Protonation States Of Residues Research Articles

Phbuilder: A Tool for Efficiently Setting up Constant pH Molecular Dynamics Simulations in GROMACS.

Constant pH molecular dynamics (MD) is a powerful technique that allows the protonation state of residues to change dynamically, thereby enabling the study of pH dependence in a manner that has not been possible before. Recently, a constant pH implementation was incorporated into the GROMACS MD package. Although this implementation provides good accuracy and performance, manual modification and the preparation of simulation input files are required, which can be complicated, tedious, and prone to errors. To simplify and automate the setup process, we present phbuilder, a tool that automatically prepares constant pH MD simulations for GROMACS by modifying the input structure and topology as well as generating the necessary parameter files. phbuilder can prepare constant pH simulations from both initial structures and existing simulation systems, and it also provides functionality for performing titrations and single-site parametrizations of new titratable group types. The tool is freely available at www.gitlab.com/gromacs-constantph. We anticipate that phbuilder will make constant pH simulations easier to set up, thereby making them more accessible to the GROMACS user community.

Characterizing Protein Protonation Microstates Using Monte Carlo Sampling.

Proteins are polyelectrolytes with acidic and basic amino acids Asp, Glu, Arg, Lys, and His, making up ≈25% of the residues. The protonation state of residues, cofactors, and ligands defines a "protonation microstate". In an ensemble of proteins some residues will be ionized and others neutral, leading to a mixture of protonation microstates rather than in a single one as is often assumed. The microstate distribution changes with pH. The protein environment also modifies residue proton affinity so microstate distributions change in different reaction intermediates or as ligands are bound. Particular protonation microstates may be required for function, while others exist simply because there are many states with similar energy. Here, the protonation microstates generated in Monte Carlo sampling in MCCE are characterized in HEW lysozyme as a function of pH and bacterial photosynthetic reaction centers (RCs) in different reaction intermediates. The lowest energy and highest probability microstates are compared. The ΔG, ΔH, and ΔS between the four protonation states of Glu35 and Asp52 in lysozyme are shown to be calculated with reasonable precision. At pH 7 the lysozyme charge ranges from 6 to 10, with 24 accepted protonation microstates, while RCs have ≈50,000. A weighted Pearson correlation analysis shows coupling between residue protonation states in RCs and how they change when the quinone in the QB site is reduced. Protonation microstates can be used to define input MD parameters and provide insight into the motion of protons coupled to reactions.

Electrostatic Perturbations in the Substrate-Binding Pocket of Taurine/α-Ketoglutarate Dioxygenase Determine its Selectivity.

Taurine/α‐ketoglutarate dioxygenase is an important enzyme that takes part in the cysteine catabolism process in the human body and selectively hydroxylates taurine at the C1‐position. Recent computational studies showed that in the gas‐phase the C2−H bond of taurine is substantially weaker than the C1−H bond, yet no evidence exists of 2‐hydroxytaurine products. To this end, a detailed computational study on the selectivity patterns in TauD was performed. The calculations show that the second‐coordination sphere and the protonation states of residues play a major role in guiding the enzyme to the right selectivity. Specifically, a single proton on an active site histidine residue can change the regioselectivity of the reaction through its electrostatic perturbations in the active site and effectively changes the C1−H and C2−H bond strengths of taurine. This is further emphasized by many polar and hydrogen bonding interactions of the protein cage in TauD with the substrate and the oxidant that weaken the pro‐R C1−H bond and triggers a chemoselective reaction process. The large cluster models reproduce the experimental free energy of activation excellently.

Protonation states of central amino acids in a zinc metalloprotease complexed with inhibitor: Molecular mechanics optimizations and ab initio molecular orbital calculations

The zinc-metalloprotease pseudolysin (PLN) secreted from bacteria degrades extracellular proteins to produce bacterial nutrition. Since PLN has a Zn ion at the inhibitor-binding site, the interactions between Zn and PLN residues as well as inhibitor can be significantly changed depending on the protonation states of PLN residues at the inhibitor-binding site. To determine stable protonation states of these residues, we here considered different protonation states for Glu and His residues located around Zn and investigated the electronic states of the PLN + inhibitor complex, using ab initio molecular simulations. The protonation state of His223 was found to significantly affect the specific interactions between PLN and the inhibitor.

