Hierarchical domain‐motion analysis of conformational changes in sarcoplasmic reticulum Ca2+‐ATPase

Sarco(endo)plasmic reticulum Ca²⁺‐ATPase transports two Ca²⁺ per ATP‐hydrolyzed across biological membranes against a large concentration gradient by undergoing large conformational changes. Structural studies with X‐ray crystallography revealed functional roles of coupled motions between the cytoplasmic domains and the transmembrane helices in individual reaction steps. Here, we employed “Motion Tree (MT),” a tree diagram that describes a conformational change between two structures, and applied it to representative Ca²⁺‐ATPase structures. MT provides information of coupled rigid‐body motions of the ATPase in individual reaction steps. Fourteen rigid structural units, “common rigid domains (CRDs)” are identified from seven MTs throughout the whole enzymatic reaction cycle. CRDs likely act as not only the structural units, but also the functional units. Some of the functional importance has been newly revealed by the analysis. Stability of each CRD is examined on the morphing trajectories that cover seven conformational transitions. We confirmed that the large conformational changes are realized by the motions only in the flexible regions that connect CRDs. The Ca²⁺‐ATPase efficiently utilizes its intrinsic flexibility and rigidity to response different switches like ligand binding/dissociation or ATP hydrolysis. The analysis detects functional motions without extensive biological knowledge of experts, suggesting its general applicability to domain movements in other membrane proteins to deepen the understanding of protein structure and function. Proteins 2015; 83:746–756. © 2015 Wiley Periodicals, Inc.     

Role of conformational fluctuations in the enzymatic reaction of HIV-1 protease

The emergence of compensatory drug-resistant mutations in HIV-1 protease challenges the common view of the reaction mechanism of this enzyme. Here, we address this issue by performing classical and ab initio molecular dynamics simulations (MD) on a complex between the enzyme and a peptide substrate. The classical MD calculation reveals large-scale protein motions involving the flaps and the cantilever. These motions modulate the conformational properties of the substrate at the cleavage site. The ab initio calculations show in turn that substrate motion modulates the activation free energy barrier of the enzymatic reaction dramatically. Thus, the catalytic power of the enzyme does not arise from the presence of a pre-organized active site but from the protein mechanical fluctuations. The implications of this finding for the emergence of drug-resistance are discussed.     

The dynamic role of distal side residues in heme hydroperoxidase catalysis. Interplay between X-ray crystallography and ab initio MD simulations

The enzymatic cycle of hydroperoxidases involves the resting Fe(III) state of the enzyme and the high-valent iron intermediates Compound I and Compound II. These states might be characterized by X-ray crystallography and the transition pathways between each state can be investigated using atomistic simulations. Here we review our recent work in the modeling of two key steps of the enzymatic reaction of hydroperoxidases: the formation of Cpd I in peroxidase and the reduction of Cpd I in catalase. It will be shown that small conformational motions of distal side residues (His in peroxidases and His/Asn in catalases), not,or only partially, revealed by the available X-ray structures, play an important role in the catalytic processes examined.     

Conformational State Distributions and Catalytically Relevant Dynamics of a Hinge-Bending Enzyme Studied by Single-Molecule FRET and a Coarse-Grained Simulation

Over the last few decades, a view has emerged showing that multidomain enzymes are biological machines evolved to harness stochastic kicks of solvent particles into highly directional functional motions. These intrinsic motions are structurally encoded, and Nature makes use of them to catalyze chemical reactions by means of ligand-induced conformational changes and states redistribution. Such mechanisms align reactive groups for efficient chemistry and stabilize conformers most proficient for catalysis. By combining single-molecule Förster resonance energy transfer measurements with normal mode analysis and coarse-grained mesoscopic simulations, we obtained results for a hinge-bending enzyme, namely phosphoglycerate kinase (PGK), which support and extend these ideas. From single-molecule Förster resonance energy transfer, we obtained insight into the distribution of conformational states and the dynamical properties of the domains. The simulations allowed for the characterization of interdomain motions of a compact state of PGK. The data show that PGK is intrinsically a highly dynamic system sampling a wealth of conformations on timescales ranging from nanoseconds to milliseconds and above. Functional motions encoded in the fold are performed by the PGK domains already in its ligand-free form, and substrate binding is not required to enable them. Compared to other multidomain proteins, these motions are rather fast and presumably not rate-limiting in the enzymatic reaction. Ligand binding slightly readjusts the orientation of the domains and feasibly locks the protein motions along a preferential direction. In addition, the functionally relevant compact state is stabilized by the substrates, and acts as a prestate to reach active conformations by means of Brownian motions.     

