Developing polarized protein-specific charges for protein dynamics: MD free energy calculation of pKa shifts for Asp26/Asp20 in thioredoxin

Ab initio quantum mechanical calculation of protein in solution is carried out to generate polarized protein-specific charge(s) (PPC) for molecular dynamics (MD) stimulation of protein. The quantum calculation of protein is made possible by developing a fragment-based quantum chemistry approach in combination with the implicit continuum solvent model. The computed electron density of protein is utilized to derive PPCs that represent the polarized electrostatic state of protein near the native structure. These PPCs are atom-centered like those in the standard force fields and are thus computationally attractive for molecular dynamics simulation of protein. Extensive MD simulations have been carried out to investigate the effect of electronic polarization on the structure and dynamics of thioredoxin. Our study shows that the dynamics of thioredoxin is stabilized by electronic polarization through explicit comparison between MD results using PPC and AMBER charges. In particular, MD free-energy calculation using PPCs accurately reproduced the experimental value of pK_a shift for ionizable residue Asp²⁶ buried inside thioredoxin, whereas previous calculations using standard force fields overestimated pK_a shift by twice as much. Accurate prediction of pK_a shifts by rigorous MD free energy simulation for ionizable residues buried inside protein has been a significant challenge in computational biology for decades. This study presented strong evidence that electronic polarization of protein plays an important role in protein dynamics.     

On the Permeation by Dioxygen of Urate Oxidase from Aspergillus flavus in Complex with Xanthine Anion: Dioxygen Pathways and a Portrait of the Enzyme Cavities from Molecular Dynamics Simulations in Water Solution

This work describes molecular dynamics (MD) simulations in aqueous media for the complex of the homotetrameric urate oxidase (UOX) from Aspergillus flavus with xanthine anion ( 5 ) in the presence of dioxygen (O₂). After 196.6 ns of trajectory from unrestrained MD, a O₂ molecule was observed leaving the bulk solvent to penetrate the enzyme between two subunits, A/C. From here, the same O₂ molecule was observed migrating, across subunit C, to the hydrophobic cavity that shares residue V227 with the active site. The latter was finally attained, after 378.3 ns of trajectory, with O₂ at a bonding distance from 5 . The reverse same O₂ pathway, from 5 to the bulk solvent, was observed as preferred pathway under random acceleration MD (RAMD), where an external, randomly oriented force was acting on O₂. Both MD and RAMD simulations revealed several cavities populated by O₂ during its migration from the bulk solvent to the active site or backwards. Paying attention to the last hydrophobic cavity that apparently serves as O₂ reservoir for the active site, it was noticed that its volume undergoes ample fluctuations during the MD simulation, as expected from the thermal motion of a flexible protein, independently from the particular subunit and no matter whether the cavity is filled or not by O₂.     

On Dioxygen Permeation of MaL Laccase from the Thermophilic Fungus Melanocarpus albomyces: An all‐Atom Molecular Dynamics Investigation

In this article, biased molecular dynamics (MD) simulations of O₂ egress from the active center of MaL laccase toward the bulk solvent were described. Parameterization of the set of four Cu(II) ions, in the framework of CHARMM‐36 FF, was carried out on a recent dummy‐atom model that takes into account the Jahn–Teller effect. By carrying out a number of statistically relevant MD simulations, under a tiny randomly oriented external force applied to the molecule O₂, three preferred gates for O₂ egress from the enzyme and a few intermediate binding pockets (BPs) for O₂ were visible; all the gates and pockets were located on two of the three domains. This wide distribution of preferred gates notwithstanding the molecule O₂ was seen to follow specific pathways, exploiting consistently the interstices created by the enzyme thermal fluctuations. These are features that can be imagined to have evolved to make MaL laccase extremely efficient as catalysts in various reactions that require O₂.     

