The mechanism of ligand binding to the periplasmic C4-dicarboxylate binding protein (DctP) from Rhodobacter capsulatus.

The kinetics of ligand binding to the periplasmic C4-dicarboxylate binding protein (DctP) from Rhodobacter capsulatus were investigated by exploiting the changes in the intrinsic fluorescence of the protein upon binding ligands. Steady state measurements have shown that L-malate, succinate, and fumarate are all bound with sub-micromolar Kd values, whereas D-malate is bound 2 orders of magnitude more weakly. Stopped-flow studies have revealed that the binding process involves at least three steps. In the absence of ligand, the protein is in equilibrium between an essentially nonbinding form, BP1, and the binding form, BP2. Ligands bind to the BP2 form, shifting the equilibrium toward the BP2-L conformation, and also inducing a further isomerization of the protein, to the BP3-L form. The kinetic properties of the four different conformational states of the DctP protein identified in this study would be consistent with their identification as the closed-conformation, the open-conformation, an open-liganded conformation, and a closed-liganded conformation. The latter three states have been identified by x-ray crystallographic studies of binding proteins, but no kinetic or structural data have been presented previously to support the possibility of a closed but unliganded conformation.     

Crystal structures of two Bordetella pertussis periplasmic receptors contribute to defining a novel pyroglutamic acid binding DctP subfamily

Gram-negative bacteria have developed several different transport systems for solute uptake. One of these, the tripartite ATP independent periplasmic transport system (TRAP-T), makes use of an extracytoplasmic solute receptor (ESR) which captures specific solutes with high affinity and transfers them to their partner permease complex located in the bacterial inner membrane. We hereby report the structures of DctP6 and DctP7, two such ESRs from Bordetella pertussis. These two proteins display a high degree of sequence and structural similarity and possess the "Venus flytrap" fold characteristic of ESRs, comprising two globular alpha/beta domains hinged together to form a ligand binding cleft. DctP6 and DctP7 both show a closed conformation due to the presence of one pyroglutamic acid molecule bound by highly conserved residues in their respective ligand binding sites. BLAST analyses have revealed that the DctP6 and DctP7 residues involved in ligand binding are strictly present in a number of predicted TRAP-T ESRs from other bacteria. In most cases, the genes encoding these TRAP-T systems are located in the vicinity of a gene coding for a pyroglutamic acid metabolising enzyme. Both the high degree of conservation of these ligand binding residues and the genomic context of these TRAP-T-coding operons in a number of bacterial species, suggest that DctP6 and DctP7 constitute the prototypes of a novel TRAP-T DctP subfamily involved in pyroglutamic acid transport.     

CAMP-dissociation kinetics in hormone-dependent and -independent rat mammary tumor cytosols

Cytosols from 7, 12-dimethylbenz (alpha) anthracene-induced rat mammary tumors which exhibit different hormone-responsiveness were compared with respect to their cAMP-dissociation kinetics. At 22 degree C, pH 4.5, 1 micrometer cAMP, hormone-dependent mammary tumors exhibited monophasic dissociation rates with a rate constant of k-1 = 0.06 min-1. In contrast, hormone-independent mammary tumors exhibited biphasic dissociation curves with rate constants of k-1 = 0.47 and k-2 = 0.06 min-1. The binding of cAMP was completely reversible; radio-labeled ligand was completely dissociated by 1mM nonradioactive cAMP; the binding protein could be reassociated to its original binding level after dextran-coated charcoal adsorption. The mammary cytosols exhibited specific binding for cAMP which could be displaced partially by cGMP but not by ATP, ADP, AMP, or adenosine. Receptor inactivation during the course of incubation was negligible. Both mammary tissue cytosols exhibited similar association rates at 22 degree C, pH 4.5, 1 micrometer cAMP (k+1 = 5-7 x 10(5)M-1 min-1). These data indicate that mammary tissues exhibit 2 cAMP dissociation rates. Hormone-dependent mammary tumors exhibit a dissociation constant of a high affinity binding site (k-1/k+1 = 0.07 micrometer) whereas hormone-independent mammary tumors exhibit dissociation constants of one high affinity (k-1/k+1 = 0.07 micrometer) and a second low affinity site (k-1/k+1 = 0.05 micrometer).     

