Carbonic Anhydrases: Different Active Sites, Same Metal Selectivity Rules.

Carbonic anhydrases are mononuclear metalloenzymes catalyzing the reversible hydration of carbon dioxide in organisms belonging to all three domains of life. Although the mechanism of the catalytic reaction is similar, different families of carbonic anhydrases do not have a common ancestor nor do they exhibit significant resemblance in the amino acid sequence or the structure and composition of the metal-binding sites. Little is known about the physical principles determining the metal affinity and selectivity of the catalytic centers, and how well the native metal is protected from being dislodged by other metal species from the local environment. Here, we endeavor to shed light on these issues by studying (via a combination of density functional theory calculations and polarizable continuum model computations) the thermodynamic outcome of the competition between the native metal cation and its noncognate competitor in various metal-binding sites. Typical representatives of the competing cations from the cellular environments of the respective classes of carbonic anhydrases are considered. The calculations reveal how the Gibbs energy of the metal competition changes when varying the metal type, structure, composition, and solvent exposure of the active center. Physical principles governing metal competition in different carbonic anhydrase metal-binding sites are delineated.

Enhanced Solubility of Ibuprofen by Complexation with ß-Cyclodextrin and Citric Acid.

The ability of ß-CD to form inclusion complexes with ibuprofen (IBU) and at the same time to make a two-phase system with citric acid was explored in the present study for achieving improved solubility and dissolution rate of IBU. Mechanical milling as well as mechanical milling combined with thermal annealing of the powder mixtures were applied as synthetic methods. Solubility and dissolution kinetics of the complexes were studied in compliance with European Pharmacopoeia (ICH Q4B). ß-CD and citric acid (CA) molecules were shown to interact by both ball milling (BM), thermal annealing, as well as BM with subsequent annealing. Complexes were also formed by milling the three compounds (ß-CD, CA and IBU) simultaneously, as well as by a consecutive first including IBU into ß-CD and then binding the formed ß-CD/IBU inclusion complex with CA. As a result, ternary ß-CD/IBU/CA complex formed by initial incorporation of ibuprofen into ß-CD, followed by successive formation of a two-phase mixture with CA, exhibited notably improved dissolution kinetics compared to the pure ibuprofen and slightly better compared to the binary ß-CD/IBU system. Although the addition of CA to ß-CD/IBU does not significantly increase the solubility rate of IBU, it must be considered that the amount of ß-CD is significantly less in the ternary complex compared to the binary ß-CD/IBU.

In Silico Analysis of the Ga3+/Fe3+ Competition for Binding the Iron-Scavenging Siderophores of P. aeruginosa-Implementation of Three Gallium-Based Complexes in the "Trojan Horse" Antibacterial Strategy.

The emergence of multidrug-resistant (MDR) microorganisms combined with the ever-draining antibiotic pipeline poses a disturbing and immensely growing public health challenge that requires a multidisciplinary approach and the application of novel therapies aimed at unconventional targets and/or applying innovative drug formulations. Hence, bacterial iron acquisition systems and bacterial Fe2+/3+-containing enzymes have been identified as a plausible target of great potential. The intriguing "Trojan horse" approach deprives microorganisms from the essential iron. Recently, gallium's potential in medicine as an iron mimicry species has attracted vast attention. Different Ga3+ formulations exhibit diverse effects upon entering the cell and thus supposedly have multiple targets. The aim of the current study is to specifically distinguish characteristics of great significance in regard to the initial gallium-based complex, allowing the alien cation to effectively compete with the native ferric ion for binding the siderophores pyochelin and pyoverdine secreted by the bacterium P. aeruginosa. Therefore, three gallium-based formulations were taken into consideration: the first-generation gallium nitrate, Ga(NO3)3, metabolized to Ga3+-hydrated forms, the second-generation gallium maltolate (tris(3-hydroxy-2-methyl-4-pyronato)gallium), and the experimentally proven Ga carrier in the bloodstream-the protein transferrin. We employed a reliable in silico approach based on DFT computations in order to understand the underlying biochemical processes that govern the Ga3+/Fe3+ rivalry for binding the two bacterial siderophores.

