Mutations in alpha-helical solvent-exposed sites of eglin c have long-range effects: evidence from molecular dynamics simulations

Eglin c is a small protease inhibitor whose structural and thermodynamic properties have been well studied. Previous thermodynamic measurements on mutants at solvent-accessible positions in the protein's helix have shown the unexpected result that the data could be best fit by the inclusion of residue- and position-specific parameters to the model. To explore the origins of this surprising result, long molecular dynamics simulations in explicit solvent have been performed. These simulations indicate specific long-range interactions between the solvent-exposed residues in the eglin c alpha-helix and binding loop, an unexpected observation for such a small protein. The residues involved in the interaction are on opposite sides of the protein, about 25 A apart. Simulations of alanine substitutions at the solvent-exposed helix positions, arginine 22, glutamic acid 23, threonine 26, and leucine 27, show both small and large perturbations of eglin c dynamics. Two mutations exhibit large impacts on the long-range helix-loop interactions. Previous stability measurements (Yi et al., Biochemistry 2003;42:7594-7603) had indicated that an alanine substitution at position 27 was less stabilizing than at other solvent-exposed positions in the helix. The L27A mutation effects observed in these simulations suggest that the position-dependent loss of stability measured in wet bench experiments is derived from changes in dynamics that involve long-range interactions; thus, these simulations support the hypothesis that solvent-exposed positions in helices are not always equivalent.     

Accuracy and precision of NMR relaxation experiments and MD simulations for characterizing protein dynamics

Model-free parameters obtained from nuclear magnetic resonance (NMR) relaxation experiments and molecular dynamics (MD) simulations commonly are used to describe the intramolecular dynamical properties of proteins. To assess the relative accuracy and precision of experimental and simulated model-free parameters, three independent data sets derived from backbone ¹⁵N NMR relaxation experiments and two independent data sets derived from MD simulations of Escherichia coli ribonuclease HI are compared. The widths of the distributions of the differences between the order parameters for pairs of NMR data sets are congruent with the uncertainties derived from statistical analyses of individual data sets; thus, current protocols for analyzing NMR data encapsulate random uncertainties appropriately. Large differences in order parameters for certain residues are attributed to systematic differences between samples for intralaboratory comparisons and unknown, possibly magnetic field-dependent, experimental effects for interlaboratory comparisons. The widths of distributions of the differences between the order parameters for two NMR sets are similar to widths of distributions for an NMR and an MD set or for two MD sets. The linear correlations between the order parameters for an MD set and an NMR set are within the range of correlations observed between pairs of NMR sets. These comparisons suggest that the NMR and MD generalized order parameters for the backbone amide N—H bond vectors are of comparable accuracy for residues exhibiting motions on a fast time scale (<100 ps). Large discrepancies between NMR and MD order parameters for certain residues are attributed to the occurrence of “rare” motional events over the simulation trajectories, the disruption of an element of secondary structure in one of the simulations, and lack of consensus among the experimental data sets. Consequently, (easily detectable) severe distortions of local protein structure and infrequent motional events in MD simulations appear to be the most serious artifacts affecting the accuracy and precision, respectively, of MD order parameters relative to NMR values. In addition, MD order parameters for motions on a fast (<100 ps) timescale are more precisely determined than their NMR counterparts, thereby permitting more detailed dynamic characterization of biologically important residues by MD simulation than is sometimes possible by experimental methods. Proteins 28:481–493, 1997. © 1997 Wiley-Liss, Inc.     

Sequence-specific 1H NMR assignments and secondary structure of eglin c

Sequence-specific nuclear magnetic resonance assignments were obtained for eglin c, a polypeptide inhibitor of the granulocytic proteinases elastase and cathepsin G and some other proteinases. The protein consists of a single polypeptide chain of 70 residues. All proton resonances were assigned except for some labile protons of arginine side chains. The patterns of nuclear Overhauser enhancements and coupling constants and the observation of slow hydrogen exchange were used to characterize the secondary structure of the protein. The results indicate that the solution structure of the free inhibitor is very similar to the crystal structure reported for the same protein in the complex with subtilisin Carlsberg. However, a part of the binding loop seems to have a significantly different conformation in the free protein.     