How maltose influences structural changes to bind to maltose‐binding protein: Results from umbrella sampling simulation

A well‐studied periplasmic‐binding protein involved in the abstraction of maltose is maltose‐binding protein (MBP), which undergoes a ligand‐induced conformational transition from an open (ligand‐free) to a closed (ligand‐bound) state. Umbrella sampling simulations have been us to estimate the free energy of binding of maltose to MBP and to trace the potential of mean force of the unbinding event using the center‐of‐mass distance between the protein and ligand as the reaction coordinate. The free energy thus obtained compares nicely with the experimentally measured value justifying our theoretical basis. Measurement of the domain angle (N‐terminal‐domain – hinge – C‐terminal‐domain) along the unbinding pathway established the existence of three different states. Starting from a closed state, the protein shifts to an open conformation during the initial unbinding event of the ligand then resides in a semi‐open conformation and later resides predominantly in an open‐state. These transitions along the ligand unbinding pathway have been captured in greater depth using principal component analysis. It is proposed that in mixed‐model, both conformational selection and an induced‐fit mechanism combine to the ligand recognition process in MBP. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Correlation of acid-induced conformational transition of ferricytochrome <Emphasis Type="Italic">c</Emphasis> with cyanide binding kinetics

A relation between pH-induced conformational transitions of horse heart ferricytochrome c and the kinetics of external ligand coordination to heme iron was investigated by optical spectroscopy, circular dichroism and viscometry. The dependencies of both the association, k (a), and dissociation rate constants of cyanide binding on pH were determined from kinetic measurements. The association rate constant exhibits a bell-shaped form of dependence on pH in the region where this protein unfolds. The maximum of the dependence of k (a) on pH is found to be coincident with the pK values of conformational transitions of ferricytochrome c in solutions with both low and high ionic strengths. This observation is explained in terms of ferricytochrome c unfolding, which is characterized by two processes: the gradual opening of the heme crevice accompanied by the detachment of the axial Met80 and its replacement with a water molecule. The former process enhances the rate, whereas the latter results in the inhibition of the rate of cyanide binding.     

Transferrins, the mechanism of iron release by ovotransferrin.

Iron release from ovotransferrin in acidic media (3 < pH < 6) occurs in at least six kinetic steps. The first is a very fast (相似文献   

Stopped-flow fluorescence kinetic studies of Glu-plasminogen. Conformational changes triggered by AH-site ligand binding

Binding of 6-aminohexanoic acid to the AH-site, a weak lysine binding site in Glu-plasminogen, alters the conformation of the molecule. The kinetics of the binding and the accompanying conformational change are investigated at pH 7.8, 25 degrees C. Changes of intrinsic protein fluorescence were measured as a function of time after rapid mixing in a stopped-flow apparatus. The results reflect a two-step reaction mechanism: Rapid association of Glu-plasminogen and 6-aminohexanoic acid (K1 = 44 mM) followed by the conformational change (k2 = 69 s-1 and k-2 = 3 s-1) with an overall dissociation constant Kd = 2.0 mM. Thus the conformational change is rather fast, t12 = 0.01 s. Its importance for the rates of Glu-plasminogen activation reactions is discussed.     

Kinetics and mechanism for the conformational transition in p-guanidinobenzoate bovine trypsinogen induced by the isoleucine-valine dipeptide

The interaction between p-guanidinobenzoate-trypsinogen and the isoleucine-valine dipeptide has been investigated by temperature-jump relaxation spectrometry. Using the absorbance at 281 nm the concentration dependence of the relaxation parameters is consistent with the conventional induced-fit model: rapid ligand binding coupled to a slower intramolecular change; some alternative mechanisms can be excluded. At 296 K, 0.1 M Tris HCl, pH = 7.4, the dissociation equilibrium constant for the overall process is K = 5.1(+/- 0.2) X 10(-5) M; for the binding step K1 = 2.3(+/- 0.3) X 10(-3) M and the rate constants for the structural change are k2 = 26(+/-6)s-1 and k-2 = 0.61(+/- 0.04)s-1; the overall dissociation reaction enthalpy is delta H0 = 26(+/-6)KJmol-1 and the reactiom entropy is delta S0 = 4(+/- 20) kJ-1 mol-1. In combination with CD and X-ray crystallographic data, the results of this study suggest that the binding of the dipeptide to a trypsinogen-like, partially disordered conformation induces a transition to a trypsin-like highly ordered structure.     