N-Methyl- and N-Phenylpiperazine Functionalized Styryl Dyes Inside Cucurbiturils: Theoretical Assessment of the Factors Governing the Host-Guest Recognition.

The family of cucurbiturils (CBs), the unique pumpkin-shaped macrocycles, has received great attention over the past four decades owing to their remarkable recognition properties. They have found diverse applications including biosensing and drug delivery technologies. The cucurbituril complexation of guest molecules can modulate their pKas, improve their solubility in aqueous solution, and reduce the adverse effects of the drugs, as well as enhance the stability and/or enable targeted delivery of the drug molecule. Employing twelve cationic styryl dyes with N-methyl- and N-phenylpiperazine functionality as probes, we attempted to understand the factors that govern the host-guest complexation of such molecules within CB[7] and CB[8] host systems. Various key factors determining the process were recognized, such as the pH and dielectric constant of the medium, the cavity size of the host, the chemical characteristics of the substituents in the guest entity, and the presence/absence of metal cations. The presented results add to our understanding (at the molecular level) of the mechanism of encapsulation of styryl dyes by cucurbiturils, thus shedding new light on various aspects of the intriguing complexation chemistry and the underlying recognition processes.

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+.

Electrostatic interactions - key determinants of the metal selectivity in La3+ and Ca2+ binding proteins.

Nearly half of all known proteins contain metal co-factors. In the course of evolution two dozen metal cations (mostly monovalent and divalent species) have been selected to participate in processes of vital importance for living organisms. Trivalent metal cations have also been selected, although to a lesser extent as compared with their mono- and divalent counterparts. Notably, factors governing the metal selectivity in trivalent metal centers in proteins are less well understood than those in the respective divalent metal centers. Thus, the source of high La3+/Ca2+ selectivity in lanthanum-binding proteins, as compared with that of calcium-binding proteins (i.e., calmodulin), is still shrouded in mystery. The well-calibrated thermochemical calculations, performed here, reveal the dominating role of electrostatic interactions in shaping the metal selectivity in La3+-binding centers. The calculations also disclose other (second-order) determinants of metal selectivity in these systems, such as the rigidity and extent of solvent exposure of the binding site. All these factors are also implicated in shaping the metal selectivity in Ca2+-binding proteins.

Cu+/Ag+ Competition in Type I Copper Proteins (T1Cu).

Due to the similarity in the basic coordination behavior of their mono-charged cations, silver biochemistry is known to be linked to that of copper in biological systems. Still, Cu+/2+ is an essential micronutrient in many organisms, while no known biological process requires silver. In human cells, copper regulation and trafficking is strictly controlled by complex systems including many cytosolic copper chaperones, whereas some bacteria exploit the so-called "blue copper" proteins. Therefore, evaluating the controlling factors of the competition between these two metal cations is of enormous interest. By employing the tools of computational chemistry, we aim to delineate the extent to which Ag+ might be able to compete with the endogenous copper in its Type I (T1Cu) proteins, and where and if, alternatively, it is handled uniquely. The effect of the surrounding media (dielectric constant) and the type, number, and composition of amino acid residues are taken into account when modelling the reactions in the present study. The obtained results clearly indicate the susceptibility of the T1Cu proteins to a silver attack due to the favorable composition and geometry of the metal-binding centers, along with the similarity between the Ag+/Cu+-containing structures. Furthermore, by exploring intriguing questions of both metals' coordination chemistry, an important background for understanding the metabolism and biotransformation of silver in organisms is provided.

Lanthanides as Calcium Mimetic Species in Calcium-Signaling/Buffering Proteins: The Effect of Lanthanide Type on the Ca2+/Ln3+ Competition.

