NMR Analysis of Apo Glutamine‐Binding Protein Exposes Challenges in the Study of Interdomain Dynamics

Glutamine‐binding protein (GlnBP) displays an apo, “open” and a holo, “closed” crystal form, mutually related by a rigid‐body reorientation of its domains. A fundamental question about such large‐scale conformational transitions, whether the closed state exists in the absence of ligand, is controversial in the case of GlnBP. NMR observations have indicated no evidence of the closed form, whereas experimentally validated computations have suggested a remarkable ca. 40 % population. Herein, a paramagnetic NMR strategy designed to detect the putative apo‐closed species shows that a major population of the latter is highly improbable. Further, NMR residual dipolar couplings collected under three anisotropic conditions do not reveal differential domain alignment and establish that the average solution conformation is satisfied by the apo‐open crystal structure. Our results indicate that the computational prediction of large‐scale interdomain motions is not trivial and may lead to erroneous conclusions without proper experimental validation.     

(Thermo)dynamic Role of Receptor Flexibility,Entropy, and Motional Correlation in Protein–Ligand Binding

The binding of 2‐amino‐5‐methylthiazole to the W191G cavity mutant of cytochrome c peroxidase is an ideal test case to investigate the entropic contribution to the binding free energy due to changes in receptor flexibility. The dynamic and thermodynamic role of receptor flexibility are studied by 50 ns‐long explicit‐solvent molecular dynamics simulations of three separate receptor ensembles: W191G binding a K⁺ ion, W191G–2a5mt complex with a closed 190–195 gating loop, and apo with an open loop. We employ a method recently proposed to estimate accurate absolute single‐molecule configurational entropies and their differences for systems undergoing conformational transitions. We find that receptor flexibility plays a generally underestimated role in protein–ligand binding (thermo)dynamics and that changes of receptor motional correlation determine such large entropy contributions.     

An approximate method in using molecular mechanics simulations to study slow protein conformational changes

The broad range of characteristic motions in proteins has limited the applicability of molecular dynamics simulations in studying large-scale conformational transitions. We present an approximate method, making use of standard MD simulations and using a much larger integration time step, to obtain the structural changes for slow systematic motions of large complex systems. We show the applicability of this method by simulating the open to closed Calmodulin calcium binding domain conformational changes. Starting with the Ca2+-bound X-ray structure, and after the removal of the Ca2+ ions, our calculation yielded intermediate conformations during the rearrangement of helices in each Ca2+ binding pocket, leading to a structure with a lowest rmsd of 1.56 A compared to the NMR apo-calmodulin structure.     

Structural dissimilarity sampling with dynamically self‐guiding selection

Structural dissimilarity sampling (SDS) has been proposed as an enhanced conformational sampling method for reproducing the structural transitions of a given protein. SDS consists of cycles of two steps: (1) Selections of initial structures with structural dissimilarities by referring to a measure. (2) Conformational resampling by restarting short‐time molecular dynamics (MD) simulations from the initial structures. In the present study, an efficient measure is proposed as a dynamically self‐guiding selection to accelerate the structural transitions from a reactant state to a product state as an extension to the original SDS. In the extended SDS, the inner product (IP ) between the reactant and the snapshots generated by short‐time MD simulations are evaluated and ranked according to the IP s at every cycle. Then, the snapshots with low IP s are selected as initial structures for the short‐time MD simulations. This scheme enables one to choose dissimilar and distant initial structures from the reactant, and thus the initial structures dynamically head towards the product, promoting structural transitions from the reactant. To confirm the conformational sampling efficiency, the extended SDS was applied to maltodextrin binding protein (MBP), and we successfully reproduced the structural transition from the open to closed states with submicrosecond‐order simulation times. However, a conventional long‐time MD simulation failed to reproduce the same structural transition. We also compared the performance with that obtained by the ordinary SDS and other sampling techniques that have been developed by us to characterize the possible utility of the extended SDS for actual applications. © 2017 Wiley Periodicals, Inc.     

A Designed Conformational Shift To Control Protein Binding Specificity

In a conformational selection scenario, manipulating the populations of binding‐competent states should be expected to affect protein binding. We demonstrate how in silico designed point mutations within the core of ubiquitin, remote from the binding interface, change the binding specificity by shifting the conformational equilibrium of the ground‐state ensemble between open and closed substates that have a similar population in the wild‐type protein. Binding affinities determined by NMR titration experiments agree with the predictions, thereby showing that, indeed, a shift in the conformational equilibrium enables us to alter ubiquitin’s binding specificity and hence its function. Thus, we present a novel route towards designing specific binding by a conformational shift through exploiting the fact that conformational selection depends on the concentration of binding‐competent substates.     