Conformational State Distributions and Catalytically Relevant Dynamics of?a Hinge-Bending Enzyme Studied by Single-Molecule FRET and a Coarse-Grained Simulation

Over the last few decades, a view has emerged showing that multidomain enzymes are biological machines evolved to harness stochastic kicks of solvent particles into highly directional functional motions. These intrinsic motions are structurally encoded, and Nature makes use of them to catalyze chemical reactions by means of ligand-induced conformational changes and states redistribution. Such mechanisms align reactive groups for efficient chemistry and stabilize conformers most proficient for catalysis. By combining single-molecule Förster resonance energy transfer measurements with normal mode analysis and coarse-grained mesoscopic simulations, we obtained results for a hinge-bending enzyme, namely phosphoglycerate kinase (PGK), which support and extend these ideas. From single-molecule Förster resonance energy transfer, we obtained insight into the distribution of conformational states and the dynamical properties of the domains. The simulations allowed for the characterization of interdomain motions of a compact state of PGK. The data show that PGK is intrinsically a highly dynamic system sampling a wealth of conformations on timescales ranging from nanoseconds to milliseconds and above. Functional motions encoded in the fold are performed by the PGK domains already in its ligand-free form, and substrate binding is not required to enable them. Compared to other multidomain proteins, these motions are rather fast and presumably not rate-limiting in the enzymatic reaction. Ligand binding slightly readjusts the orientation of the domains and feasibly locks the protein motions along a preferential direction. In addition, the functionally relevant compact state is stabilized by the substrates, and acts as a prestate to reach active conformations by means of Brownian motions.     

Placing single-molecule T4 lysozyme enzymes on a bacterial cell surface: toward probing single-molecule enzymatic reaction in living cells

The T4 lysozyme enzymatic hydrolyzation reaction of bacterial cell walls is an important biological process, and single-molecule enzymatic reaction dynamics have been studied under physiological condition using purified Escherichia coli cell walls as substrates. Here, we report progress toward characterizing the T4 lysozyme enzymatic reaction on a living bacterial cell wall using a combined single-molecule placement and spectroscopy. Placing a dye-labeled single T4 lysozyme molecule on a targeted bacterial cell wall by using a hydrodynamic microinjection approach, we monitored single-molecule rotational motions during binding, attachment to, and dissociation from the cell wall by tracing single-molecule fluorescence intensity time trajectories and polarization. The single-molecule attachment duration of the T4 lysozyme to the cell wall during enzymatic reactions was typically shorter than the photobleaching time under physiological conditions. Applying single-molecule fluorescence polarization measurements to characterize the binding and motions of the T4 lysozyme molecules, we observed that the motions of wild-type and mutant T4 lysozyme proteins are essentially the same whether under an enzymatic reaction or not. The changing of the fluorescence polarization suggests that the motions of the T4 lysozyme are associated with orientational rotations. This observation also suggests that the T4 lysozyme binding-unbinding motions on cell walls involve a complex mechanism beyond a single-step first-order rate process.     

Dynamic model for enzyme action

Background: Protein thermodynamic structure theory is an integrated approach to the study of protein dynamics and the mechanisms of enzyme catalysis. In this paper, a hypothesis arising from this theory is examined. The timescale of an enzymatic reaction (TER) gives a key to characterizing enzyme conformational changes. The aspects of timescale important in our approach are: (i) it is logically related to internal motions of the main chain of a protein; (ii) it sets the upper limit on the size or scope of protein conformational changes. Feature (i) is linked to the dynamic properties of enzyme-reactant complexes. Feature (ii) is linked to the dynamic sites of the main chain (promoting motion) involved in enzyme activity. Conclusion: Our analysis shows that a comprehensive understanding of enzymology can be established on the basis of protein thermodynamic structure theory.     

Conformational dynamics of a multidomain protein by neutron scattering and computational analysis