Automated NMR determination of protein backbone dihedral angles from cross-correlated spin relaxation

The simultaneous interpretation of a suite of dipole-dipole and dipole-CSA cross-correlation rates involving the backbone nuclei ¹³C, ¹H,¹³CO, ¹⁵N and ¹H^N can be used to resolve the ambiguities associated with each individual cross-correlation rate. The method is based on the transformation of experimental cross-correlation rates via calculated values based on standard peptide plane geometry and solid-state ¹³CO CSA parameters into a dihedral angle probability surface. Triple resonance NMR experiments with improved sensitivity have been devised for the quantification of relaxation interference between ¹H(i)-¹³C(i)/¹⁵N(i)-¹H^N(i) and ¹H(i–1)-¹³C(i–1)/¹⁵N(i)-¹H^N(i) dipole-dipole mechanisms in ¹⁵N,¹³C-labeled proteins. The approach is illustrated with an application to ¹³C,¹⁵N-labeled ubiquitin.     

Starting structure dependence of NMR order parameters derived from MD simulations: implications for judging force-field quality

Comparing experimental generalized N-H S² order parameters to those calculated from molecular dynamics trajectories is increasingly used to judge force-field quality and completeness of sampling. Herein we demonstrate for the well-investigated system hen egg white lysozyme that different experimental starting structures can lead to significant differences in molecular-dynamics-derived S² parameters that can be even larger than S² parameter deviations due to different force fields. Caution should thus be taken in general when simulated S² parameters are compared to experimental data with the aim of judging force-field quality. We show that adequately sampling flexible regions (∼100 ns) and only calculating S² parameters averaged over short time windows proved necessary to obtain consistent results irrespective of the starting structure.     

Quantization of pH: evidence for acidic activity of triglyceride lipases

Here we present a study of lipolytic activity of lipases from Fusarium solani pisi (cutinase), Rhizomucor miehei, Pseudomonas cepacia, and Humicola lanuginosa. Their activities toward triolein provide clear evidence for considerable enzymatic activity under acidic conditions. The activity was followed using Fourier transform infrared attenuated total reflection (FTIR-ATR) and nuclear magnetic resonance (NMR). Using these approaches, all the lipases that were studied exhibited lipolytic activity down to pH 4. The common model for the catalytic activity of the F. solani pisi cutinase, and lipases in general, requires the deprotonation of the active site histidine. Measurements using (13)C NMR spectroscopy showed a pK(a) value in the absence of substrate that is not consistent with the detected acid activity. We propose a novel model for the electrostatics in the active site of cutinase that could explain the observed acidic activity. The active site is essentially covered with the lipid surface during catalysis, thus preventing chemical communication between the active site and the bulk solvent. We propose that the classical definition of pH in bulk solution is not applicable to the active site environment of a lipase when the active site is inaccessible to solvent. In small restricted volumes, the pH must be quantized, and since much of the biological world is dependent on compartmentalization of processes in small volumes, it becomes relevant to investigate when this mechanism comes into play. We have made a quantitative assessment of how large the restricted volume can be and still lead to quantization of pH.     

Outer membrane proteins: comparing X-ray and NMR structures by MD simulations in lipid bilayers

The structures of three bacterial outer membrane proteins (OmpA, OmpX and PagP) have been determined by both X-ray diffraction and NMR. We have used multiple (7 × 15 ns) MD simulations to compare the conformational dynamics resulting from the X-ray versus the NMR structures, each protein being simulated in a lipid (DMPC) bilayer. Conformational drift was assessed via calculation of the root mean square deviation as a function of time. On this basis the ‘quality’ of the starting structure seems mainly to influence the simulation stability of the transmembrane β-barrel domain. Root mean square fluctuations were used to compare simulation mobility as a function of residue number. The resultant residue mobility profiles were qualitatively similar for the corresponding X-ray and NMR structure-based simulations. However, all three proteins were generally more mobile in the NMR-based than in the X-ray simulations. Principal components analysis was used to identify the dominant motions within each simulation. The first two eigenvectors (which account for >50% of the protein motion) reveal that such motions are concentrated in the extracellular loops and, in the case of PagP, in the N-terminal α-helix. Residue profiles of the magnitude of motions corresponding to the first two eigenvectors are similar for the corresponding X-ray and NMR simulations, but the directions of these motions correlate poorly reflecting incomplete sampling on a ∼10 ns timescale.     