Insights into the dynamics of HIV-1 protease: a kinetic network model constructed from atomistic simulations

The conformational dynamics in the flaps of HIV-1 protease plays a crucial role in the mechanism of substrate binding. We develop a kinetic network model, constructed from detailed atomistic simulations, to determine the kinetic mechanisms of the conformational transitions in HIV-1 PR. To overcome the time scale limitation of conventional molecular dynamics (MD) simulations, our method combines replica exchange MD with transition path theory (TPT) to study the diversity and temperature dependence of the pathways connecting functionally important states of the protease. At low temperatures the large-scale flap opening is dominated by a small number of paths; at elevated temperatures the transition occurs through many structurally heterogeneous routes. The expanded conformation in the crystal structure 1TW7 is found to closely mimic a key intermediate in the flap-opening pathways at low temperature. We investigated the different transition mechanisms between the semi-open and closed forms. The calculated relaxation times reveal fast semi-open ? closed transitions, and infrequently the flaps fully open. The ligand binding rate predicted from this kinetic model increases by 38-fold from 285 to 309 K, which is in general agreement with experiments. To our knowledge, this is the first application of a network model constructed from atomistic simulations together with TPT to analyze conformational changes between different functional states of a natively folded protein.     

DIRECT‐ID: An automated method to identify and quantify conformational variations—application to β2‐adrenergic GPCR

The conformational dynamics of a macromolecule can be modulated by a number of factors, including changes in environment, ligand binding, and interactions with other macromolecules, among others. We present a method that quantifies the differences in macromolecular conformational dynamics and automatically extracts the structural features responsible for these changes. Given a set of molecular dynamics (MD) simulations of a macromolecule, the norms of the differences in covariance matrices are calculated for each pair of trajectories. A matrix of these norms thus quantifies the differences in conformational dynamics across the set of simulations. For each pair of trajectories, covariance difference matrices are parsed to extract structural elements that undergo changes in conformational properties. As a demonstration of its applicability to biomacromolecular systems, the method, referred to as DIRECT‐ID, was used to identify relevant ligand‐modulated structural variations in the β₂‐adrenergic (β₂AR) G‐protein coupled receptor. Micro‐second MD simulations of the β₂AR in an explicit lipid bilayer were run in the apo state and complexed with the ligands: BI‐167107 (agonist), epinephrine (agonist), salbutamol (long‐acting partial agonist), or carazolol (inverse agonist). Each ligand modulated the conformational dynamics of β₂AR differently and DIRECT‐ID analysis of the inverse‐agonist vs. agonist‐modulated β₂AR identified residues known through previous studies to selectively propagate deactivation/activation information, along with some previously unidentified ligand‐specific microswitches across the GPCR. This study demonstrates the utility of DIRECT‐ID to rapidly extract functionally relevant conformational dynamics information from extended MD simulations of large and complex macromolecular systems. © 2015 Wiley Periodicals, Inc.     

RNA unrestrained molecular dynamics ensemble improves agreement with experimental NMR data compared to single static structure: a test case

Nuclear magnetic resonance (NMR) provides structural and dynamic information reflecting an average, often non-linear, of multiple solution-state conformations. Therefore, a single optimized structure derived from NMR refinement may be misleading if the NMR data actually result from averaging of distinct conformers. It is hypothesized that a conformational ensemble generated by a valid molecular dynamics (MD) simulation should be able to improve agreement with the NMR data set compared with the single optimized starting structure. Using a model system consisting of two sequence-related self-complementary ribonucleotide octamers for which NMR data was available, 0.3 ns particle mesh Ewald MD simulations were performed in the AMBER force field in the presence of explicit water and counterions. Agreement of the averaged properties of the molecular dynamics ensembles with NMR data such as homonuclear proton nuclear Overhauser effect (NOE)-based distance constraints, homonuclear proton and heteronuclear ¹H–³¹P coupling constant (J) data, and qualitative NMR information on hydrogen bond occupancy, was systematically assessed. Despite the short length of the simulation, the ensemble generated from it agreed with the NMR experimental constraints more completely than the single optimized NMR structure. This suggests that short unrestrained MD simulations may be of utility in interpreting NMR results. As expected, a 0.5 ns simulation utilizing a distance dependent dielectric did not improve agreement with the NMR data, consistent with its inferior exploration of conformational space as assessed by 2-D RMSD plots. Thus, ability to rapidly improve agreement with NMR constraints may be a sensitive diagnostic of the MD methods themselves.     

