Ionization states of residues in OmpF and mutants: effects of dielectric constant and interactions between residues

To understand ion permeation, one must assign correct ionization states to titratable amino acid residues in protein channels. We report on the effects of physical and methodological assumptions in calculating the protonation states at neutral bulk pH of titratable residues lining the lumen of the native Escherichia coli OmpF channel, and five mutants. We systematically considered a wide range of assumed protein dielectric constants and all plausible combinations of protonation states for electrostatically interacting side chains, and three different levels of accounting for solute shielding: 1), full nonlinear Poisson-Boltzmann; 2), linearized Poisson-Boltzmann; and 3), neglect of solute shielding. For this system we found it acceptable to neglect solute shielding, a result we postulate to be generalizable to narrow lumens of other protein channels. For the large majority of residues, the protonation state at neutral bulk pH was found to be independent of the assumed dielectric constant of the protein, and unambiguously determined by the calculation; for native OmpF only Asp-127 has a protonation state that is sensitive to the assumed protein dielectric constant. Our results are significant for understanding two published experimental observations: the structure of the narrow part of the channel, and the ionic selectivity of OmpF mutants.     

Two distinct proton binding sites in the ATP synthase family

The F1F0 ATP synthase utilizes energy stored in an electrochemical gradient of protons (or Na+ ions) across the membrane to synthesize ATP from ADP and phosphate. Current models predict that the protonation/deprotonation of specific acidic c ring residues is at the core of the proton translocation mechanism by this enzyme. To probe the mode of proton binding, we measured the covalent modification of the acidic c ring residues with the inhibitor dicyclohexylcarbodiimide (DCCD) over the pH range from 5 to 11. With the H+-translocating ATP synthase from the archaeum Halobacterium salinarium or the Na+-translocating ATP synthase from Ilyobacter tartaricus, the pH profile of DCCD labeling followed a titration curve with a pKa around neutral, reflecting protonation of the acidic c ring residues. However, with the ATP synthases from Escherichia coli, mitochondria, or chloroplasts, a clearly different, bell-shaped pH profile for DCCD labeling was observed which is not compatible with carboxylate protonation but might be explained by the coordination of a hydronium ion as proposed earlier [Boyer, P. D. (1988) Trends Biochem. Sci. 13, 5-7]. Upon site-directed mutagenesis of single binding site residues of the structurally resolved c ring, the sigmoidal pH profile for DCCD labeling could be converted to a more bell-shaped one, demonstrating that the different ion binding modes are based on subtle changes in the amino acid sequence of the protein. The concept of two different binding sites in the ATP synthase family is supported by the ATP hydrolysis pH profiles of the investigated enzymes.     

Multiple pH regime molecular dynamics simulation for pK calculations

Ionisation equilibria in proteins are influenced by conformational flexibility, which can in principle be accounted for by molecular dynamics simulation. One problem in this method is the bias arising from the fixed protonation state during the simulation. Its effect is mostly exhibited when the ionisation behaviour of the titratable groups is extrapolated to pH regions where the predetermined protonation state of the protein may not be statistically relevant, leading to conformational sampling that is not representative of the true state. In this work we consider a simple approach which can essentially reduce this problem. Three molecular dynamics structure sets are generated, each with a different protonation state of the protein molecule expected to be relevant at three pH regions, and pK calculations from the three sets are combined to predict pK over the entire pH range of interest. This multiple pH molecular dynamics approach was tested on the GCN4 leucine zipper, a protein for which a full data set of experimental data is available. The pK values were predicted with a mean deviation from the experimental data of 0.29 pH units, and with a precision of 0.13 pH units, evaluated on the basis of equivalent sites in the dimeric GCN4 leucine zipper.     

Constant pH molecular dynamics (CpHMD) and molecular docking studies of CquiOBP1 pH-induced ligand releasing mechanism

The odorant binding protein of Culex quinquefasciatus (CquiOBP1), expressed on the insect antenna, is crucial for the investigation of trapping baited with oviposition semi-chemicals and controlling mosquito populations. The acidic titratable residues pKa prediction and the ligand binding poses investigation in two systems (pH 7 and pH 5) are studied by constant pH molecular dynamics (CpHMD) and molecular docking methods. Research results reveal that the change of the protonation states would disrupt some important H-bonds, such as Asp 66-Asp 70, Glu 105-Asn 102, etc. The cleavage of these H-bonds leads to the movement of the relative position of hydrophobic tunnel, N- and C- termini loops and pH-sensing triad (His23-Tyr54-Val125) in acid solution. Ligand MOP has lower affinity and shows different binding poses to protein CquiOBP1 at pH 5. This ligand may be released from another tunnel between helices α3 and α4 in acidic environment. However, it would bind to the protein with high affinity in neutral environment. This work could provide more penetrating understanding of the pH-induced ligand-releasing mechanism.     