Lanthanides, the 14 4f-block elements plus Lanthanum, have been extensively used to study the structure and biochemical properties of metalloproteins. The characteristics of lanthanides within the lanthanide series are similar, but not identical. The present research offers a systematic investigation of the ability of the entire Ln3+ series to substitute for Ca2+ in biological systems. A well-calibrated DFT/PCM protocol is employed in studying the factors that control the metal selectivity in biological systems by modeling typical calcium signaling/buffering binding sites and elucidating the thermodynamic outcome of the competition between the "alien" La3+/Ln3+ and "native" Ca2+, and La3+ - Ln3+ within the lanthanide series. The calculations performed reveal that the major determinant of the Ca2+/Ln3+ selectivity in calcium proteins is the net charge of the calcium binding pocket; the more negative the charge, the higher the competitiveness of the trivalent Ln3+ with respect to its Ca2+ contender. Solvent exposure of the binding site also influences the process; buried active centers with net charge of -4 or -3 are characterized by higher Ln3+ over Ca2+ selectivity, whereas it is the opposite for sites with overall charge of -1. Within the series, the competition between La3+ and its fellow lanthanides is determined by the balance between two competing effects: electronic (favoring heavier lanthanides) and solvation (generally favoring the lighter lanthanides).

Metal-Assisted Complexation of Fluorogenic Dyes by Cucurbit[7]uril and Cucurbit[8]uril: A DFT Evaluation of the Key Factors Governing the Host-Guest Recognition.

With the emergence of host-guest systems, a novel branch of complexation chemistry has found wide application in industries such as food, pharmacy, medicine, environmental protection and cosmetics. Along with the extensively studied cyclodextrins and calixarenes, the innovative cucurbiturils (CB) have enjoyed increased popularity among the scientific community as they possess even better qualities as cavitands as compared to the former molecules. Moreover, their complexation abilities could further be enhanced with the assistance of metal cations, which can interestingly exert a dual effect on the complexation process: either by competitively binding to the host entity or cooperatively associating with the CB@guest structures. In our previous work, two metal species (Mg2+ and Ga3+) have been found to bind to CB molecules in the strongest fashion upon the formation of host-guest complexes. The current study focuses on their role in the complex formation with three dye molecules: thiazole orange, neutral red, and thioflavin T. Various key factors influencing the process have been recognized, such as pH and the dielectric constant of the medium, the cavity size of the host, Mn+ charge, and the presence/absence of hydration shell around the metal cation. A well-calibrated DFT methodology, solidly based and validated and presented in the literature experimental data, is applied. The obtained results shed new light on several aspects of the cucurbituril complexation chemistry.

Host-guest complexation of cucurbit[7]uril and cucurbit[8]uril with the antimuscarinic drugs tropicamide and atropine.

Cucurbiturils are useful excipients in eye drop formulations: they can increase the water solubility of the drug, enhance drug absorption into the eye, improve aqueous stability and reduce local irritation. Effective and safe drug delivery is, however, a challenge and the information on the host (CBs)/guest (tropicamide and atropine) interactions can help improving the existing treatments and develop novel therapies not limited only to eye diseases/conditions. Since this carrier system can easily modify the properties of the drug and ensure its delivery at the targeted ocular tissue, further insight into the intimate mechanism of the host-guest recognition is crucial. The present DFT/SMD study focuses on the role of numerous factors governing this process, namely the specific position of the guest molecule in the cavity of the cucurbituril, the ionization form (non/protonated) of the antimuscarinic drug, the dielectric constant of the medium, and the size of the cavitant pore. The obtained results are in line with experimental observations and shed light on the mechanism, at atomic resolution, of recognition between the CBs and the two parasympatholytic drugs.

Competition between Ag+ and Ni2+ in nickel enzymes: Implications for the Ag+ antibacterial activity.

