Conformational and dynamics changes induced by bile acids binding to chicken liver bile acid binding protein

The correlation between protein motions and function is a central problem in protein science. Several studies have demonstrated that ligand binding and protein dynamics are strongly correlated in intracellular lipid binding proteins (iLBPs), in which the high degree of flexibility, principally occurring at the level of helix-II, CD, and EF loops (the so-called portal area), is significantly reduced upon ligand binding. We have recently investigated by NMR the dynamic properties of a member of the iLBP family, chicken liver bile acid binding protein (cL-BABP), in its apo and holo form, as a complex with two bile salts molecules. Binding was found to be regulated by a dynamic process and a conformational rearrangement was associated with this event. We report here the results of molecular dynamics (MD) simulations performed on apo and holo cL-BABP with the aim of further characterizing the protein regions involved in motion propagation and of evaluating the main molecular interactions stabilizing bound ligands. Upon binding, the root mean square fluctuation values substantially decrease for CD and EF loops while increase for the helix-loop-helix region, thus indicating that the portal area is the region mostly affected by complex formation. These results nicely correlate with backbone dynamics data derived from NMR experiments. Essential dynamics analysis of the MD trajectories indicates that the major concerted motions involve the three contiguous structural elements of the portal area, which however are dynamically coupled in different ways whether in the presence or in the absence of the ligands. Motions of the EF loop and of the helical region are part of the essential space of both apo and holo-BABP and sample a much wider conformational space in the apo form. Together with NMR results, these data support the view that, in the apo protein, the flexible EF loop visits many conformational states including those typical of the holo state and that the ligand acts stabilizing one of these pre-existing conformations. The present results, in agreement with data reported for other iLBPs, sharpen our knowledge on the binding mechanism for this protein family.     

Molecular insights into substrate binding mechanism of undecaprenyl pyrophosphate with membrane integrated phosphatidyl glycerophosphate phosphatase B (PgpB) using molecular dynamics simulation approach

Undecaprenyl phosphate (C55-P) acts as carrier lipid in the synthesis of peptidoglycan, which is de novo synthesized from dephosphorylation of undecaprenyl pyrophosphate (C55-PP). The phosphatidylglycerol phosphate phosphatase B (PgpB) catalyzes the dephosphorylation of C55-PP and forms C55-P. As no structural study has been made regarding the binding of C55-PP to PgpB, in the current study, in silico molecular docking, followed by 150 ns molecular dynamics simulation of the putative binding complex in membrane/solvent environment has been performed to understand conformational dynamics. Results are compared with simulated apo form and PE inhibitor-bound form. Analysis of correlated residual fluctuation network in apo form, C55-PP bound and PE inhibitor-bound form suggests that difference in dynamic coupling between TM domain and α2 and α3 helix of periplasmic domain provides ligand binding to facilitate catalysis or to show inhibitory activity. Distance distribution in catalytic residual pair, H207-R104; H207-R201 and H207-D211 which stabilizes phosphate-enzyme intermediate shows a narrow peak in 2.4–3.6 Å in substrate-bound compared to apo form. Binding interactions and binding free energy analyses complement the partial inhibition of PE where PE has less binding free energy compared to the C55-PP substrate as well as the difference in binding interaction with catalytic pocket. Thus, the present study provides how substrate binding couples the movement in TM domain and periplasmic domain which might help in the understanding of active site communication in PgpB. C55-PP phosphatase interactions with a catalytic pocket of PgpB provide new insight for designing drugs against bacterial infection.     

Coenzyme A binding to the aminoglycoside acetyltransferase (3)-IIIb increases conformational sampling of antibiotic binding site

NMR spectroscopy experiments and molecular dynamics simulations were performed to describe the dynamic properties of the aminoglycoside acetyltransferase (3)-IIIb (AAC) in its apo and coenzyme A (CoASH) bound forms. The (15)N-(1)H HSQC spectra indicate a partial structural change and coupling of the CoASH binding site with another region in the protein upon the CoASH titration into the apo enzyme. Molecular dynamics simulations indicate a significant structural and dynamic variation of the long loop in the antibiotic binding domain in the form of a relatively slow (250 ns), concerted opening motion in the CoASH-enzyme complex and that binding of the CoASH increases the structural flexibility of the loop, leading to an interchange between several similar equally populated conformations.     