Exploring the Mechanism of Action of Membrane Fusion Protein IncA Using Molecular Dynamics Simulations

Infecting millions of people each year, Chlamydia trachomatis is currently the leading cause of non‐congenital blindness and pelvic inflammatory disease. Incapable of replicating on its own, this bacterium survives by entering a host cell through endocytosis and subsequently modifying the vacuole in which it is enclosed to allow for replication. The newly formed replicative compartment, referred to as an inclusion, has numerous inclusion membrane proteins (Inc proteins) that promote growth and maintenance of the developing C. trachomatis bodies. One Inc in particular, known as IncA, exponentially enhances growth of C. trachomatis by initiating the homotypic fusion of multiple inclusions within a host cell. Although the structure and significance of IncA are known, its mechanism of action remains a mystery. The present research aims to elucidate the structure‐function relationship of IncA; offering insight into fusion protein functionality and potential targets for drug development. Analysis of IncA was accomplished by running Constant pH Molecular Dynamics (CpH MD) simulations with a refined focus on aspartic acids, glutamic acids, and histidines. Similar to conventional MD, CpH MD creates conditions that mimic the natural environment of IncA but with the additional capability to explore protonation states of residues. By running the CpH MD system across a range of pHs, the necessary data are generated to determine dissociation constants for specific residues. Combining proton dissociation with tests examining the solvent accessibility of IncA residues, results suggested that there is a correlation between accessibility of a residue and its proton dissociation. Further analysis of residue fluctuation at varying pHs shows that there appears to be a relationship between a residue's protonation state and the degree to which that residue fluctuated throughout a simulation. These findings demonstrate that the location of titratable residues influence the activity of IncA and furthermore, that IncA's titratable residues affect inclusion membrane fusion.Support or Funding InformationNational Science Foundation, Grant No. 1560325.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of a pH Gradient on the Protonation States of Cytochrome c Oxidase: A Continuum Electrostatics Study.

Cytochrome c oxidase (CcO) couples the reduction of dioxygen to water with transmembrane proton pumping, which leads to the generation of an electrochemical gradient. In this study we analyze how one of the components of the electrochemical gradient, the difference in pH across the membrane, or ΔpH, influences the protonation states of residues in CcO. We modified our continuum electrostatics/Monte Carlo (CE/MC) method in order to include the ΔpH and applied it to the study of CcO, in what is, to our best knowledge, the first CE/MC study of CcO in the presence of a pH gradient. The inclusion of a transmembrane pH gradient allows for the identification of residues whose titration behavior depends on the pH on both sides of the membrane. Among the several residues with unusual titration profiles, three are well-known key residues in the proton transfer process of CcO: E286I, Y288I, and K362I. All three residues have been previously identified as being critical for the catalytic or proton pumping functions of CcO. Our results suggest that when the pH gradient increases, these residues may be part of a regulatory mechanism to stem the proton flow.

The Increasingly Complex Mechanism of HMG-CoA Reductase

HMG-CoA reductase (HMGR) is the target of statins, cholesterol-lowering drugs prescribed to millions of patients worldwide. More recent research indicates that HMGR could be a useful target in the development of antimicrobial agents. Over the last seven decades, researchers have proposed a series of increasingly complex reaction mechanisms for this biomedically important enzyme. The maturation of the mechanistic proposals for HMGR have paralleled advances in a diverse set of research areas, such as molecular biology and computational chemistry. Thus, the development of the HMGR mechanism provides a useful case study for following the advances in state-of-the-art methods in enzyme mechanism research. Similarly, the questions raised by these mechanism proposals reflect the limitations of the methods used to develop them. The mechanism of HMGR, a four-electron oxidoreductase, is unique and far more complex than originally thought. The reaction contains multiple chemical steps, coupled to large-scale domain motions of the homodimeric enzyme. The first proposals for the HMGR mechanism were based on kinetic and labeling experiments, drawing analogies to the mechanism of known dehydrogenases. Advances in molecular biology and bioinformatics enabled researchers to use site-directed mutagenesis experiments and protein sequencing to identify catalytically important glutamate, aspartate, and histidine residues. These studies, in turn, have generated new and more complicated mechanistic proposals. With the development of protein crystallography, researchers solved HMGR crystal structures to reveal an unexpected lysine residue at the center of the active site. The many crystal structures of HMGR led to increasingly complex mechanistic proposals, but the inherent limitations of the protein crystallography left a number of questions unresolved. For example, the protonation state of the glutamate residue within the active site cannot be clearly determined from the crystal structure. The differing protonation state of this residue leads to different proposed mechanisms for the enzyme. As computational analysis of large biomolecules has become more feasible, the application of methods such as hybrid quantum mechanics/molecular mechanics (QM/MM) calculations to the HMGR mechanism have led to the most detailed mechanistic proposal yet. As these methodologies continue to improve, they prove to be very powerful for the study of enzyme mechanisms in conjunction with protein crystallography. Nevertheless, even the most current mechanistic proposal for HMGR remains incomplete due to limitations of the current computational methodologies. Thus, HMGR serves as a model for how the combination of increasingly sophisticated experimental and computational methods can elucidate very complex enzyme mechanisms.

