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An aqua ligand bridges metal cations in a wide variety of enzymes, many of which are drug targets for various diseases. However, the factors affecting its protonation state and thus biological roles remain elusive. By computing the free energy for replacing the bridging H2O by OH- in various model Mg2+ sites, we have evaluated how the nature of an aqua bridge depends on the site's net charge (i.e., the number of charged ligands in the first and second shell and the number of metal cations), the site's solvent exposure, the ligand's charge-donating ability, the bridging oxygen's hydrogen-bonding interactions, intramolecular proton transfer from the bridging H2O to a nearby carboxylate, and the metal coordination number. The results reveal the key factors dictating the protonation state of bridging H2O and provide guidelines in predicting whether H2O or OH- bridges two Mg2+ in polynuclear sites. This helps to elucidate the nucleophile in the enzyme-catalyzed reaction and the net charge of the metal complex (metal cation and first-shell ligands), which plays a critical role in binding. [ABSTRACT FROM AUTHOR]

Amphitropic proteins, such as the virulence factor phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis, often depend on lipid-specific recognition of target membranes. However, the recognition mechanisms for zwitterionic lipids, such as phosphatidylcholine, which is enriched in the outer leaflet of eukaryotic cells, are not well understood. A 500 ns long molecular dynamics simulation of PI-PLC at the surface of a lipid bilayer revealed a strikingly high number of interactions between tyrosines at the interfacial binding site and lipid choline groups with structures characteristic of cation-π interactions. Membrane affinities of PI-PLC tyrosine variants mostly tracked the simulation results, falling into two classes: (i) those with minor losses in affinity, Kd(mutant)/Kd(wild-type) ≤ 5 and (ii) those where the apparent Kd was 50-200 times higher than wild-type. Estimating ΔΔG for these Tyr/PC interactions from the apparent Kd values reveals that the free energy associated with class I is ~1 kcal/mol, comparable to the value predicted by the Wimley–White hydrophobicity scale. In contrast, removal of class II tyrosines has a higher energy cost: ~2.5 kcal/mol toward pure PC vesicles. These higher energies correlate well with the occupancy of the cation-π adducts throughout the MD simulation. Together, these results strongly indicate that PI-PLC interacts with PC headgroups via cation-π interactions with tyrosine residues and suggest that cation-π interactions at the interface may be a mechanism for specific lipid recognition by amphitropic and membrane proteins. [ABSTRACT FROM AUTHOR]

Strontium (Sr), an alkali metal with properties similar to calcium, in the form of soluble salts is used to treat osteoporosis. Despite the information accumulated on the role of Sr 2+ as a Ca 2+ mimetic in biology and medicine, there is no systematic study of how the outcome of the competition between the two dications depends on the physicochemical properties of (i) the metal ions, (ii) the first- and second-shell ligands, and (iii) the protein matrix. Specifically, the key features of a Ca 2+ -binding protein that enable Sr 2+ to displace Ca 2+ remain unclear. To address this, we studied the competition between Ca 2+ and Sr 2+ in protein Ca 2+ -binding sites using density functional theory combined with the polarizable continuum model. Our findings indicate that Ca 2+ -sites with multiple strong charge-donating protein ligands, including one or more bidentately bound Asp - /Glu - that are relatively buried and rigid are protected against Sr 2+ attack. On the other hand, Ca 2+ -sites crowded with multiple protein ligands may be prone to Sr 2+ displacement if they are solvent-exposed and flexible enough so that an extra backbone ligand from the outer shell can bind to Sr 2+ . In addition, solvent-exposed Ca 2+ sites with only a few weak charge-donating ligands that can rearrange to fit the strontium's coordination requirements are susceptible to Sr 2+ displacement. We provide the physical basis of these results and discuss potential novel protein targets of therapeutic Sr 2+ .