The influence of a transmembrane pH gradient on protonation probabilities of bacteriorhodopsin: the structural basis of the back-pressure effect

Bacteriorhodopsin pumps protons across a membrane using the energy of light. The proton pumping is inhibited when the transmembrane proton gradient that the protein generates becomes larger than four pH units. This phenomenon is known as the back-pressure effect. Here, we investigate the structural basis of this effect by predicting the influence of a transmembrane pH gradient on the titration behavior of bacteriorhodopsin. For this purpose we introduce a method that accounts for a pH gradient in protonation probability calculations. The method considers that in a transmembrane protein, which is exposed to two different aqueous phases, each titratable residue is accessible for protons from one side of the membrane depending on its hydrogen-bond pattern. This method is applied to several ground-state structures of bacteriorhodopsin, which residues already present complicated titration behaviors in the absence of a proton gradient. Our calculations show that a pH gradient across the membrane influences in a non-trivial manner the protonation probabilities of six titratable residues which are known to participate in the proton transfer: D85, D96, D115, E194, E204, and the Schiff base. The residues connected to one side of the membrane are influenced by the pH on the other side because of their long-range electrostatic interactions within the protein. In particular, D115 senses the pH at the cytoplasmic side of the membrane and transmits this information to D85 and the Schiff base. We propose that the strong electrostatic interactions found between D85, D115, and the Schiff base as well as the interplay of their respective protonation states under the influence of a transmembrane pH gradient are responsible for the back-pressure effect on bacteriorhodopsin.     

Specific DNA recognition by the type II restriction endonuclease MunI: the effect of pH.

To investigate the effect of pH on sequence-specific binding, a thermodynamic characterization of the interaction of the protein MunI with a specific, and a nonspecific, oligonucleotide was performed. MunI is a type II restriction endonuclease which is able to bind specifically, but loses its enzymatic activity in the absence of magnesium ions. Comparison of the specific and nonspecific interactions at 10 and 25 degrees C shows that the latter is accompanied by a small change in enthalpy, and a negligible change in constant pressure heat capacity. On going through the pH range 5.75-9.0 at 25 degrees C, the affinity of specific complex formation is reduced by 20-fold. The interaction is accompanied by the protonation of groups assumed to be on the protein. Based on the simplest model that will fit the data, two distinct protonation events are observed. At low pH, two groups per protein molecule undergo protonation with a pK(a) of 6.0 and 6.9 in the free and bound forms, respectively. At high pH, a further independent protonation occurs involving two groups with pK(a) values of 8.9 and approximately 10.7 in the free and bound forms, respectively. The change in heat capacity ranges from -2.7 to -1.7 kJ mol(-1) K(-1) in going from pH 6.5 to 8.5. This range of variation of change in heat capacity can be accounted for by the effects of protonation of the interacting molecules. The change in heat capacity, calculated from surface area burial using a previously established relationship (1.15 kJ mol(-1) K(-1)), does not correlate well with the experimentally determined values.     

Long-time molecular dynamics simulations of botulinum biotoxin type-A at different pH values and temperatures

Botulinum neurotoxins type A (BoNT/A) are highly potent toxins, but are also useful in the treatment of illnesses. We studied the properties of BoNT/A at various temperatures and pH values in order to understand its toxicity and structure variations. The pH values of the environment of BoNT/A are obtained by changing the protonation states of certain titratable residue groups. Our results show that certain parts of the protein are active at acidic pH environments or at high temperatures. The protein is more stable in neutral environments at normal human body temperature, whereas, at high temperature, the protein is more stable in acidic environments. Also, the three domains of the protein tend to have relative motion rather than within individual domains.     

The pH-induced release of iron from transferrin investigated with a continuum electrostatic model.

A reduction in pH induces the release of iron from transferrin in a process that involves a conformational change in the protein from a closed to an open form. Experimental evidence suggests that there must be changes in the protonation states of certain, as yet not clearly identified, residues in the protein accompanying this conformational change. Such changes in protonation states of residues and the consequent changes in electrostatic interactions are assumed to play a large part in the mechanism of release of iron from transferrin. Using the x-ray crystal structures of human ferri- and apo-lactoferrin, we calculated the pKa values of the titratable residues in both the closed (iron-loaded) and open (iron-free) conformations with a continuum electrostatic model. With the knowledge of a residue's pKa value, its most probable protonation state at any specified pH may be determined. The preliminary results presented here are in good agreement with the experimental observation that the binding of ferric iron and the synergistic anion bicarbonate/carbonate results in the release of approximately three H+ ions. It is suggested that the release of these three H+ ions may be accounted for, in most part, by the deprotonation of the bicarbonate and residues Tyr-92, Lys-243, Lys-282, and Lys-285 together with the protonation of residues Asp-217 and Lys-277.     