Silver's antimicrobial properties have been known for centuries, but exactly how it kills bacteria is still a mystery. Information on the competition between the native Ni2+ and abiogenic Ag+ cations in bacterial systems is also critically lacking. For example, urease, a famous nickel-containing enzyme that hydrolyzes urea into carbon dioxide and ammonia (a key step in the biogeochemical nitrogen cycle on Earth), is inhibited by Ag+ cations, but the molecular mechanism of silver's action is poorly understood. By employing density functional theory (DFT) calculations combined with the polarizable continuum model (PCM) computations we assess the susceptibility of the mono/binuclear Ni2+ binding sites in the nickel enzymatic centers to Ni2+→Ag+ substitution. The active centers in the mononuclear glyoxalase I and acireductone dioxygenase enzymes appear to be well protected against Ag+ attack and, presumably, stay functional even in its presence. On the other hand, the binuclear nickel binding site in urease appears vulnerable to silver attack - the results obtained are in line with available experimental data and give reason to assume a possible substitution of the essential Ni2+ cation from the urease metal center by Ag+.

Holo-chromodulin: competition between the native Cr3+ and other biogenic cations (Fe3+, Fe2+, Mg2+, and Zn2+) for the binding sites.

Chromodulin is an oligopeptide that has an essential role for the flawless functioning of insulin. Although the precise sequence of the constituent amino acid residues and the 3D structure of the molecule has not yet been deciphered, it is known that chromodulin contains only four amino acids in the ratio of Glu-: Gly: Cys: Asp- = 4: 2: 2: 2. An indispensable part for the integrity of the molecule in its active (holo-) form are four chromium cations (hence the name) in the oxidation state of 3+, positioned in two metal binding sites containing one and three Cr3+ ions. Experimental works provide some hints/clues concerning the structure of the metal centers, although their exact composition, type, and arrangement of metal ligating entities remain enigmatic. In the current study, we endeavor to unveil possible structure(s) of the Cr3+ loaded binding sites by strictly following the evidence provided by the experimental data. Well-calibrated in silico methodology for optimization and evaluation of Gibbs free energies is applied and gives strong premises for reliably deciphering the composition/structure of chromodulin metal binding sites. Additional computations reveal the advantage of choosing Cr3+ over other tri- (Fe3+) and divalent (Fe2+, Mg2+, and Zn2+) biogenic ions for securing maximum stability of the metal-occupied binding sites.

Theoretical Insight into the Phosphate-Targeted Silver's Antibacterial Action: Differentiation between Gram (+) and Gram (-) Bacteria.

Although silver is one of the first metals finding broad applications in everyday life, specific key points of the intimate mechanism of its bacteriostatic/bactericidal activity lack explanation. It is widely accepted that the antimicrobial potential of the silver cation depends on the composition and thickness of the bacterial external envelope: the outer membrane in Gram-negative bacteria is more prone to Ag+ attack than the cell wall in Gram-positive bacteria. The major cellular components able to interact strongly with Ag+ (teichoic acids, phospholipids, and lipopolysaccharides) contain mono/diesterified phosphate moieties. By applying a reliable DFT/SMD methodology, we modeled the reactions between the aforementioned constituents in typical Gram-positive and Gram-negative bacteria and hydrated Ag+ species, thus disclosing the factors that govern the process of metal-model ligand complexation. The conducted research indicates thermodynamically possible reactions in all cases but still a greater preference of the Ag+ toward the constituents in Gram-negative bacteria in comparison with their counterparts in Gram-positive bacteria. The observed tendencies shed light on the specific interactions of the silver cation with the modeled phosphate-containing units at the atomic level.

Complexation of trivalent metal cations (Al3+, Ga3+, In3+, La3+, Lu3+) to cucurbiturils: a DFT/SMD evaluation of the key factors governing the host-guest recognition.

Cucurbiturils (CBs), the pumpkin-shaped macrocycles, are suitable hosts for an array of neutral and cationic species. A plethora of host-guest complexes between CBs and a variety of guest molecules has been studied. However, much remains unknown, even in the complexation of very simple guests such as metal cations. In the computational study herein, DFT molecular modeling has been employed to investigate the interactions of a series of trivalent metal cations (Al3+, Ga3+, In3+, La3+, Lu3+) to cucurbit[n]urils and to evaluate the main factors controlling the host-guest complexation. The thermodynamic descriptors (Gibbs energies in the gas phase and in a water environment) of the corresponding complexation reactions have been estimated. This research is a logical continuation of an earlier study on the interaction between CB[n]s and a series of biologically essential mono- and divalent metal cations (Na+/K+ and Mg2+/Ca2+, respectively).