NMR characterization of a pH-dependent equilibrium between two folded solution conformations of the pheromone-binding protein from Bombyx mori

NMR spectroscopic changes as a function of pH in solutions of the pheromone-binding protein of Bombyx mori (BmPBP) show that BmPBP undergoes a conformational transition between pH 4.9 and 6.0. At pH below 4.9 there is a single "acid form" (A), and a homogeneous "basic form" (B) exists at pH above 6.0. Between pH 5 and 6, BmPBP exists as a mixture of A and B in slow exchange on the NMR chemical shift time scale, with the transition midpoint at pH 5.4. The form B has a well-dispersed NMR spectrum, indicating that it represents a more structured, "closed" conformation than form A, which has a significantly narrower chemical shift dispersion. Conformational transitions of the kind observed here may explain heterogeneity reported for a variety of odorant-binding proteins, and it will be of interest to further investigate possible correlations with pH-dependent regulation of ligand binding and release in the biological function of this class of proteins.     

Mechanism of the conformational transition of melittin.

It is known that, while melittin at micromolar concentrations is unfolded under conditions of low ionic strength at neutral pH, it adopts a tetrameric alpha-helical structure under conditions of high ionic strength, at alkaline pH, or at high peptide concentrations. To understand the mechanism of the conformational transition of melittin, we examined in detail the conformation of melittin under various conditions by far-UV circular dichroism at 20 degrees C. We found that the helical conformation is also stabilized by strong acids such as perchloric acid. The effects of various acids varied largely and were similar to those of the corresponding salts, indicating that the anions are responsible for the salt- or acid-induced transitions. The order of effectiveness of various monovalent anions was consistent with the electroselectivity series of anions toward anion-exchange resins, indicating that the anion binding is responsible for the salt- or acid-induced transitions. From the NaCl-, HCl-, and alkaline pH-induced conformational transitions, we constructed a phase diagram of the anion- and pH-dependent conformational transition. The phase diagram was similar in shape to that of acid-denatured apomyoglobin [Goto, Y., & Fink, A.L. (1990) J. Mol. Biol. 214, 803-805] or that of the amphiphilic Lys, Leu model polypeptide [Goto, Y., & Aimoto, S. (1991) J. Mol. Biol. 218, 387-396], suggesting a common mechanism of the conformational transition. The anion-, pH-, and peptide concentration-dependent conformational transition of melittin was explained on the basis of an equation in which the conformational transition is linked to proton and anion binding to the titratable groups.     

Kinetic evidence for two interconvertible forms of the folate transport protein from Lactobacillus casei.

Lactobacillus casei cells contain a folate transport protein which exhibits a high affinity for folate. The dissociation constant (KD) for folate derived from binding parameters at the steady state (at 0 degree C) is 0.4 nM at pH 7.5 and 0.1 nM at pH 6.0. In the present study, folate binding to this protein at pH 7.5 (and 0 degree C) was shown to follow second-order kinetics and to proceed with an association constant (k+1) of 4.9 X 10(7) liter/mol per min. K+1 was not affected by preincubation conditions which alter the energetic state of the cell. Measurements on the extent of binding showed further that (at 0 degree C) essentially all unoccupied folate-binding sites reside at or are readily accessible to the outer surface of the membrane. In contrast, after saturating the binding site with [3H]folate, the first-order rate constant (k-1) for dissociation of the bound substrate (at 0 degree C) was found to vary substantially with the conditions employed. k-1 was 0.028/min in freshly harvested cells, but it increased by 2.8-fold in cells preincubated at 23 degrees C for 60 min and by 5.4-fold in isolated membranes. In addition, the faster rate observed in preincubated cells (k-1 = 0.077/min) returned to a slower rate after brief exposure of the cells to pH 6.0 (k-1 = 0.041/min), glucose (k-1 = 0.050/min), or both (k-1 = 0.012/min). k-1 was twofold lower at pH 6.0 than at pH 7.5 and was less dependent on the preincubation conditions, although it also increased substantially (5.5-fold) when the cells were converted to plasma membranes. The proposed explanation for these results is that folate transport protein of L. casei exists in two forms which can be distinguished by the accessibility of the binding site to the external medium and whose amounts are dependent upon the presence of bound folate, the pH, and the energetic state of the cell. It is suggested that these forms are transport proteins with binding sites oriented towards the inner and outer surfaces of the membrane.     