Comprehensive Characterization of the Self-Folding Cavitand Dynamics

The conformational equilibria and guest exchange process of a resorcin[4]arene derived self-folding cavitand receptor have been characterized in detail by molecular dynamics simulations (MD) and ¹H EXSY NMR experiments. A multi-timescale strategy for exploring the fluxional behaviour of this system has been constructed, exploiting conventional MD and accelerated MD (aMD) techniques. The use of aMD allows the reconstruction of the folding/unfolding process of the receptor by sampling high-energy barrier processes unattainable by conventional MD simulations. We obtained MD trajectories sampling events occurring at different timescales from ns to s: 1) rearrangement of the directional hydrogen bond seam stabilizing the receptor, 2) folding/unfolding of the structure transiting partially open intermediates, and 3) guest departure from different folding stages. Most remarkably, reweighing of the biased aMD simulations provided kinetic barriers that are in very good agreement with those determined experimentally by ¹H NMR. These results constitute the first comprehensive characterization of the complex dynamic features of cavitand receptors. Our approach emerges as a valuable rational design tool for synthetic host-guest systems     

Investigations into conformational transitions and solvation structure of a 7-piperidino-5,9-methanobenzo[8] annulene in water

Solvation shell structure of a 7-piperidino-5,9-methanobenzo[8] annulene (PMA) in water has been investigated in ambient conditions using both molecular dynamics (MD) and Car-Parrinello molecular dynamics (CPMD) calculations. From the MD calculations, we find that this molecule exists in three major conformational states out of which two are in twist-boat forms and one in chair form. Due to the limited time scale accessible in CPMD simulations, we have studied all the three conformational states separately using CPMD. The molecular geometry, electronic charge distribution and solvation structure for all three forms are investigated. The stability order of the chair and twist-boat conformations in water solvent has been reversed when compared to the gaseous phase results and in the case of polar aprotic solvents (J. Org. Chem., 1999, 61, 5979). From the radial distribution function, we find that the solvent density around the chair form is significantly lower, which has to be directly related to the smaller solvent accessible area for this conformation and this is in complete agreement with earlier reports. Among the findings are that the solvation shell structure around the nitrogen atom in the chair form of PMA is considerably different from the open conformational forms or the twist-boat forms. The dipole moment for the closed form is found to be significantly larger when compared to the twist-boat forms.     

Inherent flexibility and protein function: The open/closed conformational transition in the N-terminal domain of calmodulin

The key to understand a protein's function often lies in its conformational dynamics. We develop a coarse-grained variational model to investigate the interplay between structural transitions, conformational flexibility, and function of the N-terminal calmodulin domain (nCaM). In this model, two energy basins corresponding to the "closed" apo conformation and "open" holo conformation of nCaM are coupled by a uniform interpolation parameter. The resulting detailed transition route from our model is largely consistent with the recently proposed EFbeta-scaffold mechanism in EF-hand family proteins. We find that the N-terminal parts of the calcium binding loops shows higher flexibility than the C-terminal parts which form this EFbeta-scaffold structure. The structural transition of binding loops I and II are compared in detail. Our model predicts that binding loop II, with higher flexibility and earlier structural change than binding loop I, dominates the open/closed conformational transition in nCaM.     

Conformational Dynamics of Minimal Elastin‐Like Polypeptides: The Role of Proline Revealed by Molecular Dynamics and Nuclear Magnetic Resonance

Previous molecular dynamics studies of the elastin‐like peptide (ELP) GVG(VPGVG) predict that this ELP undergoes a conformational transition from an open to a more compact closed state upon an increase in temperature. These structural changes occurring in this minimal elastin model at the so‐called inverse temperature transition (ITT), which takes place when elastin is heated to temperatures of about 20–40 ^oC, are investigated further in this work by means of a combined theoretical and experimental approach. To do this, additional extensive classical molecular dynamics (MD) simulations of the capped octapeptide are carried out, analyzed, and compared to data obtained from homonuclear magnetic resonance (NMR) spectroscopy of the same octapeptide. Moreover, in the previous simulations, the proline residue in the ELP is found to act as a hinge, thereby allowing for the large‐amplitude opening and closing conformational motion of the ITT. To explore the role of proline in such elastin repeating units, a point mutant (P5I), which replaces the proline residue with an isoleucine residue, is also investigated using the aforementioned theoretical and experimental techniques. The results show that the site‐directed mutation completely alters the properties of this ELP, thus confirming the importance of the highly conserved proline residue in the ITT. Furthermore, a correlation between the two different methods employed is seen. Both methods predict the mutant ELP to be present in an unstructured form and the wild type ELP to have a β‐turn‐like structure. Finally, the role of the peptidyl cis to trans isomerization of the proline hinge is assessed in detail.     