Computational Study of Absorption Spectra of the Photoconvertible Fluorescent Protein EosFP in Different Protonation States.

Absorption spectra of the green-to-red convertible fluorescent protein EosFP have been computed in a hybrid quantum mechanical/molecular mechanical (QM/MM) framework. The experimentally observed absorption maximum at ∼390 nm is well reproduced by the protein with a neutral chromophore, and the anionic form is computed to absorb close to the experimentally determined maximum at ∼500 nm. Absorption of a zwitterionic form is calculated to lie in the same spectral region; however, this species cannot be unambiguously assigned to the experimental spectra. Variation of the protonation states of residues surrounding the chromophore do not have significant impact on the positions of the absorption maxima. In particular, protonation of Glu212 leaves the calculated spectra largely unaffected. This is consistent with the spectra of the E212Q mutant, which differ from the wild-type spectra only in the intensities but not in the positions of the absorption bands.

Charge Requirements for Proton Gradient-driven Translocation of Anthrax Toxin

Anthrax lethal toxin is used as a model system to study protein translocation. The toxin is composed of a translocase channel, called protective antigen (PA), and an enzyme, called lethal factor (LF). A proton gradient (ΔpH) can drive LF unfolding and translocation through PA channels; however, the mechanism of ΔpH-mediated force generation, substrate unfolding, and establishment of directionality are poorly understood. One recent hypothesis suggests that the ΔpH may act through changes in the protonation state of residues in the substrate. Here we report the charge requirements of LF's amino-terminal binding domain (LF(N)) using planar lipid bilayer electrophysiology. We found that acidic residues are required in LF(N) to utilize a proton gradient for translocation. Constructs lacking negative charges in the unstructured presequence of LF(N) translocate independently of the ΔpH driving force. Acidic residues markedly increase the rate of ΔpH-driven translocation, and the presequence is optimized in its natural acidic residue content for efficient ΔpH-driven unfolding and translocation. We discuss a ΔpH-driven charge state Brownian ratchet mechanism for translocation, where glutamic and aspartic acid residues in the substrate are the "molecular teeth" of the ratchet. Our Brownian ratchet model includes a mechanism for unfolding and a novel role for positive charges, which we propose chaperone negative charges through the PA channel during ΔpH translocation.

Microscopic pKa Analysis of Glu286 in Cytochrome c Oxidase (Rhodobacter sphaeroides): Toward a Calibrated Molecular Model

As stringent tests for the molecular model and computational protocol, microscopic pK(a) calculations are performed for the key residue, Glu286, in cytochrome c oxidase (CcO) using a combined quantum mechanical/molecular mechanical (QM/MM) potential and a thermodynamic integration protocol. The impact of the number of water molecules in the hydrophobic cavity and protonation state of several key residues (e.g., His334, Cu(B)-bound water, and PRD(a3)) on the computed microscopic pK(a) values of Glu286 has been systematically examined. To help evaluate the systematic errors in the QM/MM-based protocol, microscopic pK(a) calculations have also been carried out for sites in a soluble protein (Asp70 in T4 lysozyme) and a better-characterized membrane protein (Asp85 in bacteriorhodopsin). Overall, the results show a significant degree of internal consistency and reproducibility that support the effectiveness of the computational framework. Although the number of water molecules in the hydrophobic cavity does not greatly influence the computed pK(a) of Glu286, the protonation states of several residues, some of which are rather far away, have more significant impacts. Adopting the standard protonation state for all titratable residues leaves a large net charge on the system and a significantly elevated pK(a) for Glu286, highlighting that any attempt to address the energetics of proton transfers in CcO at a microscopic level should carefully select the protonation state of residues, even those not in the immediate neighborhood of the active site. The calculations indirectly argue against the deprotonation of His334 for the proton pumping process, although further studies that explicitly compute its pK(a) are required for a more conclusive statement. Finally, the deprotonated Glu286 is found to be in a stable water-mediated connection with PRD(a3) for at least several nanoseconds when this presumed pumping site is protonated. This does not support the proposed role of Glu286 as a robust gating valve that prevents proton leakage, although a conclusive statement awaits a more elaborate characterization of the Glu286-PRD(a3) connectivity with free energy simulations and a protonated PRD(a3). The large sets of microscopic simulations performed here have provided useful guidance to the establishment of a meaningful molecular model and effective computational protocol for explicitly analyzing the proton transfer kinetics in CcO, which is required for answering key questions regarding the pumping function of this fascinating and complex system.