Multiple-step kinetic mechanism of DNA-independent ATP binding and hydrolysis by Escherichia coli replicative helicase DnaB protein: quantitative analysis using the rapid quench-flow method

The kinetic mechanism of DNA-independent binding and hydrolysis of ATP by the E. coli replicative helicase DnaB protein has been quantitatively examined using the rapid quench-flow technique. Single-turnover studies of ATP hydrolysis, in a non-interacting active site of the helicase, indicate that bimolecular association of ATP with the site is followed by the reversible hydrolysis of nucleotide triphosphate and subsequent conformational transition of the enzyme-product complex. The simplest mechanism, which describes the data, is a three-step sequential process defined by:?eqalign???rm Helicase+ATP?&?mathop??rightleftharpoons? ?k_1?_?k_?-1????rm (H-ATP)??mathop??rightleftharpoons? ?k_2?_?k_?-2????rm (H-ADP?cdot Pi)??cr &?mathop??rightleftharpoons? ?k_3?_?k_?-3????rm (H-ADP?cdot Pi)? *?The sequential character of the mechanism excludes conformational transitions of the DnaB helicase prior to ATP binding. Analysis of relaxation times and amplitudes of the reaction allowed us to estimate all rate and equilibrium constants of partial steps of the proposed mechanism. The intrinsic binding constant for the formation of the (H-ATP) complex is K(ATP)=(1.3+/-0.5)x10(5) M(-1). The analysis of the data indicates that a part of the ATP binding energy originates from induced structural changes of the DnaB protein-ATP complex prior to ATP hydrolysis. The equilibrium constant of the chemical interconversion is K(H)=k(2)/k(-2) approximately 2 while the subsequent conformational transition is characterized by K(3)=k(3)/k(-3) approximately 30. The low value of K(H) and the presence of the subsequent energetically favorable conformational step(s) strongly suggest that free energy is released from the enzyme-product complex in the conformational transitions following the chemical step and before the product release.The combined application of single and multiple-turnover approaches show that all six nucleotide-binding sites of the DnaB hexamer are active ATPase sites. Binding of ATP to the DnaB hexamer is characterized by the negative cooperativity parameter sigma=0.25(+/-0.1). The negative cooperative interactions predominantly affect the ground state of the enzyme-ATP complex. The significance of these results for the mechanism of the free energy transduction of the DnaB helicase is discussed.     

Kinetic mechanism of the single-stranded DNA recognition by Escherichia coli replicative helicase DnaB protein. Application of the matrix projection operator technique to analyze stopped-flow kinetics

Kinetics of the Escherichia coli primary replicative helicase DnaB protein binding to a single-stranded DNA, in the presence of the ATP non-hydrolyzable analog AMP-PNP, have been performed, using the fluorescence stopped-flow technique. This is the first direct determination of the mechanism of the ssDNA recognition by a hexameric helicase. Binding of the fluorescent etheno-derivative of a ssDNA to the enzyme is characterized by a strong increase of the nucleic acid fluorescence, which provides an excellent signal to quantitatively study the mechanism of ssDNA recognition by the helicase. The kinetic experiments have been performed with a ssDNA 20-mer, depsilonA(pepsilonA)(19), that encompasses the entire, total ssDNA-binding site of the helicase and with the 10-mer depsilonA(pepsilonA)(9), which binds exclusively to the ssDNA strong subsite within the total ssDNA-binding site. Association of the DnaB helicase with the 20-mer is characterized by three relaxation times, which indicates that the binding occurs by the minimum three-step mechanism where the bimolecular binding step is followed by two isomerization steps. This mechanism is described by the equation: Helicase+ssDNAk1/(k1)<-->(k-1)(H-ssDNA)1(k2)<-->(k-2)(H-ssDNA)2 (k3)<-->(k-3)(H-ssDNA)3. The value of the bimolecular rate constant, k(1), is four to six orders of magnitude lower than the value expected for the diffusion-controlled reaction. Moreover, quantitative amplitude analysis suggests that the major conformational change of the ssDNA takes place in the formation of the (H-ssDNA)(1). These results indicate that the determined first step includes formation of the collision and an additional transition of the protein-ssDNA complex, most probably the local opening of the protein hexamer. The data indicate that the binding mechanism reflects the interactions of the ssDNA predominantly through the strong ssDNA-binding subsite. The analysis of the stopped-flow kinetics has been performed using the matrix-projection operator technique, which provides a powerful method to address stopped-flow kinetics, particularly, the amplitudes. The method allowed us to determine the specific fluorescence changes accompanying the formation of all the intermediates. The sequential nature of the determined mechanism indicates the lack of the kinetically significant conformational equilibrium of the DnaB hexamer as well as a transient dissociation of the hexamer prior to the ssDNA binding. The significance of these results for the functioning of the DnaB helicase is discussed.     