Solid-state NMR and MD simulations of the antiviral drug amantadine solubilized in DMPC bilayers

The interactions of ¹⁵N-labeled amantadine, an antiinfluenza A drug, with DMPC bilayers were investigated by solid-state NMR and by a 12.6-ns molecular dynamics (MD) simulation. The drug was found to assume a single preferred orientation and location when incorporated in these bilayers. The experimental and MD computational results demonstrate that the long axis of amantadine is on average parallel to the bilayer normal, and the amine group is oriented toward the headgroups of the lipid bilayers. The localization of amantadine was determined by paramagnetic relaxation and by the MD simulation showing that amantadine is within the interfacial region and that the amine interacts with the lipid headgroup and glycerol backbone, while the hydrocarbon portion of amantadine interacts with the glycerol backbone and much of the fatty acyl chain as it wraps underneath the drug. The lipid headgroup orientation changes on drug binding as characterized by the anisotropy of ³¹P chemical shielding and ¹⁴N quadrupolar interactions and by the MD simulation.     

Galactose-induced dimerization of blocked ricin at acidic pH: evidence for a third galactose-binding site in ricin B-chain

Blocked ricin is a glycoconjugate formed by covalent modificationof each of the two galactose-binding sites of ricin with affinityligands derived by modification of glycopeptides containinggalactose-terminated, triantennary, N-linked oligosaccharides.Blocked ricin undergoes a pH-dependent reversible self-association,being predominantly dimeric at neutral pH and monomeric at acidicpH. The shift in the monomer-dimer equilibrium towards the monomericform at acidic pH (pH 4) is inhibited by lactose, as shown bysize-exclusion chromatography. This behavior of blocked ricincan be reproduced in studies with isolated blocked B-chain.The effect, which is dependent on the concentration of the sugar,is specific for sugars having terminal galactose moieties, orsugars having the same orientation of hydroxyl groups at C2and C4 as galactose. These results are interpreted as providingfurther support for the notion that ricin B-chain has a thirdgalactosebinding site, which may be important for the intracellulartrafficking of ricin during intoxication of cells. blocked ricin galactose-binding lectin ricin     

An NMR relaxation and spin diffusion study of cellulose structure during water adsorption

The goal of this paper is a systematic investigation of changes in the supramolecular structure of cellulose during its water uptake. The main attention is concentrated on the analysis of the mechanism of dispersion of microfibrils by proton NMR relaxation techniques. Spin diffusion NMR experiments made it possible to estimate the linear dimensions of the surface thickness of cellulose crystallites and the average depth of micropores that are formed between elementary fibrils, as well as the character of the filling of micropores during adsorption. It has been shown that when the relative water content gradually increases to 7–8%, water molecules occupy the space between cellulose microfibrils, which is accompanied by an increase in the pore sizes and their specific surface area and a simultaneous decrease in the degree of crystallinity. Upon acquiring a free induction decay signal, a magic sandwich echo sequence was used, due to which the accuracy and information value of the results were considerably improved.     

Stability of trimeric DENV envelope protein at low and neutral pH: An insight from MD study

Change in pH plays a crucial role in the stability and function of the dengue envelope (DENV) protein during conformational transition from dimeric (pre-fusion state) to trimeric form (post-fusion state). In the present study we have performed various molecular dynamics (MD) simulations of the trimeric DENV protein at different pH and ionic concentrations. We have used total binding energy to justify the stability of the complex using the MMPBSA method. We found a remarkable increase in the stability of the complex at neutral pH (pH ~ 7) due to the increment of sodium ions. However, at very low pH (pH ~ 4), the total energy of the complex becomes high enough to destabilize the complex. At a specific pH, almost at a range of 6, the stability of the complex is significantly better than the stability of the trimer at neutral pH, which connotes that the trimer is most stable at this pH (pH ~ 6).     

15N NMR relaxation studies of free and ligand-bound human acidic fibroblast growth factor

15N NMR relaxation data have been used to characterize the backbone dynamics of the human acidic fibroblast growth factor (hFGF-1) in its free and sucrose octasulfate (SOS)-bound states. (15)N longitudinal (R(1)), transverse (R(2)) relaxation rates and (1H)-(15)N steady-state nuclear Overhauser effects were obtained at 500 and 600 MHz (at 25 degrees C) for all resolved backbone amide groups using (1)H- detected two-dimensional NMR experiments. Relaxation data were fit to the extended model free dynamics for each NH group. The overall correlation time (tau(m)) for the free and SOS-bound forms were estimated to be 10.4 +/- 1.07 and 11.1 +/- 1.35 ns, respectively. Titration experiments with SOS reveals that the ligand binds specifically to the C-terminal domain of the protein in a 1:1 ratio. Binding of SOS to hFGF-1 is found to induce a subtle conformational change in the protein. Significant conformational exchange (R(ex)) is observed for several residues in the free form of the protein. However, in the SOS-bound form only three residues exhibit significant R(ex) values, suggesting that the dynamics on the micro- to millisecond time scale in the free form is coupled to the cis-trans-proline isomerization. hFGF-1 is a rigid molecule with an average generalized parameter (S(2)) value of 0.89 +/- 0.03. Upon binding to SOS, there is a marked decrease in the overall flexibility (S(2) = 0.94 +/- 0.02) of the hFGF-1 molecule. However, the segment comprising residues 103-111 shows increased flexibility in the presence of SOS. Significant correlation is found between residues that show high flexibility and the putative receptor binding sites on the protein.     