Comparison of SARS and NL63 papain-like protease binding sites and binding site dynamics: inhibitor design implications

The human severe acute respiratory syndrome coronavirus (SARS-CoV) and the NL63 coronaviruses are human respiratory pathogens for which no effective antiviral treatment exists. The papain-like cysteine proteases encoded by the coronavirus (SARS-CoV: PLpro; NL63: PLP1 and PLP2) represent potential targets for antiviral drug development. Three recent inhibitor-bound PLpro structures highlight the role of an extremely flexible six-residue loop in inhibitor binding. The high binding site plasticity is a major challenge in computational drug discovery/design efforts. From conventional molecular dynamics and accelerated molecular dynamics (aMD) simulations, we find that with conventional molecular dynamics simulation, PLpro translationally samples the open and closed conformation of BL2 loop on a picosecond-nanosecond timescale but does not reproduce the peptide bond inversion between loop residues Tyr269 and Gln270 that is observed on inhibitor GRL0617 binding. Only aMD simulation, starting from the closed loop conformation, reproduced the 180° ?-ψ dihedral rotation back to the open loop state. The Tyr-Gln peptide bond inversion appears to involve a progressive conformational change of the full loop, starting at one side, and progressing to the other. We used the SARS-CoV apo X-ray structure to develop a model of the NL63-PLP2 catalytic site. Superimposition of the PLP2 model on the PLpro X-ray structure identifies binding site residues in PLP2 that contribute to the distinct substrate cleavage site specificities between the two proteases. The topological and electrostatic differences between the two protease binding sites also help explain the selectivity of non-covalent PLpro inhibitors.     

Conformational transitions in protein-protein association: binding of fasciculin-2 to acetylcholinesterase

The neurotoxin fasciculin-2 (FAS2) is a picomolar inhibitor of synaptic acetylcholinesterase (AChE). The dynamics of binding between FAS2 and AChE is influenced by conformational fluctuations both before and after protein encounter. Submicrosecond molecular dynamics trajectories of apo forms of fasciculin, corresponding to different conformational substates, are reported here with reference to the conformational changes of loop I of this three-fingered toxin. This highly flexible loop exhibits an ensemble of conformations within each substate corresponding to its functions. The high energy barrier found between the two major substates leads to transitions that are slow on the timescale of the diffusional encounter of noninteracting FAS2 and AChE. The more stable of the two apo substates may not be the one observed in the complex with AChE. It seems likely that the more stable apo form binds rapidly to AChE and conformational readjustments then occur in the resulting encounter complex.     

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.     

Molecular dynamics studies of U1A-RNA complexes

The U1A protein binds to a hairpin RNA and an internal-loop RNA with picomolar affinities. To probe the molecular basis of U1A binding, we performed state-of-the-art nanosecond molecular dynamics simulations on both complexes. The good agreement with experimental structures supports the protocols used in the simulations. We compare the dynamics, hydrogen-bonding occupancies, and interfacial flexibility of both complexes and also describe a rigid-body motion in the U1A-internal loop complex that is not observed in the U1A-hairpin simulation. We relate these observations to experimental mutational studies and highlight their significance in U1A binding affinity and specificity.     

Lipid‐associated aggregate formation of superoxide dismutase‐1 is initiated by membrane‐targeting loops

Copper‐Zinc superoxide dismutase 1 (SOD1) is a homodimeric enzyme that protects cells from oxidative damage. Hereditary and sporadic amyotrophic lateral sclerosis may be linked to SOD1 when the enzyme is destabilized through mutation or environmental stress. The cytotoxicity of demetallated or apo‐SOD1 aggregates may be due to their ability to cause defects within cell membranes by co‐aggregating with phospholipids. SOD1 monomers may associate with the inner cell membrane to receive copper ions from membrane‐bound copper chaperones. But how apo‐SOD1 interacts with lipids is unclear. We have used atomistic molecular dynamics simulations to reveal that flexible electrostatic and zinc‐binding loops in apo‐SOD1 dimers play a critical role in the binding of 1‐octanol clusters and phospholipid bilayer, without any significant unfolding of the protein. The apo‐SOD1 monomer also associates with phospholipid bilayer via its zinc‐binding loop rather than its exposed hydrophobic dimerization interface. Our observed orientation of the monomer on the bilayer would facilitate its association with a membrane‐bound copper chaperone. The orientation also suggests how membrane‐bound monomers could act as seeds for membrane‐associated SOD1 aggregation. Proteins 2014; 82:3194–3209. © 2014 Wiley Periodicals, Inc.     

