Todor Dudev

Metal binding affinity and selectivity in metalloproteins: insights from computational studies.

This review highlights insights gained from computational studies on protein-metal recognition. We systematically dissect the various factors governing metal binding affinity and selectivity in proteins starting from (a) the intrinsic properties of the metal and neighboring metal cations (if present), to (b) the primary coordination sphere, (c) the second coordination shell, (d) the protein matrix, (e) the bulk solvent, and (f) competing non-protein ligands from the surrounding biological environment. The results herein reveal the fundamental principles and the molecular bases underlying protein-metal recognition, which serve as a guide to engineer novel metalloproteins with programmed properties.

Determinants of K+ vs Na+ Selectivity in Potassium Channels

Ion channels, specialized pore-forming proteins, are an indispensable component of the nervous system and play a crucial role in regulating cardiac, skeletal, and smooth muscle contraction. Potassium ion channels, controlling the action potential of a number of excitable cells, are characterized by a remarkable ability to select K(+) over Na(+). Although the molecular basis for this striking ion selectivity has been a subject of extensive investigations using both experimental and theoretical methods, the following outstanding questions remain: (a) To what extent is the number of water molecules bound to the permeating ion (i.e., the hydration number) important for the K(+)/Na(+) competition? (b) Are the chemical type and number of coordinating groups lining the pore critical for the selectivity process? (c) Apart from providing cation-ligating groups, do the channel walls play any other role in the selectivity process? This work reveals that the pores selectivity for K(+) over Na(+) increases with (i) increasing hydration number of K(+) relative to that of Na(+), (ii) increasing number of K(+)-coordinating dipoles, (iii) increasing number of Na(+)-coordinating dipoles, and (iv) decreasing magnitude of the coordinating dipoles provided by the pore. Thus, a high K(+)/Na(+) selectivity in K(+) channels could be achieved from a combination of several favorable factors involving the native ion, the metal-coordinating ligands, and the protein matrix, viz., (a) an octahydrated permeating K(+), (b) a pore lined with 8 carbonyl ligands, and (c) finely tuned physicomechanical properties of the channel walls providing a low dielectric medium favoring a high hydration number for the permeating K(+) and enough stiffness to force the competing Na(+) to adopt an unfavorable 8-fold coordination. This implies that optimal K(+)/Na(+) selectivity in K(+) channels generally does not arise from solely structural or energetic consideration. The factors affecting ion selectivity revealed herein help to rationalize why valinomycin and the KcsA ion channels are highly K(+)-selective, whereas the NaK channel is nonselective. The calculations predict that other pores containing a different number/chemical type of coordinating groups from those observed in potassium channels could also select K(+) over Na(+).

Factors Governing the Na+ vs K+ Selectivity in Sodium Ion Channels

Monovalent Na(+) and K(+) ion channels, specialized pore-forming proteins that play crucial biological roles such as controlling cardiac, skeletal, and smooth muscle contraction, are characterized by a remarkable metal selectivity, conducting the native cation while rejecting its monovalent contender and other ions present in the cellular/extracellular milieu. Compared to K(+) channels, the principles governing Na(+) vs K(+) selectivity in both epithelial and voltage-gated Na(+) channels are much less well understood due mainly to the lack of high-resolution 3D structures. Thus, many questions remain. It is not clear if the serines lining the pore of epithelial Na(+) channel bind to the metal cation via their backbone or side chain O atoms and why substituting the Lys lining the pore of voltage-gated Na(+) channels to another residue such as Arg drastically reduces or even reverses the Na(+)/K(+) selectivity. This work systematically evaluates the effects of various factors such as (i) the number, chemical type, and charge of the pores coordinating groups, (ii) the hydration number and coordination number of the metal cation, and (iii) the solvent exposure and the size/rigidity of the pore on the Na(+) vs K(+) selectivity in model Na(+) channel selectivity filters (the narrowest part of the pore) using a combined density functional theory/continuum dielectric approach. The results reveal that the Na(+) channels selectivity for Na(+) over K(+) increases if (1) the pore provides three rather than four protein ligands to coordinate to the metal ion, (2) the protein ligands have strong charge-donating ability such as Asp/Glu carboxylate or backbone carbonyl groups, (3) the passing Na(+) is bare or less well hydrated inside the filter than the competing K(+), and (4) the pore is relatively rigid, constricted, and solvent exposed. They also reveal that factors favoring Na(+)/K(+) selectivity in Na(+) channels generally disfavor K(+)/Na(+) selectivity in K(+) channels and vice versa. The different selectivity principles for the K(+) and Na(+) channels are consistent with the different architecture, composition, and properties of their selectivity filters. They provide clues to the metal-binding site structure in the selectivity filters of epithelial and voltage-gated Na(+) channels.