Simulating proteins at constant pH: An approach combining molecular dynamics and Monte Carlo simulation

For the structure and function of proteins, the pH of the solution is one of the determining parameters. Current molecular dynamics (MD) simulations account for the solution pH only in a limited way by keeping each titratable site in a chosen protonation state. We present an algorithm that generates trajectories at a Boltzmann distributed ensemble of protonation states by a combination of MD and Monte Carlo (MC) simulation. The algorithm is useful for pH-dependent structural studies and to investigate in detail the titration behavior of proteins. The method is tested on the acidic residues of the protein hen egg white lysozyme. It is shown that small structural changes may have a big effect on the pK(A) values of titratable residues.     

A spectroscopic study of the binding of N-7-substituted cap analogues to human protein synthesis initiation factor 4E

The binding of N-7-substituted cap analogues to eIF-4E from human erythrocytes is described. Data presented here indicate that there is a correlation between the tightness of binding of these cap analogues to eIF-4E and their potency as inhibitors of protein synthesis. This result indicates that the inhibitory activity of the cap analogues is strictly a function of the affinity of the analogue for eIF-4E under equilibrium conditions. The pH dependence of binding of the cap analogues to eIF-4E indicates that the enolate form of the cap is preferred, as originally postulated by Rhoads et al. [(1983) Biochemistry 22, 6084-6088]. Data indicate that there are differences in the mode of binding of alkyl-substituted and aryl-substituted cap analogues to eIF-4E arising from favorable interactions of the phenyl ring with the guanosine moiety. These differences may explain the enhanced recognition of the aryl-substituted cap analogues by eIF-4E.     

Calbindin D(9k): a protein optimized for calcium binding at neutral pH.

The binding of calcium ions by EF-hand proteins depends strongly on the electrostatic interactions between Ca(2+) ions and negatively charged residues of these proteins. We have investigated the pH dependence of the binding of Ca(2+) ions by calbindin D(9k). This protein offers a unique possibility for interpretation of such data since the pK(a) values of all ionizable groups are known. The binding is independent of pH between 7 and 9, where maximum calcium affinity is observed. An abrupt decrease in the binding affinity is observed at pH values below 7. This decrease is due to protonation of acidic groups, leading to modification of protein charges. The pH dependence of the product of the two macroscopic Ca(2+)-binding constants can be formally described by the involvement of two acidic groups with pK(a) = 6.6. Monte Carlo calculations show that the reduction of Ca(2+) binding is strictly determined by variable electrostatic interactions due to pH-dependent changes not only in the binding sites, but also of the overall charge of the protein.     

On the Hill plot of NMR data for titration of protein residues

The Hill plots of NMR titration data for protein residues disclose more clearly than the usual titration curves the presence of multiple weak perturbations originating from other titratable groups, and should be used whenever the conventional curve fitting is poor. For a quantitative interpretation, we derive here expressions for the Hill equation and the Hill coefficient when the titration of the observed group is perturbed by more than one titratable group. When the generalized Hill equation is fitted to the data, values of the interaction parameters between the observed group and the others are extracted provided that there are no mutual interactions between the latter groups. The method is applied to the titration data of two histidyl residues of l-arginine phosphotransferase (E.C. 2.7.3.3.) in the transition state analogue complex (enzyme-Mg²⁺-ADP-NOsk3/–l-Arg). From the Hill plots, interactions with three titratable groups are disclosed for both residues, and the fitting with the Hill equation reveals that they experience perturbations from the same three groups. Microscopic pK values are obtained for all the involved groups, indicating large changes (up to 3 pH units) upon protonation of the interacting groups. As compared to the conventional fitting procedure, the use and fitting of Hill plots yields from NMR data more information on the neighbourhood of enzyme residues and on the changes intervening therein through the steps involved in the catalysis.     

