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Because Li+ and Ca2+ differ in both charge and size, the possibility that monovalent Li+ could dislodge the bulkier, divalent Ca2+ in Ca2+ proteins had not been considered. However, our recent density functional theory/continuum dielectric calculations predicted that Li+ could displace the native Ca2+ 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 Ca2+-site in the PKCα/γ C2 domain, it is not known whether other Ca2+-sites in human proteins may be susceptible to Li+ substitution. Furthermore, it is unclear what factors determine the outcome of the competition between divalent Ca2+ 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 Ca2+ over monovalent Li+ in proteins: neutral/anionic Ca2+-carboxylate sites are protected against Li+ attack. They are further protected by outer-shell Asp−/Glu− and the protein matrix rigidifying the Ca2+-site or limiting water entry. In contrast, buried, cationic Ca2+-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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Understanding when a metal-bound water molecule in a protein is deprotonated is important as this affects the charge distribution in the metal-binding/enzyme active site and thus their interactions, the enzyme mechanism, and inhibitor design. The protonation state of the metal-bound water molecule at a given pH depends on its pKa value, which in turn depends on the properties of the cation, its ligands, and the protein environment. Here, we reveal how and the extent to which (i) the first-shell composition (type, charge, and number of ligands), (ii) the metal site's immediate surroundings (first-shell…second-shell hydrogen-bonding interactions, metal–ligand distance constraints, and ligand-binding mode) and (iii) the protein architecture and coupled solvent interactions (long-range electrostatic interactions and solvent exposure of the site) affect the Zn2+-bound water pKa. The results, which are consistent with available experimental pKa values of Zn2+-bound water, provide guidelines to predict when Zn2+-bound water would likely be deprotonated at physiological pH. [ABSTRACT FROM AUTHOR]

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.

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.