Deciphering the binding mode of Zolpidem to GABA<Subscript>A</Subscript> α<Subscript>1</Subscript> receptor – insights from molecular dynamics simulation

To investigate the binding mode of Zolpidem to GABA(A) and to delineate the conformational changes induced upon agonist binding, we carried out atomistic molecular dynamics simulation using the ligand binding domain of GABA(A) α(1) receptor. Comparative molecular dynamics simulation of the apo and the holo form of GABA(A) receptor revealed that γ(2)/α(1) interface housing the benzodiazepine binding site undergoes distinct conformational changes upon Zolpidem binding. We notice that C loop of the α(1) subunit experiences an inward motion toward the vestibule and the F loop of γ(2) sways away from the vestibule, an observation that rationalizes Zolpidem as an alpha1 selective agonist. Energy decomposition analysis carried out was able to highlight the important residues implicated in Zolpidem binding, which were largely in congruence with the experimental data. The simulation study disclosed herein provides a meaningful insight into Zolpidem-GABA(A)R interactions and helps to arrive at a binding mode hypothesis with implications for drug design.     

Dynamics of the WPD loop of the Yersinia protein tyrosine phosphatase

The bacterial protein tyrosine phosphatase YopH is an essential virulence determinant in Yersinia spp., causing gastrointestinal diseases and the plague. Like eukaryotic PTPases, YopH catalyzes the hydrolysis of the phosphate moiety of phosphotyrosine within a highly conserved binding pocket, which is also characterized by the closure of the so-called "WPD loop" upon ligand binding. In this study, we investigate the conformational changes and dynamics of the WPD loop by molecular dynamics simulations. Consistent with experimental observations, our simulations show that the WPD loop of YopH is intrinsically flexible and fluctuates between the open and closed conformation with a frequency of approximately 4 ns for the apo, native protein. The region of helix alpha4 spanning loop 384-392, which has been revealed experimentally as a second substrate-binding site in YopH, is found to be highly associated with the WPD loop, stabilizing it in the closed, active conformation, and providing a structural basis for the cooperation of the second-substrate binding site in substrate recognition. Loop L4 (residues 323-327) is shown to be involved in a parallel, correlated motion mode with the WPD loop that contributes the stabilization of a more extended open conformation. In addition, we have simulated the loop reopening in the ligand-bound protein complex by applying the locally enhanced sampling method. Finally, the dynamic behavior of the WPD loop for the C403S mutant differs from the wild-type YopH remarkably. These results shed light on the role of the WPD loop in PTPase-mediated catalysis, and are useful in structure-based design for novel, selective YopH inhibitors as antibacterial drugs.     

Conformational selection in silico: loop latching motions and ligand binding in enzymes

Ligand binding frequently induces significant conformational changes in a protein receptor. Understanding and predicting such conformational changes represent an important challenge for computational biology, including applications to structure-based drug design. We describe an approach to this problem based on the assumption that the holo state is at least transiently populated in the absence of a ligand; this hypothesis has been referred to as "conformational selection." Here, we apply a method that tests this hypothesis on a challenging class of ligand-induced conformational changes, which we refer to as loop latching: the closing of a loop around an active site that sequesters the ligand from solvent. The method uses a combination of replica exchange molecular dynamics and a loop prediction algorithm to generate low-energy loop structures, and docking to select the conformation appropriate for binding a particular ligand. On a test set of six proteins, it yields loop structures including hololike conformations, generally below 2 A RMSD from the liganded structure, for loops that span up to 15 residues. Docking serves as a stringent test of the predictions. In five of the six cases, the predicted loop conformations improve the ranks of cognate ligands relative to using the apo structure, although the results remain, in most cases, significantly worse than using a holo structure. The poses of the cognate ligands are correct in four of the six test cases, while they are correct for five of the six using a holo structure.     

Dynamic behavior of the post‐SET loop region of NSD1: Implications for histone binding and drug development

NSD1 is a SET‐domain histone methyltransferase that methylates lysine 36 of histone 3. In the crystal structure of NSD1, the post‐SET loop is in an autoinhibitory position that blocks binding of the histone peptide as well as the entrance to the lysine‐binding channel. The conformational dynamics preceding histone binding and the mechanism by which the post‐SET loop moves to accommodate the target lysine is currently unknown, although potential models have been proposed. Using molecular dynamics simulations, we have identified potential conformations of the post‐SET loop differing from those of previous studies, as well as proposed a model of peptide‐bound NSD1. Our simulations illustrate the dynamic behavior of the post‐SET loop and the presence of a few distinct conformations. In every case, the post‐SET loop remains in an autoinhibitory position blocking the peptide‐binding cleft, suggesting that another interaction is required to optimally position NSD1 in an active conformation. This finding provides initial evidence for a mechanism by which NSD1 preferentially binds nucleosomal substrates.     