Competition between Li+ and Mg2+ in Metalloproteins. Implications for Lithium Therapy

Lithium is used (in the form of soluble salts) to treat bipolar disorder and has been considered as a possible drug in treating chronic neurodegenerative diseases such as Alzheimers, Parkinsons, and Huntingtons diseases. One of the proposed mechanisms of Li(+) action involves a competition between the alien Li(+) and native Mg(2+) for metal-binding sites and subsequent inhibition of key enzymes involved in specific neurotransmission pathways, but not vital Mg(2+) proteins in the cell. This raises the following intriguing questions: Why does Li(+) replace Mg(2+) only in enzymes involved in bipolar disorder, but not in Mg(2+) proteins essential to cells? In general, what factors allow monovalent Li(+) to displace divalent Mg(2+) in proteins? Specifically, how do the composition, overall charge, and solvent exposure of the metal-binding site as well as a metal-bound phosphate affect the selectivity of Li(+) over Mg(2+)? Among the many possible factors, we show that the competition between Mg(2+) and Li(+) depends on the net charge of the metal complex, which is determined by the numbers of metal cations and negatively charged ligands, as well as the relative solvent exposure of the metal cavity. The protein itself is found to select Mg(2+) over the monovalent Li(+) by providing a solvent-inaccessible Mg(2+)-binding site lined by negatively charged Asp/Glu, whereas the cell machinery was found to select Mg(2+) among other competing divalent cations in the cellular fluids such as Ca(2+) and Zn(2+) by maintaining a high concentration ratio of Mg(2+) to its biogenic competitor in various biological compartments. The calculations reveal why Li(+) replaces Mg(2+) only in enzymes that are known targets of Li(+) therapy, but not in Mg(2+) enzymes essential to cells, and also reveal features common to the former that differ from those in the latter proteins.

Selectivity Mechanism of the Voltage-gated Proton Channel, HV1

Voltage-gated proton channels, HV1, trigger bioluminescence in dinoflagellates, enable calcification in coccolithophores, and play multifarious roles in human health. Because the proton concentration is minuscule, exquisite selectivity for protons over other ions is critical to HV1 function. The selectivity of the open HV1 channel requires an aspartate near an arginine in the selectivity filter (SF), a narrow region that dictates proton selectivity, but the mechanism of proton selectivity is unknown. Here we use a reduced quantum model to elucidate how the Asp–Arg SF selects protons but excludes other ions. Attached to a ring scaffold, the Asp and Arg side chains formed bidentate hydrogen bonds that occlude the pore. Introducing H3O+ protonated the SF, breaking the Asp–Arg linkage and opening the conduction pathway, whereas Na+ or Cl– was trapped by the SF residue of opposite charge, leaving the linkage intact, thus preventing permeation. An Asp–Lys SF behaved like the Asp–Arg one and was experimentally verified to be proton-selective, as predicted. Hence, interacting acidic and basic residues form favorable AspH0–H2O0–Arg+ interactions with hydronium but unfavorable Asp––X–/X+–Arg+ interactions with anions/cations. This proposed mechanism may apply to other proton-selective molecules engaged in bioenergetics, homeostasis, and signaling.

Competition among Ca2+, Mg2+, and Na+ for model ion channel selectivity filters: determinants of ion selectivity.

Because voltage-gated ion channels play critical biological roles, understanding how they can discriminate the native metal ion from rival cations in the milieu is of great interest. Although Ca(2+), Mg(2+), and Na(+) are present in comparable concentrations outside the cell, the factors governing the competition among these cations for the selectivity filter of voltage-gated Ca(2+) ion channel remain unclear. Using density functional theory combined with continuum dielectric methods, we evaluate the effect of (1) the number, chemical type, and charge of the ligands lining the pore, (2) the pores rigidity, size, symmetry, and solvent accessibility, and (3) the Ca(2+) hydration number outside the selectivity filter on the competition among Ca(2+), Mg(2+), and Na(+) in model selectivity filters. The calculations show how the outcome of this competition depends on the interplay between electronic and solvation effects. Selectivity for monovalent Na(+) over divalent Ca(2+)/Mg(2+) is achieved when solvation effects outweigh electrostatic effects; thus filters comprising a few weak charge-donating groups such as Ser/Thr side chains, where electrostatic effects are relatively weak and are easily overcome by solvation effects, are Na(+)-selective. In contrast, selectivity for divalent Ca(2+)/Mg(2+) over monovalent Na(+) is achieved when metal-ligand electrostatic effects outweigh solvation effects. The key differences in selectivity between Mg(2+) and Ca(2+) lie in the pore size, oligomericity, and solvent accessibility. The results, which are consistent with available experimental data, reveal how the structure and composition of the ion channel selectivity pore had adapted to the specific physicochemical properties of the native metal ion to enhance the competitiveness of the native metal toward rival cations.

Wnt and lithium: a common destiny in the therapy of nervous system pathologies?

Wnt signaling is required for neurogenesis, the fate of neural progenitors, the formation of neuronal circuits during development, neuron positioning and polarization, axon and dendrite development and finally for synaptogenesis. This signaling pathway is also implicated in the generation and differentiation of glial cells. In this review, we describe the mechanisms of action of Wnt signaling pathways and their implication in the development and correct functioning of the nervous system. We also illustrate how a dysregulated Wnt pathway could lead to psychiatric, neurodegenerative and demyelinating pathologies. Lithium, used for the treatment of bipolar disease, inhibits GSK3β, a central enzyme of the Wnt/β-catenin pathway. Thus, lithium could, to some extent, mimic Wnt pathway. We highlight the possible dialogue between lithium therapy and modulation of Wnt pathway in the treatment of the diseases of the nervous system.