The flexible conformations of a multidomain protein are responsible for its biological functions. Although MurD, a 47-kDa protein that consists of three domains, sequentially changes its domain conformation from an open form to a closed form through a semiclosed form in its enzymatic reaction, the domain dynamics in each conformation remains unclear. In this study, we verify the conformational dynamics of MurD in the corresponding three states (apo and ATP- and inhibitor-bound states) with a combination of small-angle x-ray and neutron scattering (SAXS and SANS), dynamic light scattering (DLS), neutron backscattering (NBS), neutron spin echo (NSE) spectroscopy, and molecular dynamics (MD) simulations. Applying principal component analysis of the MD trajectories, twisting and open-closed domain modes are identified as the major collective coordinates. The deviations of the experimental SAXS profiles from the theoretical calculations based on the known crystal structures become smaller in the ATP-bound state than in the apo state, and a further decrease is evident upon inhibitor binding. These results suggest that domain motions of the protein are suppressed step by step of each ligand binding. The DLS and NBS data yield collective and self-translational diffusion constants, respectively, and we used them to extract collective domain motions in nanometer and nanosecond scales from the NSE data. In the apo state, MurD shows both twisting and open-closed domain modes, whereas an ATP binding suppresses twisting domain motions, and a further reduction of open-closed mode is seen in the inhibitor-binding state. These observations are consistent with the structure modifications measured by the small-angle scattering as well as the MD simulations. Such changes in the domain dynamics associated with the sequential enzymatic reactions should be related to the affinity and reaction efficiency with a ligand that binds specifically to each reaction state.     

Substrate-dependent millisecond domain motions in DNA polymerase β

DNA polymerase β (Pol β) is a 39-kDa enzyme that performs the vital cellular function of repairing damaged DNA. Mutations in Pol β have been linked to various cancers, and these mutations are further correlated with altered Pol β enzymatic activity. The fidelity of correct nucleotide incorporation into damaged DNA is essential for Pol β repair function, and several studies have implicated conformational changes in Pol β as a determinant of this repair fidelity. In this work, the rate constants for domain motions in Pol β have been determined by solution NMR relaxation dispersion for the apo and substrate-bound, binary forms of Pol β. In apo Pol β, molecular motions, primarily isolated to the DNA lyase domain, are observed to occur at 1400 s(-1). Additional analysis suggests that these motions allow apo Pol β to sample a conformation similar to the gapped DNA-substrate-bound form. Upon binding DNA, these lyase domain motions are significantly quenched, whereas evidence for conformational motions in the polymerase domain becomes apparent. These NMR studies suggest an alteration in the dynamic landscape of Pol β due to substrate binding. Moreover, a number of the flexible residues identified in this work are also the location of residues, which upon mutation lead to cancer phenotypes in vivo, which may be due to the intimate role of protein motions in Pol β fidelity.     

Kinetics of the lactate dehydrogenase reaction in high-viscosity media

The effect of the medium viscosity on kinetics parameters of lactate dehydrogenase reaction was studied. The viscosity increase results in a sharp decline in the catalytic rate for both the pyruvate reduction and lactate oxidation reactions. It is shown that the catalytic step and its associated conformational motions is the only step which is considerably retarded when the viscosity increases. The reaction is not sensitive to changes in the dielectric properties of the medium. An inverse power function observed between the rate constant and viscosity cannot be explained by the theory of absolute reaction rates. However, it can easily be interpreted on the basis of the Kramers theory dealing with the transition over the activation barrier as a diffusional motion in the field of random forces. The influence of the medium's viscosity on the kinetic parameters indicates the existence of strong coupling between the dynamics of the solvent and the conformational motions of the protein molecule, which are correlated with the catalytic step.     

Dynamics of electron-conformational transitions in proteins and physical mechanisms of biomacromolecule function]

The proteins can be considered as a microheterogeneous structured media possessing memory and feedback properties. The conformational energy surface depends on the chemical states of protein groups. Conformational motions are local diffusion with relaxation times much longer than vibrational relaxation times in condensed media. Owing to the hierarchy of relaxation times chemical reaction rates depend on conformation parametrically. Regulation of functional activity by conformational mobility is accomplished via transmission of information in the form of changes in the distribution functions of separate groups along the conformational substates. The interpretation of drastic effects on conformational mobility needs super-stochastic approaches. A possible mechanism of sharp conformational change are discussed in terms of the catastrophe theory.     

Hydride transfer catalysed by Escherichia coli and Bacillus subtilis dihydrofolate reductase: coupled motions and distal mutations

This paper reviews the results from hybrid quantum/classical molecular dynamics simulations of the hydride transfer reaction catalysed by wild-type (WT) and mutant Escherichia coli and WT Bacillus subtilis dihydrofolate reductase (DHFR). Nuclear quantum effects such as zero point energy and hydrogen tunnelling are significant in these reactions and substantially decrease the free energy barrier. The donor-acceptor distance decreases to ca 2.7 A at transition-state configurations to enable the hydride transfer. A network of coupled motions representing conformational changes along the collective reaction coordinate facilitates the hydride transfer reaction by decreasing the donor-acceptor distance and providing a favourable geometric and electrostatic environment. Recent single-molecule experiments confirm that at least some of these thermally averaged equilibrium conformational changes occur on the millisecond time-scale of the hydride transfer. Distal mutations can lead to non-local structural changes and significantly impact the probability of sampling configurations conducive to the hydride transfer, thereby altering the free-energy barrier and the rate of hydride transfer. E. coli and B. subtilis DHFR enzymes, which have similar tertiary structures and hydride transfer rates with 44% sequence identity, exhibit both similarities and differences in the equilibrium motions and conformational changes correlated to hydride transfer, suggesting a balance of conservation and flexibility across species.     