Multiscale computational study of ligand binding pathways: Case of p38 MAP kinase and its inhibitors

Protein kinases are one of the most important drug targets in the past 10 years. Understanding the inhibitor association processes will profoundly impact new binder designs with preferred binding kinetics. However, after more than a decade of effort, a complete atomistic-level study of kinase inhibitor binding pathways is still lacking. As all kinases share a similar scaffold, we used p38 kinase as a model system to investigate the conformational dynamics and free energy transition of inhibitor binding toward kinases. Two major kinase conformations, Asp-Phe-Gly (DFG)-in and DFG-out, and three types of inhibitors, type I, II, and III, were thoroughly investigated in this work. We performed Brownian dynamics simulations and up to 340 μs Gaussian-accelerated molecular dynamics simulations to capture the inhibitor binding paths and a series of conformational transitions of the p38 kinase from its apo to inhibitor-bound form. Eighteen successful binding trajectories, including all types of inhibitors, are reported herein. Our simulations suggest a mechanism of inhibitor recruitment, a faster ligand association step to a pre-existing DFG-in/DFG-out p38 protein, followed by a slower molecular rearrangement step to adjust the protein-ligand conformation followed by a shift in the energy landscape to reach the final bound state. The ligand association processes also reflect the energetic favor of type I and type II/III inhibitor binding through ATP and allosteric channels, respectively. These different binding routes are directly responsible for the fast (type I binders) and slow (type II/III binders) kinetics of different types of p38 inhibitors. Our findings also echo the recent study of p38 inhibitor dissociation, implying that ligand unbinding could undergo a reverse path of binding, and both processes share similar metastates. This study deepens the understanding of molecular and energetic features of kinase inhibitor-binding processes and will inspire future drug development from a kinetic point of view.     

Insights from free-energy calculations: protein conformational equilibrium, driving forces, and ligand-binding modes

Accurate free-energy calculations provide mechanistic insights into molecular recognition and conformational equilibrium. In this work, we performed free-energy calculations to study the thermodynamic properties of different states of molecular systems in their equilibrium basin, and obtained accurate absolute binding free-energy calculations for protein-ligand binding using a newly developed M2 algorithm. We used a range of Asp-Phe-Gly (DFG)-in/out p38α mitogen-activated protein kinase inhibitors as our test cases. We also focused on the flexible DFG motif, which is closely connected to kinase activation and inhibitor binding. Our calculations explain the coexistence of DFG-in and DFG-out states of the loop and reveal different components (e.g., configurational entropy and enthalpy) that stabilize the apo p38α conformations. To study novel ligand-binding modes and the key driving forces behind them, we computed the absolute binding free energies of 30 p38α inhibitors, including analogs with unavailable experimental structures. The calculations revealed multiple stable, complex conformations and changes in p38α and inhibitor conformations, as well as balance in several energetic terms and configurational entropy loss. The results provide relevant physics that can aid in designing inhibitors and understanding protein conformational equilibrium. Our approach is fast for use with proteins that contain flexible regions for structure-based drug design.     

Binding free energy calculation with QM/MM hybrid methods for Abl-Kinase inhibitor

We report a Quantum mechanics/Molecular Mechanics–Poisson-Boltzmann/ Surface Area (QM/MM-PB/SA) method to calculate the binding free energy of c-Abl human tyrosine kinase by combining the QM and MM principles where the ligand is treated quantum mechanically and the rest of the receptor by classical molecular mechanics. To study the role of entropy and the flexibility of the protein ligand complex in a solvated environment, molecular dynamics calculations are performed using a hybrid QM/MM approach. This work shows that the results of the QM/MM approach are strongly correlated with the binding affinity. The QM/MM interaction energy in our reported study confirms the importance of electronic and polarization contributions, which are often neglected in classical MM-PB/SA calculations. Moreover, a comparison of semi-empirical methods like DFTB-SCC, PM3, MNDO, MNDO-PDDG, and PDDG-PM3 is also performed. The results of the study show that the implementation of a DFTB-SCC semi-empirical Hamiltonian that is derived from DFT gives better results than other methods. We have performed such studies using the AMBER molecular dynamic package for the first time. The calculated binding free energy is also in agreement with the experimentally determined binding affinity for c-Abl tyrosine kinase complex with Imatinib.     