Consequences of Sequential Ca2+ Occupancy for the Structure and Dynamics of CalbindinD9K: Computational Simulations and Comparison to Experimental Determinations in Solution

Abstract

Molecular dynamics (MD) simulations of the structures of calbindin_D9K (CAB) with different occupancies of the two Ca²⁺ binding sites were carried out to gain insight into structural and energetic consequences of sequential Ca²⁺ binding. The aim of the study is to identify effects of Ca-binding site occupancy that relate to the properties and functions of Ca-binding proteins containing EF-hand motifs. Two different models of solvation were employed, one defined by a linear, distance dependent dielectric permittivity (ε = r) and inclusion only of the 36 crystallographically observed water molecules, and the other with the protein immersed in a 9Å shell of explicit waters and ε = 1. Experimental results from x-ray crystallography, and insights from NMR and from measurements of hydrogen exchange rates in these systems served as tests and guides for assessing the quality, validity and mechanistic interpretation of the results from the computational study. The results of the MD simulations agree very well with earlier experimental observations that the structure of calbindin_D9k is rather insensitive to removal of Ca²⁺, and indicate that this insensitivity is not dependent on the order in which the ions are removed. The calculated values of the electrostatic potentials at the Ca²⁺ binding sites are very similar, in agreement with the small differences in the measured microscopic binding constants. Details of the dynamic mechanisms of molecular flexibility revealed by the MD simulations are also in good agreement with experimental findings, including the local properties identified from comparisons of hydrogen exchange rates in various parts of the structures of sequentially occupied forms of CAB. Estimation of the changes in configurational entropy from the rms fluctuations in the structures of CAB at various levels of Ca²⁺ occupancy in the EF-hands, supports earlier suggestions relating the dynamic properties of the protein to the observed cooperativity in the binding of Ca²⁺. The computational approaches and the results of the MD simulations are evaluated in relation to the study of effects of Ca²⁺ occupancy in calmodulin and troponin C where ion binding determines function and is known to trigger significant changes in structural and dynamic properties.     

Nuclear magnetic resonance‐based determination of dioxygen binding sites in protein cavities

The location and ligand accessibility of internal cavities in cysteine‐free wild‐type T4 lysozyme was investigated using O₂ gas‐pressure NMR spectroscopy and molecular dynamics (MD) simulation. Upon increasing the concentration of dissolved O₂ in solvent to 8.9 mM, O₂‐induced paramagnetic relaxation enhancements (PREs) to the backbone amide and side chain methyl protons were observed, specifically around two cavities in the C‐terminal domain. To determine the number of O₂ binding sites and their atomic coordinates from the 1/r⁶ distance dependence of the PREs, we established an analytical procedure using Akaike's Information Criterion, in combination with a grid‐search. Two O₂‐accessible sites were identified in internal cavities: One site was consistent with the xenon‐binding site in the protein in crystal, and the other site was established to be a novel ligand‐binding site. MD simulations performed at 10 and 100 mM O₂ revealed dioxygen ingress and egress as well as rotational and translational motions of O₂ in the cavities. It is therefore suggested that conformational fluctuations within the ground‐state ensemble transiently develop channels for O₂ association with the internal protein cavities.     

Determination of an unusual secondary structural element in the immunostimulating tetrapeptide rigin in aqueous environments: insights via MD simulations, 1H NMR and CD spectroscopic studies

An immunomodulating tetrapeptide, rigin (H‐Gly‐Gln‐Pro‐Arg‐OH), has been examined for its secondary structure preferences through combined use of high‐temperature unrestrained MD simulations in implicit water and 1D and 2D ¹H NMR spectroscopy. The distribution of backbone torsion angles revealed the predominance of trans conformers across the Xaa‐Pro peptide bond. The results of MD simulations revealed that of the five predicted families A–E, the predominant families, family A (92 structures), family C (63 structures) and family D (31 structures), could be complemented by extensive 1D and 2D ¹H NMR parameters acquired in aqueous PBS solution. A survey of specific inter‐ and intraresidue NOEs substantiated the predominance of an unusual type VII β‐turn structure, defined by two torsion angles, i.e. ψ_Gln ～ 155° and ?_Pro ～ ? 65° across the Gln‐Pro segment. The proposed semi‐folded kinked topology precluded formation of any intramolecular interaction, i.e. hydrogen bond or electrostatic interaction. Far‐UV CD spectral characteristics of rigin in aqueous PBS solution and non‐aqueous structure‐promoting organic solvents, TFE and TMP, revealed its strong solvent dependence. However, in aqueous PBS solution, the presence of a weak negative shoulder at nm could be ascribed to a small population with ordered, semi‐folded topology. We propose that the plausible structural attributes may be exploited for design and rigidification of the bioactive conformation of this immunomodulator toward improved immunopharmacological properties. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Microscopic mechanisms that govern the titration response and pKa values of buried residues in staphylococcal nuclease mutants