Electrostatic interactions in the acid denaturation of alpha-lactalbumin determined by NMR.

alpha-Lactalbumin (alpha-LA) undergoes a pH-dependent unfolding from the native state to a partially unfolded state (the molten globule state). To understand the role of electrostatic interactions in protein denaturation, NMR and CD pH titration experiments are performed on guinea pig alpha-LA. Variation of pH over the range of 7.0 to 2.0 simultaneously leads to the acid denaturation of the protein and the titration of individual ionizable groups. The pH titrations are interpreted in the context of these coupled events, and indicate that acid denaturation in alpha-LA is a cooperative event that is triggered by the protonation of two ionizable residues. Our NMR results suggest that the critical electrostatic interactions that contribute to the denaturation of alpha-LA are concentrated in the calcium binding region of the protein.     

Solution structures of purine base analogues 9-deazaguanine and 9-deazahypoxanthine

Deaza analogues of nucleobases are potential drugs against infectious diseases caused by parasites. A caveat is that apart from binding their target parasite enzymes, they also bind and inhibit enzymes of the host. In order to design derivatives of deaza analogues which specifically bind target enzymes, knowledge of their molecular structure, protonation state, and predominant tautomers at physiological conditions is essential. We have employed resonance Raman spectroscopy at an excitation wavelength of 260 nm, to decipher solution structure of 9-deazaguanine (9DAG) and 9-deazahypoxanthine (9DAH). These are analogues of guanine and hypoxanthine, respectively, and have been exploited to study static complexes of nucleobase binding enzymes. Such enzymes are known to perturb pK_a of their ligands, and thus, we also determined solution structures of these analogues at two, acidic and alkaline, pH. Structure of each possible protonation state and tautomer was computed using density functional theoretical calculations. Species at various pHs were identified based on isotopic shifts in experimental wavenumbers and by comparing these shifts with corresponding computed isotopic shifts. Our results show that at physiological pH, N1 of pyrimidine ring in 9DAG and 9DAH bears a proton. At lower pH, N3 is place of protonation, and at higher pH, deprotonation occurs at N1 position. The proton at N7 of purine ring remains intact even at pH 12.5. We have further compared these results with naturally occurring nucleotides. Our results identify key vibrational modes which can report on hydrogen bonding interactions, protonation and deprotonation in purine rings upon binding to the active site of enzymes.     

Assessing the acid-base and conformational properties of histidine residues in human prion protein (125-228) by means of pK(a) calculations and molecular dynamics simulations

A thorough study of the acid-base behavior of the four histidines and the other titratable residues of the structured domain of human prion protein (125-228) is presented. By using multi-tautomer electrostatic calculations, average titration curves have been built for all titratable residues, using the whole bundles of NMR structures determined at pH 4.5 and 7.0. According to our results, (1) only histidine residues are likely to be involved in the first steps of the pH-driven conformational transition of prion protein; (2) the pK(a)'s of His140 and His177 are approximately 7.0, whereas those of His155 and His187 are < 5.5. 10-ns long molecular dynamics simulations have been performed on five different models, corresponding to the most significant combinations of histidine protonation states. A critical comparison between the available NMR structures and our computational results (1) confirms that His155 and His187 are the residues whose protonation is involved in the conformational rearrangement of huPrP in mildly acidic condition, and (2) shows how their protonation leads to the destructuration of the C-terminal part of HB and to the loss of the last turn of HA that represent the crucial microscopic steps of the rearrangement.     

Estimating binding affinities by docking/scoring methods using variable protonation states

To investigate the effects of multiple protonation states on protein-ligand recognition, we generated alternative protonation states for selected titratable groups of ligands and receptors. The selection of states was based on the predicted pK(a) of the unbound receptor and ligand and the proximity of titratable groups of the receptor to the binding site. Various ligand tautomer states were also considered. An independent docking calculation was run for each state. Several protocols were examined: using an ensemble of all generated states of ligand and receptor, using only the most probable state of the unbound ligand/receptor, and using only the state giving the most favorable docking score. The accuracies of these approaches were compared, using a set of 176 protein-ligand complexes (15 receptors) for which crystal structures and measured binding affinities are available. The best agreement with experiment was obtained when ligand poses from experimental crystal structures were used. For 9 of 15 receptors, using an ensemble of all generated protonation states of the ligand and receptor gave the best correlation between calculated and measured affinities.     