Structural insights into the cellular retinaldehyde-binding protein (CRALBP)

Cellular retinaldehyde-binding protein (CRALBP) is an essential protein in the human visual cycle without a known three-dimensional structure. Previous studies associate retinal pathologies to specific mutations in the CRALBP protein. Here we use homology modeling and molecular dynamics methods to investigate the structural mechanisms by which CRALBP functions in the visual cycle. We have constructed two conformations of CRALBP representing two states in the process of ligand association and dissociation. Notably, our homology models map the pathology-associated mutations either directly in or adjacent to the putative ligand-binding cavity. Furthermore, six novel residues have been identified to be crucial for the hinge movement of the lipid-exchange loop in CRALBP. We conclude that the binding and release of retinoid involve large conformational changes in the lipid-exchange loop at the entrance of the ligand-binding cavity.     

A Model of Lipid-Free Apolipoprotein A-I Revealed by Iterative Molecular Dynamics Simulation

Apolipoprotein A-I (apo A-I), the major protein component of high-density lipoprotein, has been proven inversely correlated to cardiovascular risk in past decades. The lipid-free state of apo A-I is the initial stage which binds to lipids forming high-density lipoprotein. Molecular models of lipid-free apo A-I have been reported by methods like X-ray crystallography and chemical cross-linking/mass spectrometry (CCL/MS). Through structural analysis we found that those current models had limited consistency with other experimental results, such as those from hydrogen exchange with mass spectrometry. Through molecular dynamics simulations, we also found those models could not reach a stable equilibrium state. Therefore, by integrating various experimental results, we proposed a new structural model for lipid-free apo A-I, which contains a bundled four-helix N-terminal domain (1–192) that forms a variable hydrophobic groove and a mobile short hairpin C-terminal domain (193–243). This model exhibits an equilibrium state through molecular dynamics simulation and is consistent with most of the experimental results known from CCL/MS on lysine pairs, fluorescence resonance energy transfer and hydrogen exchange. This solution-state lipid-free apo A-I model may elucidate the possible conformational transitions of apo A-I binding with lipids in high-density lipoprotein formation.     

Crystal structures of the catalytic domain of human protein kinase associated with apoptosis and tumor suppression

We have determined X-ray crystal structures with up to 1.5 A resolution of the catalytic domain of death-associated protein kinase (DAPK), the first described member of a novel family of pro-apoptotic and tumor-suppressive serine/threonine kinases. The geometry of the active site was studied in the apo form, in a complex with nonhydrolyzable AMPPnP and in a ternary complex consisting of kinase, AMPPnP and either Mg2+ or Mn2+. The structures revealed a previously undescribed water-mediated stabilization of the interaction between the lysine that is conserved in protein kinases and the beta- and gamma-phosphates of ATP, as well as conformational changes at the active site upon ion binding. Comparison between these structures and nucleotide triphosphate complexes of several other kinases disclosed a number of unique features of the DAPK catalytic domain, among which is a highly ordered basic loop in the N-terminal domain that may participate in enzyme regulation.     

Structural and dynamic roles of permanent water molecules in ligand molecular recognition by chicken liver bile acid binding protein

Chicken liver bile acid binding protein (cL-BABP) crystallizes with water molecules in its binding site. To obtain insights on the role of internal water, we performed two 100 ns molecular dynamics (MD) simulations in explicit solvent for cL-BABP, as apo form and as a complex with two molecules of cholic acid, and analyzed in detail the dynamics properties of all water molecules. The diffusion coefficients of the more persistent internal water molecules are significantly different from the bulk, but similar between the two protein forms. A different number of molecules and a different organization are observed for apo- and holo-cL-BABP. Most water molecules identified in the binding site of the apo-crystal diffuse to the bulk during the simulation. In contrast, almost all the internal waters of the holo-crystal maintain the same interactions with internal sidechains and ligands, which suggests they have a relevant role in protein-ligand molecular recognition. Only in the presence of these water molecules we were able to reproduce, by a classical molecular docking approach, the structure of the complex cL-BABP::cholic acid with a low ligand root mean square deviation (RMSD) with respect to its reference positioning. Literature data reported a conserved pattern of hydrogen bonds between a single water molecule and three amino acid residues of the binding site in a series of crystallized FABP. In cL-BABP, the interactions between this conserved water molecule and the three residues are present in the crystal of both apo- and holo-cL-BABP but are lost immediately after the start of molecular dynamics. Copyright (c) 2008 John Wiley & Sons, Ltd.