To probe the microscopic mechanisms that govern the titration behavior of buried ionizable groups, microsecond explicit solvent molecular dynamics simulations are carried out for several mutants of Staphylococcal nuclease using both fixed charge and polarizable force fields. While the ionization of Asp 66, Glu 66, and Lys 125 lead to enhanced structural fluctuations and partial unfolding of adjacent α‐helical regions, the ionization of Lys 25 causes local unfolding of adjacent β sheets. Using the sampled conformational ensembles, good agreement with experimental pK_a values is obtained with Poisson–Boltzmann calculations using a protein dielectric constant of 2–4 for V66D/E; slightly larger dielectric constants are needed for Lys mutants especially L25K, suggesting that structural responses beyond microseconds are involved in ionization of Lys 25. Overall, the set of unbiased simulations provides insights into the spatial and temporal scales of protein and solvent motions that dictate the diverse titration behaviors of buried protein residues. Proteins 2017; 85:268–281. © 2016 Wiley Periodicals, Inc.     

Thermodynamics calculation of protein–ligand interactions by QM/MM polarizable charge parameters

The calculation of protein–ligand binding free energy (ΔG) is of great importance for virtual screening and drug design. Molecular dynamics (MD) simulation has been an attractive tool to investigate this scientific problem. However, the reliability of such approach is affected by many factors including electrostatic interaction calculation. Here, we present a practical protocol using quantum mechanics/molecular mechanics (QM/MM) calculations to generate polarizable QM protein charge (QMPC). The calculated QMPC of some atoms in binding pockets was obviously different from that calculated by AMBER ff03, which might significantly affect the calculated ΔG. To evaluate the effect, the MD simulations and MM/GBSA calculation with QMPC for 10 protein–ligand complexes, and the simulation results were then compared to those with the AMBER ff03 force field and experimental results. The correlation coefficient between the calculated ΔΔG using MM/GBSA under QMPC and the experimental data is .92, while that with AMBER ff03 force field is .47 for the complexes formed by streptavidin or its mutants and biotin. Moreover, the calculated ΔΔG with QMPC for the complexes formed by ERβ and five ligands is positively related to experimental result with correlation coefficient of .61, while that with AMBER ff03 charge is negatively related to experimental data with correlation coefficient of .42. The detailed analysis shows that the electrostatic polarization introduced by QMPC affects the electrostatic contribution to the binding affinity and thus, leads to better correlation with experimental data. Therefore, this approach should be useful to virtual screening and drug design.     

Melting studies of short DNA hairpins: Influence of loop sequence and adjoining base pair identity on hairpin thermodynamic stability

Spectroscopic and calorimetric melting studies of 28 DNA hairpins were performed. These hairpins form by intramolecular folding of 16 base self‐complementary DNA oligomer sequences. Sequence design dictated that the hairpin structures have a six base pair duplex linked by a four base loop and that the first five base pairs in the stem are the same in every molecule. Only loop sequence and identity of the duplex base pair closing the loop vary for the set of hairpins. For these DNA samples, melting studies were carried out to investigate effects of the variables on hairpin stability. Stability of the 28 oligomers was ascertained from their temperature‐induced melting transitions in buffered 115 mM Na⁺ solvent, monitored by ultraviolet absorbance and differential scanning calorimetry (DSC). Experiments revealed the melting temperatures of these molecules range from 32.4 to 60.5°C and are concentration independent over strand concentrations of 0.5 to 260 μM; thus, as expected for hairpins, the melting transitions are apparently unimolecular. Model independent thermodynamic transition parameters, ΔH_cal, ΔS_cal, and ΔG_cal, were determined from DSC measurements. Model dependent transition parameters, ΔH_vH, ΔS_vH, and ΔG_vH were estimated from a van't Hoff (two‐state) analysis of optical melting transitions. Results of these studies reveal a significant sequence dependence to DNA hairpin stability. Thermodynamic parameters evaluated by either procedure reveal the transition enthalpy, ΔH_cal (ΔH_vH) can differ by as much as 20 kcal/mol depending on sequence. Similarly, values of the transition entropy ΔS_cal (ΔS_vH) can differ by as much as 60 cal/Kmol (eu) for different molecules. Differences in free energies ΔG_cal (ΔG_vH) are as large as 4 kcal/mol for hairpins with different sequences. Comparisons between the model independent calorimetric values and the thermodynamic parameters evaluated assuming a two‐state model reveal that 10 of the 28 hairpins display non‐two‐state melting behavior. The database of sequence‐dependent melting free energies obtained for the hairpins was employed to extract a set of n‐n (nearest‐neighbor) sequence dependent loop parameters that were able to reproduce the input data within error (with only two exceptions). Surprisingly, this suggests that the thermodynamic stability of the DNA hairpins can in large part be reasonably represented in terms of sums of appropriate nearest‐neighbor loop sequence parameters. © 1999 John Wiley & Sons, Inc. Biopoly 50: 425–442, 1999     