A DFT/PCM Study on the Affinity of Salinomycin to Bind Monovalent Metal Cations.

The affinity of the polyether ionophore salinomycin to bind IA/IB metal ions was accessed using the Gibbs free energy of the competition reaction between SalNa (taken as a reference) and its rival ions: [M+-solution] + [SalNa] → [SalM] + [Na+-solution] (M = Li, K, Rb, Cs, Cu, Ag, Au). The DFT/PCM computations revealed that the ionic radius, charge density and accepting ability of the competing metal cations, as well as the dielectric properties of the solvent, have an influence upon the selectivity of salinomycin. The optimized structures of the monovalent metal complexes demonstrate the flexibility of the ionophore, allowing the coordination of one or two water ligands in SalM-W1 and SalM-W2, respectively. The metal cations are responsible for the inner coordination sphere geometry, with coordination numbers spread between 2 (Au+), 4 (Li+ and Cu+), 5/6 (Na+, K+, Ag+), 6/7 (Rb+) and 7/8 (Cs+). The metals' affinity to salinomycin in low-polarity media follows the order of Li+ > Cu+ > Na+ > K+ > Au+ > Ag+ > Rb+ > Cs+, whereas some derangement takes place in high-dielectric environment: Li+ ≥ Na+ > K+ > Cu+ > Au+ > Ag+ > Rb+ > Cs+.

How Theoretical Evaluations Can Generate Guidelines for Designing/Engineering Metalloproteins with Desired Metal Affinity and Selectivity.

Almost half of all known proteins contain metal co-factors. Crucial for the flawless performance of a metalloprotein is the selection with high fidelity of the cognate metal cation from the surrounding biological fluids. Therefore, elucidating the factors controlling the metal binding and selectivity in metalloproteins is of particular significance. The knowledge thus acquired not only contributes to better understanding of the intimate mechanism of these events but, also, significantly enriches the researcher's toolbox that could be used in designing/engineering novel metalloprotein structures with pre-programmed properties. A powerful tool in aid of deciphering the physical principles behind the processes of metal recognition and selectivity is theoretical modeling of metal-containing biological structures. This review summarizes recent findings in the field with an emphasis on elucidating the major factors governing these processes. The results from theoretical evaluations are discussed. It is the hope that the physical principles evaluated can serve as guidelines in designing/engineering of novel metalloproteins of interest to both science and industry.

Ca2+/Sr2+ Selectivity in Calcium-Sensing Receptor (CaSR): Implications for Strontium's Anti-Osteoporosis Effect.

The extracellular calcium-sensing receptor (CaSR) controls vital bone cell functions such as cell growth, differentiation and apoptosis. The binding of the native agonist (Ca2+) to CaSR activates the receptor, which undergoes structural changes that trigger a cascade of events along the cellular signaling pathways. Strontium (in the form of soluble salts) has been found to also be a CaSR agonist. The activation of the receptor by Sr2+ is considered to be the major mechanism through which strontium exerts its anti-osteoporosis effect, mostly in postmenopausal women. Strontium-activated CaSR initiates a series of signal transduction events resulting in both osteoclast apoptosis and osteoblast differentiation, thus strengthening the bone tissue. The intimate mechanism of Sr2+ activation of CaSR is still enigmatic. Herewith, by employing a combination of density functional theory (DFT) calculations and polarizable continuum model (PCM) computations, we have found that the Ca2+ binding sites 1, 3, and 4 in the activated CaSR, although possessing a different number and type of protein ligands, overall structure and charge state, are all selective for Ca2+ over Sr2+. The three binding sites, regardless of their structural differences, exhibit almost equal metal selectivity if they are flexible and have no geometrical constraints on the incoming Sr2+. In contrast to Ca2+ and Sr2+, Mg2+ constructs, when allowed to fully relax during the optimization process, adopt their stringent six-coordinated octahedral structure at the expense of detaching a one-backbone carbonyl ligand and shifting it to the second coordination layer of the metal. The binding of Mg2+ and Sr2+ to a rigid/inflexible calcium-designed binding pocket requires an additional energy penalty for the binding ion; however, the price for doing so (to be paid by Sr2+) is much less than that of Mg2+. The results obtained delineate the key factors controlling the competition between metal cations for the receptor and shed light on some aspects of strontium's therapeutic effects.