Because Li + and Ca 2+ differ in both charge and size, the possibility that monovalent Li + could dislodge the bulkier, divalent Ca 2+ in Ca 2+ proteins had not been considered. However, our recent density functional theory/continuum dielectric calculations predicted that Li + could displace the native Ca 2+ from the C2 domain of cytosolic PKCα/γ. This would reduce electrostatic interactions between the Li + -bound C2 domain and the membrane, consistent with experimental studies showing that Li + can inhibit the translocation of cytoplasmic PKC to membranes. Besides the trinuclear Ca 2+ -site in the PKCα/γ C2 domain, it is not known whether other Ca 2+ -sites in human proteins may be susceptible to Li + substitution. Furthermore, it is unclear what factors determine the outcome of the competition between divalent Ca 2+ and monovalent Li + . Here we show that the net charge of residues in the first and second coordination shell is a key determinant of the selectivity for divalent Ca 2+ over monovalent Li + in proteins: neutral/anionic Ca 2+ -carboxylate sites are protected against Li + attack. They are further protected by outer-shell Asp - /Glu - and the protein matrix rigidifying the Ca 2+ -site or limiting water entry. In contrast, buried, cationic Ca 2+ -sites surrounded by Arg + /Lys + , which are found in the C2 domains of PKCα/γ, as well as certain synaptotagmins, are prone to Li + attack.

Di/triphosphates perform a multitude of essential tasks, being important components of many vital organic cofactors such as adenosine/guanosine di/triphosphate (ADP/GDP, ATP/GTP), flavin adenine dinucleotide, and nicotinamide adenine dinucleotide and its phosphate derivative. They are generally bound to cations inside cells, in particular Mg 2+ in the case of ATP/GTP. Yet how their metal-binding modes depend on the number, charge, and solvent exposure of the polyphosphate group and how Mg 2+ and Ca 2+ dications that coexist in cellular fluids compete for di/triphosphates in biological systems remain elusive. Using density functional theory calculations combined with a polarizable continuum model, we have determined the relative free energies and stabilities of the different binding modes of di- and triphosphate groups to Mg 2+ and Ca 2+ . We show that the thermodynamic outcome of the competition between Mg 2+ and Ca 2+ for cellular di/triphosphates depends mainly on the oligomericity/charge and metal-binding mode of the phosphate ligand as well as the solvent exposure of the binding site. Increasing the charge and thus denticity of the phosphate ligand from bi- to tridentate in a buried binding pocket enhances the affinity of the host system for the stronger charge acceptor, Mg 2+ . The cellular di/triphosphates's intrinsic properties and the protein matrix allowing them to bind a dication bi/tridentately, along with the higher cytosolic concentration of Mg 2+ compared to Ca 2+ , enables Mg 2+ to outcompete Ca 2+ in binding to these highly charged anions. This suggests an explanation for why nature has chosen Mg 2+ but not Ca 2+ to perform most of the essential tasks associated with biological triphosphates.

In Zn-proteins, structural Zn-sites are mostly Cys-rich lined by two or more Cys residues, whereas catalytic Zn-sites usually contain His or Asp/Glu residues and a water molecule. Here, we reveal many examples outside this trend with Zn 2+ bound to ligands commonly found in both structural and catalytic Zn-sites, namely, Zn-CC(C/H) x ( x = D, E, or H 2 O) sites. We show that these atypical Zn-sites are found in all known life forms (i.e., eukaryotes, bacteria, archaea, and viruses) and can serve structural roles in some proteins but catalytic roles in others. By calculating the physical properties of these atypical Zn-binding sites, we elucidate why Zn-CC(C/H) x sites of the same composition can serve structural and catalytic roles in proteins. Furthermore, we found new sequence/structural motifs characteristic of catalytic Zn-CCHw sites and provide guidelines to predict the structural/catalytic role of atypical Zn-CC(C/H) x sites of unknown function. We discuss how our results could help to design inhibitors targeting catalytic Zn-CC(C/H) H 2 O sites.

At a given pH, whether a metal-bound water molecule is deprotonated or not can be determined if the pKa of the metal-bound water molecule (denoted pKw) is known. Although protocols/tools to predict the protonation states of titratable amino acid residues and small molecules have been developed, an efficient and accurate method to predict the absolute pKw values of metal complexes is lacking. Here, we present calibrated methods for optimizing the geometries and computing the absolute pKw values of a wide range of Zn2+-complexes containing protein-like ligating groups. We tested 18 different geometry-optimization methods on 19 ultra high-resolution structures of Zn2+ complexes of varying coordination numbers and ligating atoms and 98 methods in reproducing 36 experimental pKw values of diverse Zn2+ complexes in the absence and presence of explicit water molecules. The results underscore the importance of estimating the Zn2+-bound water/hydroxide solvation properly, whereas correcting for the basis set superposition error was not found to be important. The protocol presented can be used to (i) evaluate the geometries of the different Zn2+-sites found in proteins and (ii) to dissect the individual contributions of the various factors modulating the pKw in Zn2+-sites found in proteins. Predicting absolute pKw values in various environments with efficiency and accuracy will indicate when a Zn2+-bound water molecule is deprotonated, thus providing physical insight into the mechanisms of enzyme-catalyzed reactions and the design of drug candidates that can displace a metal-bound water molecule.