On the similarity of properties in solution or in the crystalline state: A molecular dynamics study of hen lysozyme

As protein crystals generally possess a high water content, it is assumed that the behaviour of a protein in solution and in crystal environment is very similar. This assumption can be investigated by molecular dynamics (MD) simulation of proteins in the different environments. Two 2ns simulations of hen egg white lysozyme (HEWL) in crystal and solution environment are compared to one another and to experimental data derived from both X-ray and NMR experiments, such as crystallographic B-factors, NOE atom–atom distance bounds, ³J_{H N}-coupling constants, and ¹H-¹⁵N bond vector order parameters. Both MD simulations give very similar results. The crystal simulation reproduces X-ray and NMR data slightly better than the solution simulation.     

Comparison of end-point continuum-solvation methods for the calculation of protein-ligand binding free energies

We have compared the predictions of ligand‐binding affinities from several methods based on end‐point molecular dynamics simulations and continuum solvation, that is, methods related to MM/PBSA (molecular mechanics combined with Poisson–Boltzmann and surface area solvation). Two continuum‐solvation models were considered, viz., the Poisson–Boltzmann (PB) and generalised Born (GB) approaches. The nonelectrostatic energies were also obtained in two different ways, viz., either from the sum of the bonded, van der Waals, nonpolar solvation energies, and entropy terms (as in MM/PBSA), or from the scaled protein–ligand van der Waals interaction energy (as in the linear interaction energy approach, LIE). Three different approaches to calculate electrostatic energies were tested, viz., the sum of electrostatic interaction energies and polar solvation energies, obtained either from a single simulation of the complex or from three independent simulations of the complex, the free protein, and the free ligand, or the linear‐response approximation (LRA). Moreover, we investigated the effect of scaling the electrostatic interactions by an effective internal dielectric constant of the protein (?_int). All these methods were tested on the binding of seven biotin analogues to avidin and nine 3‐amidinobenzyl‐1H‐indole‐2‐carboxamide inhibitors to factor Xa. For avidin, the best results were obtained with a combination of the LIE nonelectrostatic energies with the MM+GB electrostatic energies from a single simulation, using ?_int = 4. For fXa, standard MM/GBSA, based on one simulation and using ?_int = 4–10 gave the best result. The optimum internal dielectric constant seems to be slightly higher with PB than with GB solvation. © Proteins 2012; © 2012 Wiley Periodicals, Inc.     

Effect of guanidine and arginine on protein–ligand interactions in multimodal cation‐exchange chromatography

