing for structural transitions along an induced fit pathway.

Crystallographic snapshots along a protein-induced DNA-bending pathway.

Two new high-resolution cocrystal structures of EcoRV endonuclease bound to DNA show that a large variation in DNA-bending angles is sampled in the ground state binary complex. Together with previous structures, these data reveal a contiguous series of protein conformational states delineating a specific trajectory for the induced-fit pathway. Rotation of the DNA-binding domains, together with movements of two symmetry-related helices binding in the minor groove, causes base unstacking at a key base-pair step and propagates structural changes that assemble the active sites. These structures suggest a complex mechanism for DNA bending that depends on forces generated by interacting protein segments, and on selective neutralization of phosphate charges along the inner face of the bent double helix.

Scanning curves and kinetics of the acetylcholine/acetylcholine receptor hysteresis

The high affinity binding of the neurotransmitter acetylcholine (AcCh) to the nicotinic acetylcholine receptor (nAcChR) of the electric organ of Torpedo californica exhibits a pronounced hysteresis and scanning loops. Dialysis conditions lead to an equilibrium binding curve which is coincident with the upper hysteresis branch; K = 5 × 10 −9 M, 4° C; and one AcCh molecule binds to the receptor (R) monomer of M r ≈ 290 000. Kinetic analysis of the changes in free and bound AcCh during the open-system conditions of dialysis, which releases the metastability, shows that AcCh (A) binding proceeds along an induced-fit pathway according to A + R h ⇌ AR h ⇌ AR vh, describing the ligand-induced transition from the high affinity state (R h; K h ≈ 10 −7 M) to the very high affinity state (R vh; K vh ⪡ k ). The rate constant of the step AR h → AR vh is k 2 = 6 × 10 −3 s −1 and that of the reverse s is k −2 = 3 × 10 −4 s −1. Direct binding of A to free R vh can be neglected. Therefore, the state R vh does not pre-exist; it is induced and only stable, as AR vh, by bound AcCh. The metastability can be described in terms of long-lived AR vh·R 1 hybrid dimers. Physiologically, the steric hindrance in the metastable hybrid may be viewed as a saving device: the functionally important, receptor channel-active R 1 conformer is, at low AcCh concentrations ([A] < 1 μ M), prevented from converting into the desensitized states R h and AR vh.

Scanning curves and kinetics of the acetylcholine / acetylcholine receptor hysteresis

The high affinity binding of the neurotransmitter acetylcholine (AcCh) to the nicotinic acetylcholine receptor (nAcChR) of the electric organ of Torpedo californica exhibits a pronounced hysteresis and scanning loops. Dialysis conditions lead to an equilibrium binding curve which is coincident with the upper hysteresis branch; K = 5 × 10 −9 M, 4° C; and one AcCh molecule binds to the receptor (R) monomer of M r ≈ 290 000. Kinetic analysis of the changes in free and bound AcCh during the open-system conditions of dialysis, which releases the metastability, shows that AcCh (A) binding proceeds along an induced-fit pathway according to A + R h ⇌ AR h ⇌ AR vh, describing the ligand-induced transition from the high affinity state (R h; K h ≈ 10 −7 M) to the very high affinity state (R vh; K vh ⪡ K ). The rate constant of the step AR h → AR vh is k 2 = 6 × 10 −3 s −1 and that of the reverse step is k −2 = 3 × 10 −4 s −1. Direct binding of A to free R vh can be neglected. Therefore, the state R vh does not pre-exist; it is induced and only stable, as AR vh, by bound AcCh. The metastability can be described in terms of long-lived AR vh · R 1 hybrid dimers. Physiologically, the steric hindrance in the metastable hybrid may be viewed as a saving device; the functionally important, receptor channel-active R 1 conformer is, at low AcCh concentrations ([A] < 1 μ M), prevented from converting into the desensitized states R h and AR vh.