Essential dynamics for the study of microstructures in liquids

Essential Dynamics (ED) is a powerful tool for analyzing molecular dynamics (MD) simulations and it is widely adopted for conformational analysis of large molecular systems such as, for example, proteins and nucleic acids. In this study, we extend the use of ED to the study of clusters of arbitrary size constituted by weakly interacting particles, for example, atomic clusters and supramolecular systems. The key feature of the method we present is the identification of the relevant atomic‐molecular clusters to be analyzed by ED for extracting the information of interest. The application of this computational approach allows a straightforward and unbiased conformational study of the local microstructures in liquids, as emerged from semiclassical MD simulations. The good performance of the method is demonstrated by calculating typical observables of liquid water, that is, NMR, NEXAFS O1s, and IR spectra, known to be rather sensitive both to the presence and to the conformational features of hydrogen‐bonded clusters. © 2014 Wiley Periodicals, Inc.     

Validation of the GROMOS 54A7 Force Field Regarding Mixed α/β‐Peptide Molecules

A molecular‐dynamics (MD) simulation study of two heptapeptides containing α‐ and β‐amino acid residues is presented. According to NMR experiments, the two peptides differ in dominant fold when solvated in MeOH: peptide 3 adopts predominantly β‐hairpin‐like conformations, while peptide 8 adopts a 14/15‐helical fold. The MD simulations largely reproduce the experimental data. Application of NOE atom? atom distance restraining improves the agreement with experimental data, but reduces the conformational sampling. Peptide 3 shows a variety of conformations, while still agreeing with the NOE and ³J‐coupling data, whereas the conformational ensemble of peptide 8 is dominated by one helical conformation. The results confirm the suitability of the GROMOS 54A7 force field for simulation or structure refinement of mixed α/β‐peptides in MeOH.     

Microsecond Timescale Dynamics of GDP‐Bound Ras Underlies the Formation of Novel Inhibitor‐Binding Pockets

The recent discovery of inhibitory compounds binding to distinct pockets on GDP‐bound Ras has renewed the view on the druggability of this crucial cancer driver. However, the origin of these pockets, which are not readily formed in the crystal structure in the absence of the compounds, is yet unclear. Herein, we explored the intrinsic flexibility of Ras?GDP on microsecond to millisecond timescales using relaxation‐based NMR experiments, and identified substantial slow dynamics with τ_ex of 34 μs at 5 °C, which maps to the regions showing a high level of correlation with those displaying conformational differences between the inhibitor‐bound and free states. These findings, which have been demonstrated in both wild‐type Ras and the oncogenic mutant (G12V), support the mechanism of extended conformational selection for Ras–inhibitor interactions where the small molecules redistribute the protein conformational ensemble favoring the final bound states.     

Temperature‐shuffled parallel cascade selection molecular dynamics accelerates the structural transitions of proteins

Parallel cascade selection molecular dynamics (PaCS‐MD) is an enhanced conformational sampling method for searching structural transition pathways from a given reactant to a product. Recently, a temperature‐aided PaCS‐MD (Vinod et al., Eur. Biophys. J. 2016, 45, 463) has been proposed as its extension, in which the temperatures were introduced as additional parameters in conformational resampling, whereas the temperature is fixed in the original PaCS‐MD. In the present study, temperature‐shuffled PaCS‐MD is proposed as a further extension of temperature‐aided PaCS‐MD in which the temperatures are shuffled among different replicas at the beginning of each cycle of conformational resampling. To evaluate their conformational sampling efficiencies, the original, temperature‐aided, and temperature‐shuffled PaCS‐MD were applied to a protein‐folding process of Trp‐cage, and their minimum computational costs to identify the native state were addressed. Through the evaluation, it was confirmed that temperature‐shuffled PaCS‐MD remarkably accelerated the protein‐folding process of Trp‐cage compared with the other methods. © 2017 Wiley Periodicals, Inc.     