The addition of fluid phase modifiers provides significant opportunities for increasing the selectivity of multimodal chromatography. In order to optimize this selectivity, it is important to understand the fundamental interactions between proteins and these modifiers. To this end, molecular dynamics (MD) simulations were first performed to study the interactions of guanidine and arginine with three proteins. The simulation results showed that both guanidine and arginine interacted primarily with the negatively charged regions on the proteins and that these regions could be readily predicted using electrostatic potential maps. Protein surface characterization was then carried out using computationally efficient coarse‐grained techniques for a broader set of proteins which exhibited interesting chromatographic retention behavior upon the addition of these modifiers. It was shown that proteins exhibiting an increased retention in the presence of guanidine possessed hydrophobic regions adjacent to negatively charged regions on their surfaces. In contrast, proteins which exhibited a decreased binding in the presence of guanidine did not have hydrophobic regions adjacent to negatively charged patches. These results indicated that the effect of guanidine could be described as a combination of competitive binding, charge neutralization and increased hydrophobic interactions for certain proteins. In contrast, arginine resulted in a significant decrease in protein retention times primarily due to competition for the resin and steric effects, with minimal accompanying increase in hydrophobic interactions. The approach presented in this paper which employs MD simulations to guide the application of coarse‐grained approaches is expected to be extremely useful for methods development in downstream bioprocesses. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:435–447, 2017     

Structure of the Sm Binding Site From Human U4 snRNA Derived From a 3 ns PME Molecular Dynamics Simulation

Abstract

A molecular dynamics simulation of the Sm binding site from human U4 snRNA was undertaken to determine the conformational flexibility of this region and to identify RNA conformations that were important for binding of the Sm proteins. The RNA was fully-solvated (>9,000 water molecules) and charge neutralized by inclusion of potassium ions. A three nanosecond MD simulation was conducted using AMBER with long-range electrostatic forces considered using the particle mesh Ewald summation method. The initial model of the Sm binding site region had the central and 3′ stem-loops that flanked the Sm site co-axial with one another, and with the single-stranded Sm binding site region ([I] conformation). During the course of the trajectory, the axes of the 3′ stem-loop, and later the central stem-loop, became roughly orthogonal from their original anti-parallel orientation. As these conformational changes occurred, the snRNA adopted first an [L] conformation, and finally a [U] conformation. The [U] conformation was more stable than either the [I] or [L] conformations, and persisted for the final 1 ns of the trajectory. Analysis of the structure resulting from the MD simulations revealed the bulged nucleotide, U₁₁₄, and the mismatched A₉₁-G₁₁₀ base pair provided distinctive structural features that may enhance Sm protein binding. Based on the results of the MD simulation and the available experimental data, we proposed a mechanism for the binding of the Sm protein sub-complexes to the snRNA. In this model, the D₁/D₂ and E/F/G Sm protein sub-complexes first bind the snRNA in the [U] conformation, followed by conformational re-arrangement to the [I] conformation and binding of the D₃/B Sm protein sub-complex.     

Coarse‐grained modeling of the intrinsically disordered protein Histatin 5 in solution: Monte Carlo simulations in combination with SAXS

Monte Carlo simulations and coarse‐grained modeling have been used to analyze Histatin 5, an unstructured short cationic salivary peptide known to have anticandidical properties. The calculated scattering functions have been compared with intensity curves and the distance distribution function P(r) obtained from small angle X‐ray scattering (SAXS), at both high and low salt concentrations. The aim was to achieve a molecular understanding and a physico‐chemical insight of the obtained SAXS results and to gain information of the conformational changes of Histatin 5 due to altering salt content, charge distribution, and net charge. From a modeling perspective, the accuracy of the electrostatic interactions are of special interest. The used coarse‐grained model was based on the primitive model in which charged hard spheres differing in charge and in size represent the ionic particles, and the solvent only enters the model through its relative permittivity. The Hamiltonian of the model comprises three different contributions: (i) excluded volumes, (ii) electrostatic, and (iii) van der Waals interactions. Even though the model can be considered as gross omitting all atomistic details, a great correspondence is obtained with the experimental results. Proteins 2016; 84:777–791. © 2016 Wiley Periodicals, Inc.     