Kinetics of ligand binding to the type 1 Fc epsilon receptor on mast cells

Rates of association and dissociation of several specific monovalent ligands to and from the type I Fc epsilon receptor (Fc epsilon RI) were measured on live mucosal type mast cells of the rat line RBL-2H3. The ligands employed were a monoclonal murine IgE and Fab fragments prepared from three different, Fc epsilon RI-specific monoclonal IgG class antibodies. These monoclonals (designated H10, J17, and F4) were shown previously to trigger mediator secretion by RBL-2H3 mast cells upon binding to and dimerization of the Fc epsilon RI. Analysis of the kinetics shows that the minimal mechanism to which all data can be fitted involves two consecutive steps: namely, ligand binding to a low-affinity state of the receptor, followed by a conformational transition into a second, higher affinity state h of the receptor-ligand complex. These results resolve the recently noted discrepancy between the affinity of IgE binding to the Fc epsilon RI as determined by means of binding equilibrium measurements [Ortega et al. (1988) EMBO J. 7, 4101] and the respective parameter derived from the ratio of the rate constant of rat IgE dissociation and the initial rate of rat IgE association [Wank et al. (1983) Biochemistry 22, 954]. The probability of undergoing the conformational transition differs for the four different Fc epsilon RI-ligand complexes: while binding of Fab-H10 and IgE favors the h state, binding of Fab-J17 and Fab-F4 preferentially maintains the low-affinity 1 state (at 25 degrees C). The temperature dependence of the ligand interaction kinetics with the Fc epsilon RI shows that the activation barrier for ligand association is determined by positive enthalpic and entropic contributions. The activation barrier of the 1----h transition, however, has negative enthalpic contributions counteracted by a decrease in activation entropy. The h----1 transition encounters a barrier that is predominantly entropic and similar for all ligands employed, thus suggesting that the Fc epsilon RI undergoes a similar conformational transition upon binding any of the ligands.     

Conformational selection or induced fit? A critical appraisal of the kinetic mechanism

For almost five decades, two competing mechanisms of ligand recognition, conformational selection and induced fit, have dominated our interpretation of ligand binding in biological macromolecules. When binding-dissociation events are fast compared to conformational transitions, the rate of approach to equilibrium, k(obs), becomes diagnostic of conformational selection or induced fit based on whether it decreases or increases, respectively, with the ligand concentration, [L]. However, this simple conclusion based on the rapid equilibrium approximation is not valid in general. Here we show that conformational selection is associated with a rich repertoire of kinetic properties, with k(obs) decreasing or increasing with [L] depending on the relative magnitude of the rate of ligand dissociation, k(off), and the rate of conformational isomerization, k(r). We prove that, even for the simplest two-step mechanism of ligand binding, a decrease in k(obs) with [L] is unequivocal evidence of conformational selection, but an increase in k(obs) with [L] is not unequivocal evidence of induced fit. Ligand binding to glucokinase, thrombin, and its precursor prethrombin-2 are used as relevant examples. We conclude that conformational selection as a mechanism for a ligand binding to its target may be far more common than currently believed.     

A kinetic study of protein-protein interactions.

Kinetic studies have been carried out of the monomer-dimer interaction of insulin, beta-lactoglobulin, and alpha-chymotrypsin using stopped-flow and temperature-jump techniques. The pH indicators bromothymol blue, bromophenol blue, and phenol red were used to monitor pH changes associated with the monomer-dimer interaction. In all three cases a kinetic process was observed which could be attributed to a simple monomer-dimer equilibrium, and association (k1) and dissociation (k-1) rate constants were determined. The results obtained are as follows: for insulin at 23 degrees C, pH 6.8, 0.125 M KNO3, k1 = 1.14 X 10(8) M-1 s-1, k-1 - 1.48 X 10(4)s(-1); for beta-lactoglobulin AB at 35 degrees C, pH 3.7, 0.025 M KNO3, d1 = 4.7 X 10(4) M-1 s-1, k-1 = 2.1 s-1; for alpha-chymotrypsin at 25 degreesC, pH 4.3, 0.05 M KNO3 k1 - 3.7 X 10(3) M-1 s-1, k-1 - 0.68 s-1. The kinetic behavior of the separated beta-lactoglobulin A and B was similar to that of the mixture. In the case of chymotrypsin, bromophenol blue was found to activate the enzyme catalyzed hydrolysis of p-nitrophenyl acetate, and a rate process was observed with the temperature jump which could be attributed to a conformational change of the indicator-protein complex. The association rate constant for dimer formation of insulin approaches the value expected for a diffusion-controlled process, while the values obtained for the other two proteins are below those expected for a diffusion-controlled reaction unless unusally large steric and electrostatic effects are present.     