Hamiltonian and distance replica exchange method studies of Met-enkephalin

The conformational states of the zwitterionic form of the pentapeptide Met-enkephalin were explored with the use of explicit solvent molecular dynamics (MD). The N and C termini are ionized, as appropriate to polar solvent conditions, and consequently, there is a competition between open forms driven by polar solvation of the ammonium and carboxylate groups and closed forms driven by their salt-bridge formation. Normal MD started from an open state does not sample closed conformations. Sampling was enhanced with a distance replica exchange method (DREM) and with a Hamiltonian replica exchange method (HREM). The potential of mean force (PMF) along an end-to-end distance reaction coordinate was obtained with the DREM. The PMF shows a stable salt-bridge state and the presence of a large region of open states, as hypothesized for conformationally promiscuous small opiate peptides. The HREM systems differ by scaling the peptide-peptide and peptide-solvent electrostatic and Lennard-Jones potentials, with the goal of improving the sampling efficiency with a limited number of systems. A small number of systems were found to be sufficient to sample closed and open states. A principal component analysis (PCA) shows that the HREM-generated fluctuations are dominated by the first two principal modes. The first corresponds to the end-to-end reaction coordinate found in the DREM, and the first mode PMF is similar to the DREM PMF. The second mode describes the presence of two conformations, both of which correspond to the salt-bridge state distance. The conformers differ in the values of neighboring psi and phi dihedral angles, since such psi/phi compensation can still produce the same end-to-end distance. The two-dimensional PMF constructed from the first two PCA modes captures most of the significant backbone conformational space of Met-enkephalin.     

Enhanced conformational sampling method for proteins based on the TaBoo SeArch algorithm: Application to the folding of a mini‐protein,chignolin

The conformational samplings are indispensible for obtaining reliable canonical ensembles, which provide statistical averages of physical quantities such as free energies. However, the samplings of vast conformational space of biomacromolecules by conventional molecular dynamics (MD) simulations might be insufficient, due to their inadequate accessible time‐scales for investigating biological functions. Therefore, the development of methodologies for enhancing the conformational sampling of biomacromolecules still remains as a challenging issue in computational biology. To tackle this problem, we newly propose an efficient conformational search method, which is referred as TaBoo SeArch (TBSA) algorithm. In TBSA, an inverse energy histogram is used to select seeds for the conformational resampling so that states with high frequencies are inhibited, while states with low frequencies are efficiently sampled to explore the unvisited conformational space. As a demonstration, TBSA was applied to the folding of a mini‐protein, chignolin, and automatically sampled the native structure (C_α root mean square deviation < 1.0 Å) with nanosecond order computational costs started from a completely extended structure, although a long‐time 1‐µs normal MD simulation failed to sample the native structure. Furthermore, a multiscale free energy landscape method based on the conformational sampling of TBSA were quantitatively evaluated through free energy calculations with both implicit and explicit solvent models, which enable us to find several metastable states on the folding landscape. © 2015 Wiley Periodicals, Inc.     

Conformational Flexibility and Dynamics of Two (1→6)‐Linked Disaccharides Related to an Oligosaccharide Epitope Expressed on Malignant Tumour Cells

The conformational flexibility and dynamics of two (1→6)‐linked disaccharides that are related to the action of the glycosyl transferase GnT‐V have been investigated. NMR NOE and T‐ROE spectroscopy experiments, conformation‐dependent coupling constants and molecular dynamics (MD) simulations were used in the analyses. To facilitate these studies, the compounds were synthesised as α‐d‐ [6‐¹³C]‐Manp‐OMe derivatives, which reduced the ¹H NMR spectral overlap and facilitated the determination of two‐ and three‐bond ¹H,¹H, ¹H,¹³C and ¹³C,¹³C‐coupling constants. The population distribution for the glycosidic ω torsion angle in α‐d‐ Manp‐(1→6)‐α‐d‐ Manp‐OMe for gt/gg/tg was equal to 45:50:5, whereas in α‐d‐ Manp‐OMe it was determined to be 56:36:8. The dynamic model that was generated for β‐d‐ GlcpNAc‐(1→6)‐α‐d‐ Manp‐OMe by MD simulations employing the PARM22/SU01 CHARMM‐based force field was in very good agreement with experimental observations. β‐d‐ GlcpNAc‐(1→6)‐α‐d‐ Manp‐OMe is described by an equilibrium of populated states in which the ? torsion angle has the exo‐anomeric conformation, the ψ torsion angle an extended antiperiplanar conformation and the ω torsion angle a distribution of populations predominantly between the gauche–trans and the gauche–gauche conformational states (i.e., gt/gg/tg) is equal to 60:35:5, respectively. The use of site‐specific ¹³C labelling in these disaccharides leads to increased spectral dispersion, thereby making NMR spectroscopy based conformational analysis possible that otherwise might be difficult to attain.