A comparative study of the thermal stability of plastocyanin, cytochrome c6 and Photosystem I in thermophilic and mesophilic cyanobacteria

Cytochrome c₆ (Cyt) from the thermophilic cyanobacterium Phormidium laminosum has been purified and characterized. It is a mildly acidic protein, with physicochemical properties very similar to those of plastocyanin (Pc). This is in agreement with the functional interchangeability of the two metalloproteins as electron donors to Photosystem I (PS I). The kinetic analyses of the interaction of Pc and Cyt with Photosystem I show that both metalloproteins reduce PS I with similar efficiencies, according to an oriented collisional kinetic model involving repulsive electrostatic interactions. The thermostability study of the Phormidium Pc/PS I system compared with those from mesophilic cyanobacteria (Synechocystis, Anabaena and Pseudanabaena) reveals that Pc is the partner limiting the thermostability of the Phormidium couple. The cross-reactions between Pc and PS I from different organisms demonstrate not only that Phormidium Pc enhances the stability of the Pc/PS I system using PS I from mesophilic cyanobacteria, but also that Phormidium PS I possesses a higher thermostability than the other photosystems. This revised version was published online in June 2006 with corrections to the Cover Date.     

Experimental evidence for the role of buried polar groups in determining the reduction potential of metalloproteins: the S79P variant of Chromatium vinosum HiPIP

 The amide group between residues 78 and 79 of Chromatium vinosum high-potential iron-sulfur protein (HiPIP) is in close proximity to the Fe₄S₄ cluster of this protein and interacts via a hydrogen bond with Sγ of Cys77, one of the cluster ligands. The reduction potential of the S79P variant was 104±3 mV lower than that of the recombinant wild-type (rcWT) HiPIP (5 mM phosphate, 100 mM NaCl, pH 7, 293 K), principally due to a decrease in the enthalpic term which favors the reduction of the rcWT protein. Analysis of the variant protein by NMR spectroscopy indicated that the substitution has little effect on the structure of the HiPIP or on the electron distribution in the oxidized cluster. Potential energy calculations indicate that the difference in reduction potential between rcWT and S79P variant HiPIPs is due to the different electrostatic properties of amide 79 in these two proteins. These results suggest that the influence of amide group 79 on the reduction potential of C. vinosum HiPIP is a manifestation of a general electrostatic effect rather than a specific interaction. More generally, these results provide experimental evidence for the importance of buried polar groups in determining the reduction potentials of metalloproteins. Received: 26 April 1999 / Accepted: 24 August 1999     

A long lifetime component in the tryptophan fluorescence of some proteins

The tryptophan fluorescence of two membrane proteins (outer membrane protein A and lactose permease), a 21-residue hydrophobic peptide, three soluble proteins (rat serum albumin, ribonuclease T_I, and azurin), and N-acetyltryptophanamide (NATA) was investigated by time-resolved measurements extended over 65 ns. A long lifetime component with a characteristic time of 25 ns and an amplitude below 1% was found for outer membrane protein A, lactose permease, the peptide in lipid membranes, and azurin in water, but not for rat serum albumin, ribonuclease T_I, and NATA in water. When outer membrane protein A was dissolved and unfolded in guanidinum hydrochloride, the long lifetime component disappeared. Hence, a hydrophobic environment seems to be a necessary requirement for the long lifetime component to be present. However, NATA dissolved in butanol does not exhibit the long lifetime component, while the peptide dissolved in the same solvent under conditions which preserve its helical structure does show the long lifetime. Thus, a regular secondary structure for the polypetide chain to which the tryptophan residue belongs seems to be a second necessary requirement for the long lifetime component to be present. The long lifetime component may therefore be seen in the context of protein substates.Abbreviations OmpA outer membrane protein A - LP lactose permease - RSA rat serum albumin - RNAse TI ribonuclease TI - P21 21-residue peptide - NATA N-acetyltryptophanamide - PTP paraterphenyl - POPE palmitoyloleoylphosphatidylethanolamine - POPC palmitoyloleoylphosphatidylcholine - POPG palmitoyloleoylphosphatidylglycerol - GdHCI guanidinium hydrochloride Correspondence to: F. Jähnig     