Oligonucleotide conformations. (5) NMR and relaxation studies on GpU and UpG at neutral pH.

The average conformation of GpU and UpG in neutral aqueous solutions has been investigated by proton chemical shifts and coupling measurements as well as T1 relaxation time experiments. The proportion of the N and S pseudorotational conformers of the ribose ring has been derived from the vicinal coupling constants. The relaxation data provide information about the syn--anti equilibrium of the orientation of the base about the glycosidic bond. This orientation is predominantly syn for the Guo base in both dinucleoside phosphates, that of Urd is anti in the case of GpU and shows an almost equivalent syn and anti character for UpG.     

Stabilization of Microtubules by Taxane Diterpenoids: Insight from Docking and MD simulations

Microtubules are formed from the molecules of tubulin, whose dynamics is important for many functions in a cell, the most dramatic of which is mitosis. Taxol is known to interact within a specific site on tubulin and also believed to block cell-cycle progression during mitosis by binding to and stabilizing microtubules. Along with the tremendous potential that taxol has shown as an anticancer drug, clinical problems exist with solubility, toxicity, and development of drug resistance. The crystal structure of taxane diterpenoids, namely, 10, 13-deacetyl-abeo-baccatin-IV (I), 5-acetyl-2-deacetoxydecinnamoyl-taxinine-0.29hydrate (II), 7, 9-dideacetyltaxayuntin (III), and Taxawallin-K (IV), are very similar to the taxol molecule. Considerable attention has been given to such molecules whose archetype is taxol but do not posses long aliphatic chains, to be developed as a substitute for taxol with fewer side effects. In the present work, the molecular docking of these taxane diterpenoids has been carried out with the tubulin alpha-beta dimer (1TUB) and refined microtubule structure (1JFF) using Glide-XP, in order to assess the potential of tubulin binding of these cytotoxic agents. Results show that all the ligands dock into the classical taxol binding site of tubulin. Taxol shows the best binding capabilities. On the basis of docking energy and interactions, apart from taxol, molecule II has a better tendency of binding with 1TUB while molecule I shows better binding capability with bovine tubulin 1JFF. To validate the binding capabilities, molecular dynamics (MD) simulations of the best docked complexes of ligands with 1JFF have been carried out for 15.0 ns using DESMOND. Average RMSD variations and time line study of interactions and contacts indicate that these complexes remain stable during the course of the dynamics. However, taxol and molecule II prevail over other taxoids.

Electronic supplementary material

The online version of this article (doi:10.1007/s10867-014-9369-5) contains supplementary material, which is available to authorized users.     

Effect of sterol structure on the bending rigidity of lipid membranes: A H NMR transverse relaxation study

The effect of incorporation of 3-43 mol% sterol on the lipid order and bilayer rigidity has been investigated for model membranes of dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine. ²H NMR spectra and spin-lattice relaxation rates were measured for macroscopically aligned bilayers. The characteristics of spectra obtained at temperatures between 0-60 °C are interpreted in terms of a two-phase coexistence of the liquid disordered and the liquid ordered phases and the data is found to be in agreement with the phase diagram published by Vist and Davis (Biochemistry 29 (1990), pp. 451-464). The bending modulus of the bilayers was calculated from plots of relaxation rate vs. the square of the order parameter at 44 °C. Clear differences were obtained in the efficiency of the sterols to increase the stiffness of the bilayers. These differences are correlated to the ability of the sterols to induce the liquid ordered phase in binary as well as in ternary systems; the only exception being ergosterol, which was found to be unable to induce l_o phases and also had a relatively weak effect on the bilayer stiffness in contrast to earlier reports.     