Characterization of mutations in crucial residues around the Qo binding site of the cytochrome bc1 complex from Paracoccus denitrificans

The protonation state of residues around the Q(o) binding site of the cytochrome bc(1) complex from Paracoccus denitrificans and their interaction with bound quinone(s) was studied by a combined electrochemical and FTIR difference spectroscopic approach. Site-directed mutations of two groups of conserved residues were investigated: (a) acidic side chains located close to the surface and thought to participate in a water chain leading up to the heme b(L) edge, and (b) residues located in the vicinity of this site. Interestingly, most of the mutants retain a high degree of catalytic activity. E295Q, E81Q and Y297F showed reduced stigmatellin affinity. On the basis of electrochemically induced FTIR difference spectra, we suggest that E295 and D278 are protonated in the oxidized form or that their mutation perturbs protonated residues. Mutations Y302, Y297, E81 and E295, directly perturb signals from the oxidized quinone and of the protein backbone. By monitoring the interaction with the inhibitor stigmatellin for the wild-type enzyme at various redox states, interactions of the bound stigmatellin with amino acid side chains such as protonated acidic residues and the backbone were observed, as well as difference signals arising from the redox active inhibitor itself and the replaced quinone. The infrared difference spectra of the above Q(o) site mutations in the presence of stigmatellin confirm the previously established role of E295 as a direct interaction partner in the enzyme from P.denitrificans as well. The protonated residue E295 is proposed to change the hydrogen-bonding environment upon stigmatellin binding in the oxidized form, and is deprotonated in the reduced form. Of the residues located close to the surface, D278 remains protonated and unperturbed in the oxidized form but its frequency shifts in the reduced form. The mechanistic implications of our observations are discussed, together with previous inhibitor binding data, and referred to the published X-ray structures.

Complexity in Modeling and Understanding Protonation States: Computational Titration of HIV‐1‐Protease–Inhibitor Complexes

The computational-titration (CT) algorithm based on the 'natural' Hydropathic INTeractions (HINT) force field is described. The HINT software model is an empirical, non-Newtonian force field derived from experimentally measured partition coefficients for solvent transfer between octanol and H(2)O (log P(o/w)). The CT algorithm allows the identification, modeling, and optimization of multiple protonation states of residues and ligand functional groups at the protein-ligand active site. The importance of taking into account pH and ionization states of residues, which strongly affect the process of ligand binding, for correctly predicting binding free energies is discussed. The application of the CT protocol to a set of six cyclic inhibitors in their complexes with HIV-1 protease is presented, and the advance of HINT as a virtual-screening tool is outlined.

Influence of the Protein Environment on the Redox Potentials of Flavodoxins from Clostridium beijerinckii

The flavin mononucleotide (FMN) quinones in flavodoxin have two characteristic redox potentials, namely, Em(FMNH./FMNH-) for the one-electron reduction of the protonated FMN (E1) and Em(FMN/FMNH.) for the proton-coupled one-electron reduction (E2). These redox potentials in native and mutant flavodoxins obtained from Clostridium beijerinckii were calculated by considering the protonation states of all titratable sites as well as the energy contributed at the pKa value of FMN during protonation at the N5 nitrogen (pKa(N5)). E1 is sensitive to the subtle differences in the protein environments in the proximity of FMN. The protein dielectric volume that prevents the solvation of charged FMN quinones is responsible for the downshift of 130-160 mV of the E1 values with respect to that in an aqueous solution. The influence of the negatively charged 5'-phosphate group of FMN quinone on E1 could result in a maximum shift of 90 mV. A dramatic difference of 130 mV in the calculated E2 values of FMN quinone of the native and G57T mutant flavodoxins is due to the difference in the pKa(N5) values. This is due to the difference in the influence exerted by the carbonyl group of the protein backbone at residue 57.

