Protein Ca2+-Sites Prone to Sr2+ Substitution: Implications for Strontium Therapy.

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 Sr2+ as a Ca2+ 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 Ca2+-binding protein that enable Sr2+ to displace Ca2+ remain unclear. To address this, we studied the competition between Ca2+ and Sr2+ in protein Ca2+-binding sites using density functional theory combined with the polarizable continuum model. Our findings indicate that Ca2+-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 Sr2+ attack. On the other hand, Ca2+-sites crowded with multiple protein ligands may be prone to Sr2+ displacement if they are solvent-exposed and flexible enough so that an extra backbone ligand from the outer shell can bind to Sr2+. In addition, solvent-exposed Ca2+ sites with only a few weak charge-donating ligands that can rearrange to fit the strontium's coordination requirements are susceptible to Sr2+ displacement. We provide the physical basis of these results and discuss potential novel protein targets of therapeutic Sr2+.

A strategy for evaluating potential antiviral resistance to small molecule drugs and application to SARS-CoV-2.

Alterations in viral fitness cannot be inferred from only mutagenesis studies of an isolated viral protein. To-date, no systematic analysis has been performed to identify mutations that improve virus fitness and reduce drug efficacy. We present a generic strategy to evaluate which viral mutations might diminish drug efficacy and applied it to assess how SARS-CoV-2 evolution may affect the efficacy of current approved/candidate small-molecule antivirals for Mpro, PLpro, and RdRp. For each drug target, we determined the drug-interacting virus residues from available structures and the selection pressure of the virus residues from the SARS-CoV-2 genomes. This enabled the identification of promising drug target regions and small-molecule antivirals that the virus can develop resistance. Our strategy of utilizing sequence and structural information from genomic sequence and protein structure databanks can rapidly assess the fitness of any emerging virus variants and can aid antiviral drug design for future pathogens.

From quantum-derived principles underlying cysteine reactivity to combating the COVID-19 pandemic.

The COVID-19 pandemic poses a challenge in coming up with quick and effective means to counter its cause, the SARS-CoV-2. Here, we show how the key factors governing cysteine reactivity in proteins derived from combined quantum mechanical/continuum calculations led to a novel multi-targeting strategy against SARS-CoV-2, in contrast to developing potent drugs/vaccines against a single viral target such as the spike protein. Specifically, they led to the discovery of reactive cysteines in evolutionary conserved Zn2+-sites in several SARS-CoV-2 proteins that are crucial for viral polypeptide proteolysis as well as viral RNA synthesis, proofreading, and modification. These conserved, reactive cysteines, both free and Zn2+-bound, can be targeted using the same Zn-ejector drug (disulfiram/ebselen), which enables the use of broad-spectrum anti-virals that would otherwise be removed by the virus's proofreading mechanism. Our strategy of targeting multiple, conserved viral proteins that operate at different stages of the virus life cycle using a Zn-ejector drug combined with other broad-spectrum anti-viral drug(s) could enhance the barrier to drug resistance and antiviral effects, as compared to each drug alone. Since these functionally important nonstructural proteins containing reactive cysteines are highly conserved among coronaviruses, our proposed strategy has the potential to tackle future coronaviruses. This article is categorized under:Structure and Mechanism > Reaction Mechanisms and CatalysisStructure and Mechanism > Computational Biochemistry and BiophysicsElectronic Structure Theory > Density Functional Theory.

How the Local Environment of Functional Sites Regulates Protein Function.

Proteins form complex biological machineries whose functions in the cell are highly regulated at both the cellular and molecular levels. Cellular regulation of protein functions involves differential gene expressions, post-translation modifications, and signaling cascades. Molecular regulation, on the other hand, involves tuning an optimal local protein environment for the functional site. Precisely how a protein achieves such an optimal environment around a given functional site is not well understood. Herein, by surveying the literature, we first summarize the various reported strategies used by certain proteins to ensure their correct functioning. We then formulate three key physicochemical factors for regulating a protein's functional site, namely, (i) its immediate interactions, (ii) its solvent accessibility, and (iii) its conformational flexibility. We illustrate how these factors are applied to regulate the functions of free/metal-bound Cys and Zn sites in proteins.