On the validity of the quasi-steady state approximation of bimolecular reactions in solution

Two-step binding kinetics are extensively used to study the relative importance of diffusion in biochemical reactions. Classical analysis of this problem assumes ad hoc that the encounter complex is at quasi-steady state (QSS). Using scaling arguments we derive a criterion for the validity of this assumption in the limit of irreversible product formation. We find that the QSS approximation (QSSA) of two-step binding is only valid if the total ligand and receptor concentrations are much smaller than (k2+k-1)/k1, where k1 and k-1 are, respectively, the forward and reverse diffusion encounter rate constants and k2 is the chemical association rate constant. This criterion can be shown to imply that the average time between encounters is much longer than the half-life of the encounter complex and also guarantees that the concentration of the encounter complex is negligible compared to the reactant and product concentrations. Numerical examples of irreversible and reversible cases corroborate our analysis and illustrate that the QSS may be invalid even if k-2相似文献   

Probing the dynamics of a His73-heme alkaline transition in a destabilized variant of yeast iso-1-cytochrome c with conformationally gated electron transfer methods

The alkaline transition of cytochrome c involves substitution of the Met80 heme ligand of the native state with a lysine ligand from a surface Ω-loop (residues 70 to 85). The standard mechanism for the alkaline transition involves a rapid deprotonation equilibrium followed by the conformational change. However, recent work implicates multiple ionization equilibria and stable intermediates. In previous work, we showed that the kinetics of formation of a His73-heme alkaline conformer of yeast iso-1-cytochrome c requires ionization of the histidine ligand (pK(HL) ~ 6.5). Furthermore, the forward and backward rate constants, k(f) and k(b), respectively, for the conformational change are modulated by two auxiliary ionizations (pK(H1) ~ 5.5, and pK(H2) ~ 9). A possible candidate for pK(H1) is His26, which has a strongly shifted pK(a) in native cytochrome c. Here, we use the AcH73 iso-1-cytochrome c variant, which contains an H26N mutation, to test this hypothesis. pH jump experiments on the AcH73 variant show no change in k(obs) for the His73-heme alkaline transition from pH 5 to 8, suggesting that pK(H1) has disappeared. However, direct measurement of k(f) and k(b) using conformationally gated electron transfer methods shows that the pH independence of k(obs) results from coincidental compensation between the decrease in k(b) due to pK(H1) and the increase in k(f) due to pK(HL). Thus, His26 is not the source of pK(H1). The data also show that the H26N mutation enhances the dynamics of this conformational transition from pH 5 to 10, likely as a result of destabilization of the protein.     

Mapping the Dynamics Landscape of Conformational Transitions in Enzyme: The Adenylate Kinase Case

Conformational transition describes the essential dynamics and mechanism of enzymes in pursuing their various functions. The fundamental and practical challenge to researchers is to quantitatively describe the roles of large-scale dynamic transitions for regulating the catalytic processes. In this study, we tackled this challenge by exploring the pathways and free energy landscape of conformational changes in adenylate kinase (AdK), a key ubiquitous enzyme for cellular energy homeostasis. Using explicit long-timescale (up to microseconds) molecular dynamics and bias-exchange metadynamics simulations, we determined at the atomistic level the intermediate conformational states and mapped the transition pathways of AdK in the presence and absence of ligands. There is clearly chronological operation of the functional domains of AdK. Specifically in the ligand-free AdK, there is no significant energy barrier in the free energy landscape separating the open and closed states. Instead there are multiple intermediate conformational states, which facilitate the rapid transitions of AdK. In the ligand-bound AdK, the closed conformation is energetically most favored with a large energy barrier to open it up, and the conformational population prefers to shift to the closed form coupled with transitions. The results suggest a perspective for a hybrid of conformational selection and induced fit operations of ligand binding to AdK. These observations, depicted in the most comprehensive and quantitative way to date, to our knowledge, emphasize the underlying intrinsic dynamics of AdK and reveal the sophisticated conformational transitions of AdK in fulfilling its enzymatic functions. The developed methodology can also apply to other proteins and biomolecular systems.