Self‐guided Langevin dynamics study of regulatory interactions in NtrC

Multiple self‐guided Langevin dynamics (SGLD) simulations were performed to examine structural and dynamical properties of the receiver domain of nitrogen regulatory protein C (NtrC^r). SGLD and MD simulations of the phosphorylated active form structure suggest a mostly stable but broad structural ensemble of this protein. The finite difference Poisson–Boltzmann calculations of the pK_a values of the active site residues suggest an increase in the pK_a of His‐84 on phosphorylation of Asp‐54. In SGLD simulations of the phosphorylated active form with charged His‐84, the average position of the regulatory helix α4 is found closer to the starting structure than in simulations with the neutral His‐84. To model the transition pathway, the phosphate group was removed from the simulations. After 7 ns of simulations, the regulatory helix α4 was found approximately halfway between positions in the NMR structures of the active and inactive forms. Removal of the phosphate group stimulated loss of helix α4, suggesting that the pathway of conformational transition may involve partial unfolding mechanism. The study illustrates the potential utility of the SGLD method in studies of the coupling between ligand binding and conformational transitions. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Global fold of human cannabinoid type 2 receptor probed by solid‐state 13C‐, 15N‐MAS NMR and molecular dynamics simulations

The global fold of human cannabinoid type 2 (CB₂) receptor in the agonist‐bound active state in lipid bilayers was investigated by solid‐state ¹³C‐ and ¹⁵N magic‐angle spinning (MAS) NMR, in combination with chemical‐shift prediction from a structural model of the receptor obtained by microsecond‐long molecular dynamics (MD) simulations. Uniformly ¹³C‐ and ¹⁵N‐labeled CB₂ receptor was expressed in milligram quantities by bacterial fermentation, purified, and functionally reconstituted into liposomes. ¹³C MAS NMR spectra were recorded without sensitivity enhancement for direct comparison of C_α, C_β, and C?O bands of superimposed resonances with predictions from protein structures generated by MD. The experimental NMR spectra matched the calculated spectra reasonably well indicating agreement of the global fold of the protein between experiment and simulations. In particular, the ¹³C chemical shift distribution of C_α resonances was shown to be very sensitive to both the primary amino acid sequence and the secondary structure of CB₂. Thus the shape of the C_α band can be used as an indicator of CB₂ global fold. The prediction from MD simulations indicated that upon receptor activation a rather limited number of amino acid residues, mainly located in the extracellular Loop 2 and the second half of intracellular Loop 3, change their chemical shifts significantly (≥1.5 ppm for carbons and ≥5.0 ppm for nitrogens). Simulated two‐dimensional ¹³C_α(i)? ¹³C?O(i) and ¹³C?O(i)? ¹⁵NH(i + 1) dipolar‐interaction correlation spectra provide guidance for selective amino acid labeling and signal assignment schemes to study the molecular mechanism of activation of CB₂ by solid‐state MAS NMR. Proteins 2014; 82:452–465. © 2013 Wiley Periodicals, Inc.     

Computer Aided Study of Ligand Binding With Catalytic Domain of Avian Sarcoma Virus Integrase and Its Ligand Binding Loops

Abstract

We have carried out 1 nanosecond (ns) Molecular Dynamics (MD) simulations of the drug Y3 (4-acetylamino-5-hydroxynaphthalene-2, 7-disulfonic acid) complexed with catalytic domain of Avian sarcoma virus Integrase (ASV-IN), both in vacuum and in the presence of explicit solvent. Starting models were obtained on the basis of PDB co-ordinates (1A5X) of ASV-IN-Y3 complex, by Lubkowski et al [1]. Mn²⁺ cation was present in the active site. To neutralize the positive charge in the presence of explicit solvent, eight Cl^? anions were added. Energy Minimization (EM) and MD simulations, for both the systems, were carried out using Sander's module of AMBER5.0 [2] with all atom force field. Analysis of ligand- protein interaction in both environments is discussed in the paper. We also carried out 1 ns MD simulation on two flexible loops—L1 (Gly54-Gln62) and L2 (Trp138-Met155) playing crucial role in interaction of IN with the drug [3], under differing environmental conditions (vacuum, aqueous and organic solvent methanol). Comparison of the conformational changes in the loops, monomer and dimer is presented in the paper. Our results showed that the conformation of the loop region was closest to crystallographic data in case of monomer and constrained loops in aqueous environment. However, the dimer in vacuum was more stable than monomer. The β sheet structure of the monomer in aqueous environment was unstable. Latter also took long time for equilibration. The box formed by loops L1 and L2 from two sub units IINA and INB) of the dimer satisfies prerequisites for ligand recognition site and seems to be the functional biological unit.