Free and Bound Therapeutic Lithium in Brain Signaling.

Lithium, a first-line therapy for bipolar disorder, is effective in preventing suicide and new depressive/manic episodes. Yet, how this beguilingly simple monocation with only two electrons could yield such profound therapeutic effects remains unclear. An in-depth understanding of lithium's mechanisms of actions would help one to develop better treatments limiting its adverse side effects and repurpose lithium for treating traumatic brain injury and chronic neurodegenerative diseases. In this Account, we begin with a comparison of the physicochemical properties of Li+ and its key native rivals, Na+ and Mg2+, to provide physical grounds for their competition in protein binding sites. Next, we review the abnormal signaling pathways and proteins found in bipolar patients, who generally have abnormally high intracellular Na+ and Ca2+ concentrations, high G-protein levels, and hyperactive phosphatidylinositol signaling and glycogen synthase kinase-3ß (GSK3ß) activity. We briefly summarize experimental findings on how lithium, at therapeutic doses, modulates these abnormal signaling pathways and proteins. Following this survey, we address the following aspects of lithium's therapeutic actions: (1) Can Li+ displace Na+ from the allosteric Na+-binding sites in neurotransmitter transporters and G-protein coupled receptors (GPCRs); if so, how would this affect the host protein's function? (2) Why are certain Mg2+-dependent enzymes targeted by Li+? (3) How does Li+ binding to Mg2+-bound ATP/GTP (denoted as NTP) in solution affect the cofactor's conformation and subsequent recognition by the host protein? (4) How do NTP-Mg-Li complexes modulate the properties of the respective cellular receptors and signal-transducing proteins? We show that Li+ may displace Na+ from allosteric Na+-binding sites in certain GPCRs and stabilize inactive conformations, preventing these receptors from relaying signal to the respective G-proteins. It may also displace Mg2+ in enzymes containing highly cationic Mg2+-binding sites such as GSK3ß, but not in enzymes containing Mg2+-binding sites with low or zero charge. We further show that Li+ binding to Mg2+-NTP in water does not alter the NTP conformation, which is locked by all three phosphates binding to Mg2+. However, bound lithium in the form of [NTP-Mg-Li]2- dianions can activate or inhibit the host protein depending on the NTP-binding pocket's shape, which determines the metal-binding mode: The ATP-binding pocket's shape in the P2X receptor is complementary to the native ATP-Mg solution conformation and nicely fits [ATP-Mg-Li]2-. However, since the ATP ßγ phosphates bind Li+, bimetallic [ATP-Mg-Li]2- may be more resistant to hydrolysis than the native cofactor, enabling ATP to reside longer in the binding site and elicit a prolonged P2X response. In contrast, the elongated GTP-binding pockets in G-proteins allow only two GTP phosphates to bind Mg2+, so the GTP conformation is no longer "triply-locked". Consequently, Li+ binding to GTP-Mg can significantly alter the native cofactor's structure, lowering the activated G-protein level, thus attenuating hyperactive G-protein-mediated signaling in bipolar patients. In summary, we have presented a larger "connected" picture of lithium's diverse effects based on its competition as a free monocation with native cations or as a phosphate-bound polyanionic complex modulating the host protein function.

How First Shell-Second Shell Interactions and Metal Substitution Modulate Protein Function.

Hydrogen bonds to metal-ligands in proteins play a vital role in biological function. They help to stabilize/protect the metal complex and enhance metal-binding affinity/specificity, enzyme-substrate recognition, and enzyme activation. Yet, knowledge of the preferred hydrogen-bonding partners of metal ligands in different metalloproteins is lacking. Using well-calibrated methods, we have determined the preferred hydrogen-bonding partners of Cys- bound to native Zn2+ or xenobiotic Cd2+ in Zn-fingers of varying net charge and solvent accessibility as well as the key factors underlying the observed preference. We show how secondary hydrogen-bonding interactions with metal-bound thiolates might exert a significant impact on Zn2+→Cd2+ substitution and thus protein function. Knowing which Zn-fingers may be vulnerable to structural deformation by Cd2+ is important since this would lead to their inactivation, which might impair cell growth, differentiation, cell-cycle control, and DNA repair.