Metal-Binding Affinity and Selectivity of Nonstandard Natural Amino Acid Residues from DFT/CDM Calculations

Unnatural amino acid residues are increasingly being used in metalloprotein design and engineering to expand the repertoire of protein structures/folds and functions. However, natural but nonstandard amino acid residues (not in the basic set of 20) possessing metal-ligating groups such as selenocysteine (Sec), pyrrolysine (Pyl), and gamma-carboxyglutamic acid (Gla) have attracted little attention, and their potential as metal-binding entities in metalloprotein engineering has not been assessed. In particular, the metal-binding affinity/selectivity of these three rare residues remains unclear. Herein, the metal-binding affinity/selectivity of the Gla, Pyl, and Sec side chains have been systematically studied using a combined density functional theory and continuum dielectric method. The calculations reveal an advantage of using these noncanonical protein building blocks instead of the standard 20 amino acid residues. Gla2-, Pyl0, and Sec- have greater potential in trapping the metal cation than their standard amino acid counterparts. They prefer binding to Zn2+ rather than to Mg2+ or Ca2+ in a protein cavity due to the better electron-accepting ability and lower coordination number preference of Zn2+, as compared to Mg2+ and Ca2+. Between Ca2+ and Mg2+, Gla2- prefers Ca2+, whereas Pyl0 and Sec- poorly discriminate between the two metal cations. The results herein suggest that Gla2-, Pyl0, and Sec- could be employed as very efficient metal-binding entities in engineering metalloproteins with preprogrammed properties.

1H and 13C NMR study and AM1 calculations of some azobenzenes and N-benzylideneanilines: effect of substituents on the molecular planarity

Abstract Azobenzenes and N-benzylideneanilines with para -dimethylamino group at aromatic ring and nine various para substituents to another phenyl have been studied by 1 H and 13 C NMR spectroscopy followed by semiempirical AM1 MO calculations. All molecules exhibit trans -configuration, with the exception of cis -4′-nitro-N-4-dimethylaminobenzylideneaniline, only small amounts of trans form coexists in solution. N,N-dimethylaminoazobenzenes are planar, there is a correlation between NMR chemical shifts and the Hammett σ constants of substituents and the resonance mechanism of transferring the substituent effects dominates. Torsion angle of 28 to 35.6° of the aromatic ring bearing the substituents was calculated for N-dimethylaminobenzylideneanilines, the stronger the electron withdrawing properties of the substituent, the larger the angle. However, there is no relationship between chemical shifts and Hammett constants.

Ion Selectivity Strategies of Sodium Channel Selectivity Filters

CONSPECTUS: Sodium ion channels selectively transport Na(+) cations across the cell membrane. These integral parts of the cell machinery are implicated in regulating the cardiac, skeletal and smooth muscle contraction, nerve impulses, salt and water homeostasis, as well as pain and taste perception. Their malfunction often results in various channelopathies of the heart, brain, skeletal muscles, and lung; thus, sodium channels are key drug targets for various disorders including cardiac arrhythmias, heart attack, stroke, migraine, epilepsy, pain, cancer, and autoimmune disorders. The ability of sodium channels to discriminate the native Na(+) among other competing ions in the surrounding fluids is crucial for proper cellular functions. The selectivity filter (SF), the narrowest part of the channels open pore, lined with amino acid residues that specifically interact with the permeating ion, plays a major role in determining Na(+) selectivity. Different sodium channels have different SFs, which vary in the symmetry, number, charge, arrangement, and chemical type of the metal-ligating groups and pore size: epithelial/degenerin/acid-sensing ion channels have generally trimeric SFs lined with three conserved neutral serines and/or backbone carbonyls; eukaryotic sodium channels have EKEE, EEKE, DKEA, and DEKA SFs with an invariant positively charged lysine from the second or third domain; and bacterial voltage-gated sodium (Nav) channels exhibit symmetrical EEEE SFs, reminiscent of eukaryotic voltage-gated calcium channels. How do these different sodium channel SFs achieve high selectivity for Na(+) over its key rivals, K(+) and Ca(2+)? What factors govern the metal competition in these SFs and which of these factors are exploited to achieve Na(+) selectivity in the different sodium channel SFs? The free energies for replacing K(+) or Ca(2+) bound inside different model SFs with Na(+), evaluated by a combination of density functional theory and continuum dielectric calculations, have shed light on these questions. The SFs of epithelial and eukaryotic Nav channels select Na(+) by providing an optimal number and ligating strength of metal ligands as well as a rigid pore whose size fits the cognate Na(+) ideally. On the other hand, the SFs of bacterial Nav channels select Na(+), as the protein matrix attenuates ion-protein interactions relative to ion-solvent interactions by enlarging the pore and allowing water to enter, so the ion interacts indirectly with the conserved glutamates via bridging water molecules. This shows how these various SFs have adapted to the specific physicochemical properties of the native ion, using different strategies to select Na(+) among its contenders.