Discovering conformational sub-states relevant to protein function

Background

Internal motions enable proteins to explore a range of conformations, even in the vicinity of native state. The role of conformational fluctuations in the designated function of a protein is widely debated. Emerging evidence suggests that sub-groups within the range of conformations (or sub-states) contain properties that may be functionally relevant. However, low populations in these sub-states and the transient nature of conformational transitions between these sub-states present significant challenges for their identification and characterization.

Methods and Findings

To overcome these challenges we have developed a new computational technique, quasi-anharmonic analysis (QAA). QAA utilizes higher-order statistics of protein motions to identify sub-states in the conformational landscape. Further, the focus on anharmonicity allows identification of conformational fluctuations that enable transitions between sub-states. QAA applied to equilibrium simulations of human ubiquitin and T4 lysozyme reveals functionally relevant sub-states and protein motions involved in molecular recognition. In combination with a reaction pathway sampling method, QAA characterizes conformational sub-states associated with cis/trans peptidyl-prolyl isomerization catalyzed by the enzyme cyclophilin A. In these three proteins, QAA allows identification of conformational sub-states, with critical structural and dynamical features relevant to protein function.

Conclusions

Overall, QAA provides a novel framework to intuitively understand the biophysical basis of conformational diversity and its relevance to protein function.     

Correlated conformational fluctuations during enzymatic catalysis: Implications for catalytic rate enhancement

Correlated enzymatic conformational fluctuations are shown to contribute to the rate of enhancement achieved during catalysis. Cytidine deaminase serves as a model system. Crystallographic temperature factor data for this enzyme complexed with substrate analog, transition-state analog, and product are available, thereby establishing a measure of atomic scale conformational fluctuations along the (approximate) reaction coordinate. First, a neural network-based algorithm is used to visualize the decreased conformational fluctuations at the transition state. Second, a dynamic diffusion equation along the reaction coordinate is solved and shows that the flux velocity through the associated enzymatic conformation space is greatest at the transition state. These results suggest (1) that there are both dynamic and energetic restrictions to conformational fluctuations at the transition state, (2) that enzymatic catalysis occurs on a fluctuating potential energy surface, and (3) a form for the potential energy. The Michaelis-Menten equations are modified to describe catalysis on this fluctuating potential energy profile, leading to enhanced catalytic rates when fluctuations along the reaction coordinate are appropriately correlated. This represents a dynamic tuning of the enzyme for maximally effective transformation of the ES complex into EP.     

Folding funnels and conformational transitions via hinge-bending motions

In this article we focus on presenting a broad range of examples illustrating low-energy transitions via hinge-bending motions. The examples are divided according to the type of hinge-bending involved; namely, motions involving fragments of the protein chains, hinge-bending motions involving protein domains, and hinge-bending motions between the covalently unconnected subunits. We further make a distinction between allosterically and nonallosterically regulated proteins. These transitions are discussed within the general framework of folding and binding funnels. We propose that the conformers manifesting such swiveling motions are not the outcome of “induced fit” binding mechanism; instead, molecules exist in an ensemble of conformations that are in equilibrium in solution. These ensembles, which populate the bottoms of the funnels,a priori contain both the “open” and the “closed” conformational isomers. Furthermore, we argue that there are no fundamental differences among the physical principles behind the folding and binding funnels. Hence, there is no basic difference between funnels depicting ensembles of conformers of single molecules with fragment, or domain motions, as compared to subunits in multimeric quaternary structures, also showing such conformational transitions. The difference relates only to the size and complexity of the system. The larger the system, the more complex its corresponding fused funnel(s). In particular, funnels associated with allosterically regulated proteins are expected to be more complicated, because allostery is frequently involved with movements between subunits, and consequently is often observed in multichain and multimolecular complexes. This review centers on the critical role played by flexibility and conformational fluctuations in enzyme activity. Internal motions that extend over different time scales and with different amplitudes are known to be essential for the catalytic cycle. The conformational change observed in enzyme-substrate complexes as compared to the unbound enzyme state, and in particular the hinge-bending motions observed in enzymes with two domains, have a substantial effect on the enzymatic catalytic activity. The examples we review span the lipolytic enzymes that are particularly interesting, owing to their activation at the water-oil interface; an allosterically controlled dehydrogenase (lactate dehydrogenase); a DNA methyltransferase, with a covalently-bound intermediate; large-scale flexible loop motions in a glycolytic enzyme (TIM); domain motion in PGK, an enzyme which is essential in most cells, both for ATP generation in aerobes and for fermentation in anaerobes; adenylate kinase, showing large conformational changes, owing to their need to shield their catalytic centers from water; a calcium-binding protein (calmodulin), involved in a wide range of cellular calcium-dependent signaling; diphtheria toxin, whose large domain motion has been shown to yield “domain swapping” the hexameric glutamate dehydrogenase, which has been studied both in a thermophile and in a mesophile; an allosteric enzyme, showing subunit motion between the R and the T states (aspartate transcarbamoylase), and the historically well-studied lac represoor. Nonallosteric subunit transitions are also addressed with some examples (aspartate receptor andBamHI endonuclease). Hence, using this enzyme-catalysis-centered discussion, we address energy funnel landscapes of large-scale conformational transitions, rather than the faster, quasi-harmonic, thermal fluctuations.     