Coupling of drug protonation to the specific binding of aminoglycosides to the A site of 16 S rRNA: elucidation of the number of drug amino groups involved and their identities

2-Deoxystreptamine (2-DOS) aminoglycoside antibiotics bind specifically to the central region of the 16S rRNA A site and interfere with protein synthesis. Recently, we have shown that the binding of 2-DOS aminoglycosides to an A site model RNA oligonucleotide is linked to the protonation of drug amino groups. Here, we extend these studies to define the number of amino groups involved as well as their identities. Specifically, we use pH-dependent 15N NMR spectroscopy to determine the pK(a) values of the amino groups in neomycin B, paromomycin I, and lividomycin A sulfate, with the resulting pK(a) values ranging from 6.92 to 9.51. For each drug, the 3-amino group was associated with the lowest pK(a), with this value being 6.92 in neomycin B, 7.07 in paromomycin I, and 7.24 in lividomycin A. In addition, we use buffer-dependent isothermal titration calorimetry (ITC) to determine the number of protons linked to the complexation of the three drugs with the A site model RNA oligomer at pH 5.5, 8.8, or 9.0. At pH 5.5, the binding of the three drugs to the host RNA is independent of drug protonation effects. By contrast, at pH 9.0, the RNA binding of paromomycin I and neomycin B is coupled to the uptake of 3.25 and 3.80 protons, respectively, with the RNA binding of lividomycin A at pH 8.8 being coupled to the uptake of 3.25 protons. A comparison of these values with the protonation states of the drugs predicted by our NMR-derived pK(a) values allows us to identify the specific drug amino groups whose protonation is linked to complexation with the host RNA. These determinations reveal that the binding of lividomycin A to the host RNA is coupled to the protonation of all five of its amino groups, with the RNA binding of paromomycin I and neomycin B being linked to the protonation of four and at least five amino groups, respectively. For paromomycin I, the protonation reactions involve the 1-, 3-, 2'-, and 2"'-amino groups, while, for neomycin B, the binding-linked protonation reactions involve at least the 1-, 3-, 2', 6'-, and 2"'-amino groups. Our results clearly identify drug protonation reactions as important thermodynamic participants in the specific binding of 2-DOS aminoglycosides to the A site of 16S rRNA.     

Calcium-binding properties of cardiac and skeletal troponin C as determined by circular dichroism and ultraviolet difference spectroscopy.

Calcium titration of the conformational change in cardiac and skeletal troponin C (TN-C) was followed by circular dichroism (CD) at pH values in the range from 5.2 to 7.4. Computer analysis was used to resolve the contributions from the different classes of Ca2+ -binding sites. At pH 6.94 in skeletal TN-C, apparent affinity constants for calcium of 1.8 x 10(7) and 4.5 x 10(5) M-1 were determined for the two classes of binding sites. The more sophisticated computer analysis of the data has revealed a substantial CD contribution from the low-affinity sites (approximately 30% of the high affinity contribution at pH 6.94) and suggests that skeletal TN-C with Ca2+ bound at the low-affinity sites is in a different conformation from that when just the high-affinity sites are occupied, in agreement with a recent nuclear magnetic resonance (NMR) study on this system (Seaman, K. B., Hartshorne, D. J. & Bothener-By, A. A. (1977) Biochemistry 16,4039-4046). With the cardiac protein at pH 7.07, an apparent affinity constant for calcium of 2.0 x 10(7) M-1 was calculated while no low-affinity site at this pH was detected by CD. On the other hand, at lower pH values, such as 6.05, a CD contribution from the cardiac low-affinity Ca2+ -binding site is detected with an apparent binding constant of 3.7 +/- 0.7 x 10(4) M-1. At the lower pH values, protonation of a class of carboxyl groups in each protein which possesses a high pKa (6.2-6.3) elicits the conformational change at the high-affinity sites with a corresponding decrease in the overall magnitude of the Ca2+ -evoked changes. The expression of a conformational change upon Ca2+ binding at the level of the low-affinity sites is enchanced by protonation of a class of carboxyls with a pKa of 6.3 in cardiac TN-C and 6.7-6.8 with the skeletal homologue. In both cases, this contribution is reduced upon protonation of carboxyls with pKa less than or equal to 5.5. It was also observed that the low-affinity sites of skeletal TN-C have a much larger role to play in the total conformational change than the low-affinity sites of cardiac TN-C, a finding probably related to the inability of site 1 in the cardiac protein to bind calcium. In the cardiac protein, the Ca2+ -induced tyrosine difference-spectrum maximum is reduced from deltaepsilonM,287nm =330M-1.cm-1 to 20M-1.cm-1 by protonation of a class of groups with a pKa of 6.4, presumably the same carboxyl groups as those invoved in the CD conformational contribution from the high-affinity binding sites. No such effect was observed for the skeletal protein where deltaepsilonM,287nm was constant at 110M-1 .cm-1 over the pH range studied. The dramatic alterations in the tyrosine environment of cardiac TN-C with pH are attributed to either or both of the tyrosines located in the two high-affinity Ca2+ -binding sites (sites 3 and 4)...