Multiconformation continuum electrostatics analysis of the NhaA Na + /H + antiporter of Escherichia coli with functional implications

Sodium proton antiporters are essential enzymes that catalyze the exchange of sodium ions for protons across biological membranes. Protonations and deprotonations of individual amino acid residues and of clusters formed by these residues play an important role in activating these enzymes and in the mechanism of transport. We have used multiconformation continuum electrostatics method to investigate the protonation states of residues in the sodium proton exchanger NhaA from Escherichia coli, the structure of which has been determined recently by x-ray crystallography. Our calculations identify four clusters of electrostatically tightly interacting residues as well as long-range interactions between residues required for activation. The importance of many of these residues has been demonstrated by the characterization of site-directed mutants. A number of residues with extreme pKa values, including several of the "pH sensor," can only undergo protonation/deprotonation reactions subsequent to conformational changes. The results of the calculations provide valuable information on the activation of the antiporter and the role of individual amino acid residues, and provide a solid framework for further experiments.

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 pK a 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 pK a 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.

Perturbed interaction between residues 85 and 204 in Tyr-185-->Phe and Asp-85-->Glu bacteriorhodopsins

According to earlier reports, residue 85 in the bacteriorhodopsin mutants D85E and Y185F deprotonates with two apparent pKa values. Additionally, in Y185F, Asp-85 becomes significantly more protonated during light adaptation. We provide a new explanation for these findings. It is based on the scheme that links the protonation state of residue 85 to the protonation state of residue 204 (S.P. Balashov, E.S. Imasheva, R. Govindjee, and T.G. Ebrey. 1996. Biophys. J. 70:473-481; H.T. Richter, L.S. Brown, R. Needleman, and J.K. Lanyi. 1996. Biochemistry. 35:4054-4062) and justified by the observation that the biphasic titration curves of D85E and Y185F are converted to monophasic when the E204Q residue change is introduced as a second mutation. Accordingly, the D85E and Y 185F mutations are not the cause of the biphasic titration, as that is a property of the wild-type protein. By perturbing the extracellular region of the protein, the mutations increase the pKa of residue 85. This increases the amplitude of the second titration component and makes the biphasic character of the curves more obvious. Likewise, a small rise in the pKa of Asp-85 when the retinal isomerizes from 13-cis, 15-syn to all-trans accounts for the changed titration behavior of Y185F after light adaptation. This mechanism simplifies and unites the interpretation of what had appeared to be complex and unrelated phenomena.

Electrostatic analysis of TEM1 beta-lactamase: effect of substrate binding, steep potential gradients and consequences of site-directed mutations.

Escherichia coli TEM1 is a penicillinase and belongs to class A beta-lactamases. Its naturally occurring mutants are responsible for bacterial resistance to beta-lactamin-based antibiotics. X-ray structure determinations show that all class A beta-lactamases are similar, but, despite the numerous kinetic investigations, the reaction mechanism of these enzymes is still debated. We address the questions of what the molecular contexts during the acylation and deacylation steps are and how they contribute to the efficiency of these penicillinases. Electrostatic analysis of the 1.8 A resolution refined X-ray structure of the wild-type enzyme, and of its modelled Michaelis and acyl-enzyme complexes, showed that substrate binding induces an upward shift in the pKa of the unprotonated Lys73 by 6.4 pH units. The amine group of Lys73 can then abstract the Ser70 hydroxyl group proton and promote acylation. In the acyl-enzyme complex, the deacylating water is situated between the carboxylate group of Glu166, within the enzyme, and the estercarbonyl carbon of the acyl-enzyme complex, in an electrostatic potential gradient amounting to 30 kTe-1 A-1. Other residues, not directly involved in catalysis, also contribute to the formation of this gradient. The deacylation rate is related to the magnitude of the gradient. The kinetic behavior of site-directed mutants that affect the protonation state of residue 73 cannot be explained on the basis of the wild-type enzyme mechanism. In the wild-type enzyme, the very high rates of acylation and deacylation of class A beta-lactamases arise from an optimal chemical setup in which the acylation reaction seems triggered by substrate binding that changes the general base property of Lys73. In site-directed mutants where Lys73 is protonated, acylation may proceed through activation of a water molecule by Glu166, and Lys73 contributes as a proton shuffle partner in this pathway.