Induction of acid tolerance at neutral pH in log-phase Escherichia coli by medium filtrates from organisms grown at acidic pH

Escherichia coli grown at pH 5·0 became acid-tolerant (acid-habituated) but, in addition, neutralized medium filtrates from cultures of E. coli grown to log-phase or stationary-phase at pH 5·0 (pH 5·0 filtrates) induced acid tolerance when added to log-phase E. coli growing at pH 7·0. In contrast, filtrates from pH 7·0-grown cultures were ineffective. The pH 5·0 filtrates were inactivated by heating in a boiling water-bath but there was less activity loss at 75 °C. Protease also inactivated such filtrates, which suggested that a heat-resistant protein (or proteins) in the filtrates was essential for the induction of acid tolerance. Filtrates from cells grown at pH 5·0 plus phosphate or adenosine 3':5'-cyclic monophosphate (cAMP) were much less effective in inducing acid tolerance, while the conversion of pH 7·0-grown log-phase cells to acid tolerance by pH 5·0 filtrates was inhibited by cAMP and bicarbonate. It seems likely that the acid tolerance response (acid habituation) involved the functioning of the extracellular protein(s) as protease reduces tolerance induction if added during acid habituation. Most inducible responses are believed to involve the functioning of only intracellular reactions and components ; the present results suggest that this is not the case for acid habituation, as an extracellular protein (or proteins) is needed for induction.     

Acid-induced loss of functional properties of bacterial cell division protein FtsZ: evidence for an alternative conformation at acidic pH

Several types of bacteria live in highly acidic environments. Since the assembly of FtsZ is important for bacterial cytokinesis, the effects of pH on the assembly and structural properties of FtsZ were examined. FtsZ retained GTP binding ability but lost GTPase activity at pH 2.5. In the presence of GTP, FtsZ formed protofilaments at pH 7 while it formed aggregates instead of protofilaments at pH 2.5, indicating that GTP hydrolysis is important for the assembly of FtsZ into protofilaments. Further, the acid-inactivated state of FtsZ recovered its structural and functional properties upon refolding at pH 7, indicating that the cellular functions of FtsZ may be restored after removal of the external stress. In addition, the affinity of 1-anilinonaphthalene-8-sulfonic acid (ANS) binding to FtsZ was found to be higher at pH 2.5 than at pH 7. FtsZ-ANS complex had a higher quantum yield and lifetime at pH 2.5 than at pH 7. However, the secondary structures of FtsZ were similar at pH 7 and 2.5, indicating that FtsZ attained an alternatively folded state (A) at pH 2.5, which has some characteristics of a molten-globule-like state. The A state was more stable than the native state (N) against urea-induced unfolding. The transition from N to A state involves the formation of aggregates of FtsZ (I). The association of FtsZ monomers occurred in the narrow pH range (3.2-2.8) and it was found to be a fully reversible process. The results suggest that a productive intermediate (I) forms in the acid-induced unfolding pathway of FtsZ and that the unfolding pathway may be minimally described as N <==> I <==> A.     

Application of cross-correlated NMR spin relaxation to the zinc-finger protein CRP2(LIM2): evidence for collective motions in LIM domains

The solution structure of quail CRP2(LIM2) was significantly improved by using an increased number of NOE constraints obtained from a 13C,15N-labeled protein sample and by applying a recently developed triple-resonance cross-correlated relaxation experiment for the determination of the backbone dihedral angle psi. Additionally, the relative orientation of the 15N(i)-1HN(i) dipole and the 13CO(i) CSA tensor, which is related to both backbone angles phi and psi, was probed by nitrogen-carbonyl multiple-quantum relaxation and used as an additional constraint for the refinement of the local geometry of the metal-coordination sites in CRP2(LIM2). The backbone dynamics of residues located in the folded part of CRP2(LIM2) have been characterized by proton-detected 13C'(i-1)-15N(i) and 15N(i)-1HN(i) multiple-quantum relaxation, respectively. We show that regions having cross-correlated time modulation of backbone isotropic chemical shifts on the millisecond to microsecond time scale correlate with residues that are structurally altered in the mutant protein CRP2(LIM2)R122A (disruption of the CCHC zinc-finger stabilizing side-chain hydrogen bond) and that these residues are part of an extended hydrogen-bonding network connecting the two zinc-binding sites. This indicates the presence of long-range collective motions in the two zinc-binding subdomains. The conformational plasticity of the LIM domain may be of functional relevance for this important protein recognition motif.