Competition between Li+ and Na+ in sodium transporters and receptors: Which Na+-Binding sites are "therapeutic" Li+ targets?

Sodium (Na+) acts as an indispensable allosteric regulator of the activities of biologically important neurotransmitter transporters and G-protein coupled receptors (GPCRs), which comprise well-known drug targets for psychiatric disorders and addictive behavior. How selective these allosteric Na+-binding sites are for the cognate cation over abiogenic Li+, a first-line drug to treat bipolar disorder, is unclear. Here, we reveal how properties of the host protein and its binding cavity affect the outcome of the competition between Li+ and Na+ for allosteric binding sites in sodium transporters and receptors. We show that rigid Na+-sites that are crowded with multiple protein ligands are well-protected against Li+ attack, but their flexible counterparts or buried Na+-sites containing only one or two protein ligands are vulnerable to Li+ substitution. These findings suggest a novel possible mode of Li+ therapeutic action: By displacing Na+ bound by ≤2 protein ligands in buried GPCR sites and stabilizing the receptor's inactive state, Li+ could prohibit conformational changes to an active state, leading to lower cytosolic levels of activated guanine nucleotide-binding proteins, which are hyperactive/overexpressed in bipolar disorder patients.

Preferred Hydrogen-Bonding Partners of Cysteine: Implications for Regulating Cys Functions.

The hydrogen-bonding interactions of cysteine, which can serve as a hydrogen-bond donor and/or acceptor, play a central role in cysteine's diverse functional roles in proteins. They affect the balance between the neutral thiol (SH) or thiolate (S-) and the charge distribution in the rate-limiting transition state of a reaction. Despite their importance, no study has determined the preferred hydrogen-bonding partners of cysteine serving as a hydrogen-bond donor or acceptor. By computing the free energy for displacing a peptide backbone hydrogen-bonded to cysteine with amino acid side chains in various protein environments, we have evaluated how the strength of the hydrogen bond to the cysteine thiol/thiolate depends on its hydrogen-bonding partner and its local environment. The predicted hydrogen-bonding partners preferred by cysteine are consistent with the hydrogen-bonding interactions made by cysteines in 9138 nonredundant X-ray structures. Our results suggest a mechanism to regulate the reactivity of cysteines and a strategy to design drugs based on the hydrogen-bonding preference of cysteine.

Factors controlling the selectivity for Na(+) over Mg(2+) in sodium transporters and enzymes.

Na(+) and Mg(2+) play different crucial roles in biological systems. Both cations are present in comparable amounts in the cytosol, but how monovalent Na(+) can compete with the divalent Mg(2+), which can better accept charge from negatively charged ligands, in sodium transporters/enzymes has not been investigated. Hence, it is not clear how Na(+) and Mg(2+)-binding sites have evolved to discriminate the "right" cation among non-cognate ones from the surrounding milieu and the physical basis governing the selectivity for Na(+) over Mg(2+). The results, which are consistent with available experimental data, reveal that in proteins, the selectivity for Na(+) over Mg(2+) in sodium-binding sites stem mainly from the size, charge, and charge-accepting ability differences between Na(+) and Mg(2+). A protein could achieve Na(+) selectivity by (i) reducing the number of metal-ligating ligands, (ii) maintaining an optimal balance of different ligating-strength ligands whose interactions in the metal-binding site would favor Na(+) over rival mono/divalent cations, (iii) increasing the solvent exposure of the metal-binding site, or (iv) increasing binding site rigidity forcing Mg(2+) to adopt the coordination distances/geometry of Na(+). Sodium-binding proteins use one or more of these factors to achieve Na(+) selectivity.

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 AspH(0)-H2O(0)-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.