Abiogenic lead (Pb 2+ ), present in the environment in elevated levels due to human activities, has detrimental effects on human health. Metal-binding sites in proteins have been identified as primary targets for lead substitution resulting in malfunction of the host protein. Although Pb 2+ is known to be a potent competitor of Ca 2+ in protein binding sites, why/how Pb 2+ can compete with Ca 2+ in proteins remains unclear, raising multiple outstanding questions, including the following: (1) What are the physicochemical factors governing the competition between Pb 2+ and Ca 2+ ? (2) Which Ca 2+ -binding sites in terms of the structure, composition, overall charge, flexibility, and solvent exposure are the most likely targets for Pb 2+ attack? Using density functional theory combined with polarizable continuum model calculations, we address these questions by studying the thermodynamic outcome of the competition between Pb 2+ and Ca 2+ in various model Ca 2+ -binding sites, including those modeling voltage-gated calcium channel selectivity filters and EF-hand and non-EF-hand Ca 2+ -binding sites. The results, which are in good agreement with experiment, reveal that the metal site's flexibility and number of amino acid ligands dictate the outcome of the competition between Pb 2+ and Ca 2+ : If the Ca 2+ -binding site is relatively rigid and crowded with protein ligands, then Pb 2+ , upon binding, preserves the native metal-binding site geometry and at low concentrations, can act as an activator of the host protein. If the Ca 2+ -binding site is flexible and consists of only a few protein ligands, then Pb 2+ can displace Ca 2+ and deform the native metal-binding site geometry, resulting in protein malfunction.

Cation-π interactions between tyrosine amino acids and compounds containing N,N,N-trimethylethanolammonium (N(CH 3 ) 3 ) are involved in the recognition of histone tails by chromodomains and in the recognition of phosphatidylcholine (PC) phospholipids by membrane-binding proteins. Yet, the lack of explicit polarization or charge transfer effects in molecular mechanics force fields raises questions about the reliability of the representation of these interactions in biomolecular simulations. Here, we investigate the nature of phenol-tetramethylammonium (TMA) interactions using quantum mechanical (QM) calculations, which we also use to evaluate the accuracy of the additive CHARMM36 and Drude polarizable force fields in modeling tyrosine-choline interactions. We show that the potential energy surface (PES) obtained using SAPT2+/aug-cc-pVDZ compares well with the large basis-set CCSD(T) PES when TMA approaches the phenol ring perpendicularly. Furthermore, the SAPT energy decomposition reveals comparable contributions from electrostatics and dispersion in phenol-TMA interactions. We then compared the SAPT2+/aug-cc-pVDZ PES obtained along various approach directions to the corresponding PES obtained with CHARMM, and we show that the force field accurately reproduces the minimum distances while the interaction energies are underestimated. The use of the Drude polarizable force field significantly improves the interaction energies but decreases the agreement on distances at energy minima. The best agreement between force field and QM PES is obtained by modifying the Lennard-Jones terms for atom pairs involved in the phenol-TMA cation-π interactions. This is further shown to improve the correlation between the occupancy of tyrosine-choline cation-π interactions obtained from molecular dynamics simulations of a bilayer-bound bacterial phospholipase and experimental affinity data of the wild-type protein and selected mutants., Competing Interests: AUTHOR INFORMATION Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

An aqua ligand bridges metal cations in a wide variety of enzymes, many of which are drug targets for various diseases. However, the factors affecting its protonation state and thus biological roles remain elusive. By computing the free energy for replacing the bridging H2O by OH(-) in various model Mg(2+) sites, we have evaluated how the nature of an aqua bridge depends on the site's net charge (i.e., the number of charged ligands in the first and second shell and the number of metal cations), the site's solvent exposure, the ligand's charge-donating ability, the bridging oxygen's hydrogen-bonding interactions, intramolecular proton transfer from the bridging H2O to a nearby carboxylate, and the metal coordination number. The results reveal the key factors dictating the protonation state of bridging H2O and provide guidelines in predicting whether H2O or OH(-) bridges two Mg(2+) in polynuclear sites. This helps to elucidate the nucleophile in the enzyme-catalyzed reaction and the net charge of the metal complex (metal cation and first-shell ligands), which plays a critical role in binding.