A Physical Picture of Protein Dynamics and Conformational Changes

A physical model is reviewed which explains different aspects of protein dynamics consistently. At low temperatures, the molecules are frozen in conformational substates. Their average energy is 3/2RT. Solid-state vibrations occur on a time scale of femtoseconds to nanoseconds. Above a characteristic temperature, often called the dynamical transition temperature, slow modes of motions can be observed occurring on a time scale between about 140 and 1 ns. These motions are overdamped, quasidiffusive, and involve collective motions of segments of the size of an α-helix. Molecules performing these types of motion are in the “flexible state”. This state is reached by thermal activation. It is shown that these motions are essential for conformational relaxation. Based on this picture, a new approach is proposed to understand conformational changes. It connects structural fluctuations and conformational transitions.     

In silico evidence for DNA polymerase-beta's substrate-induced conformational change

Structural information for mammalian DNA pol-beta combined with molecular and essential dynamics studies have provided atomistically detailed views of functionally important conformational rearrangements that occur during DNA repair and replication. This conformational closing before the chemical reaction is explored in this work as a function of the bound substrate. Anchors for our study are available in crystallographic structures of the DNA pol-beta in "open" (polymerase bound to gapped DNA) and "closed" (polymerase bound to gapped DNA and substrate, dCTP) forms; these different states have long been used to deduce that a large-scale conformational change may help the polymerase choose the correct nucleotide, and hence monitor DNA synthesis fidelity, through an "induced-fit" mechanism. However, the existence of open states with bound substrate and closed states without substrates suggest that substrate-induced conformational closing may be more subtle. Our dynamics simulations of two pol-beta/DNA systems (with/without substrates at the active site) reveal the large-scale closing motions of the thumb and 8-kDa subdomains in the presence of the correct substrate--leading to nearly perfect rearrangement of residues in the active site for the subsequent chemical step of nucleotidyl transfer--in contrast to an opening trend when the substrate is absent, leading to complete disassembly of the active site residues. These studies thus provide in silico evidence for the substrate-induced conformational rearrangements, as widely assumed based on a variety of crystallographic open and closed complexes. Further details gleaned from essential dynamics analyses clarify functionally relevant global motions of the polymerase-beta/DNA complex as required to prepare the system for the chemical reaction of nucleotide extension.     

Classification and annotation of the relationship between protein structural change and ligand binding

The causal relationship between protein structural change and ligand binding was classified and annotated for 839 nonredundant pairs of crystal structures in the Protein Data Bank—one with and the other without a bound low-molecular-weight ligand molecule. Protein structural changes were first classified into either domain or local motions depending on the size of the moving protein segments. Whether the protein motion was coupled with ligand binding was then evaluated based on the location of the ligand binding site and by application of the linear response theory of protein structural change. Protein motions coupled with ligand binding were further classified into either closure or opening motions. This classification revealed the following: (i) domain motions coupled with ligand binding are dominated by closure motions, which can be described by the linear response theory; (ii) local motions frequently accompany order-disorder or α-helix-coil conformational transitions; and (iii) transferase activity (Enzyme Commission   number 2) is the predominant function among coupled domain closure motions. This could be explained by the closure motion acting to insulate the reaction site of these enzymes from environmental water.