Calcium in Signaling: Its Specificity and Vulnerabilities toward Biogenic and Abiogenic Metal Ions.

Divalent calcium ion (Ca2+) plays an indispensable role as a second messenger in a myriad of signal transduction processes. Of utmost importance for the faultless functioning of calcium-modulated signaling proteins is their binding selectivity of the native metal cation over rival biogenic/abiogenic metal ion contenders in the intra/extracellular fluids. In this Perspective, we summarize recent findings on the competition between the cognate Ca2+ and other biogenic or abiogenic divalent cations for binding to Ca2+-signaling proteins or organic cofactors. We describe the competition between the two most abundant intracellular biogenic metal ions (Mg2+ and Ca2+) for Ca2+-binding sites in signaling proteins, followed by the rivalry between native Ca2+ and "therapeutic" Li+ as well as "toxic" Pb2+. We delineate the key factors governing the rivalry between the native and non-native cations in proteins and highlight key implications for the biological performance of the respective proteins/organic cofactors.

Trinuclear Calcium Site in the C2 Domain of PKCα/γ Is Prone to Lithium Attack.

Lithium (Li+) is the first-line therapy for bipolar disorder and a candidate drug for various diseases such as amyotrophic lateral sclerosis, multiple sclerosis, and stroke. Despite being the captivating subject of many studies, the mechanism of lithium's therapeutic action remains unclear. To date, it has been shown that Li+ competes with Mg2+ and Na+ to normalize the activity of inositol and neurotransmitter-related signaling proteins, respectively. Furthermore, Li+ may co-bind with Mg2+-loaded adenosine or guanosine triphosphate to alter the complex's susceptibility to hydrolysis and mediate cellular signaling. Bipolar disorder patients exhibit abnormally high cytosolic Ca2+ levels and protein kinase C (PKC) hyperactivity that can be downregulated by long-term Li+ treatment. However, the possibility that monovalent Li+ could displace the bulkier divalent Ca2+ and inhibit PKC activity has not been considered. Here, using density functional theory calculations combined with continuum dielectric methods, we show that Li+ may displace the native dication from the positively charged trinuclear site in the C2 domain of cytosolic PKCα/γ. This would affect the membrane-docking ability of cytosolic PKCα/γ and reduce the abnormally high membrane-associated active PKCα/γ levels, thus downregulating the PKC hyperactivity found in bipolar patients.

Strontium Binding to α-Parvalbumin, a Canonical Calcium-Binding Protein of the "EF-Hand" Family.

Strontium salts are used for treatment of osteoporosis and bone cancer, but their impact on calcium-mediated physiological processes remains obscure. To explore Sr2+ interference with Ca2+ binding to proteins of the EF-hand family, we studied Sr2+/Ca2+ interaction with a canonical EF-hand protein, α-parvalbumin (α-PA). Evaluation of the equilibrium metal association constants for the active Ca2+ binding sites of recombinant human α-PA ('CD' and 'EF' sites) from fluorimetric titration experiments and isothermal titration calorimetry data gave 4 × 109 M-1 and 4 × 109 M-1 for Ca2+, and 2 × 107 M-1 and 2 × 106 M-1 for Sr2+. Inactivation of the EF site by homologous substitution of the Ca2+-coordinating Glu in position 12 of the EF-loop by Gln decreased Ca2+/Sr2+ affinity of the protein by an order of magnitude, whereas the analogous inactivation of the CD site induced much deeper suppression of the Ca2+/Sr2+ affinity. These results suggest that Sr2+ and Ca2+ bind to CD/EF sites of α-PA and the Ca2+/Sr2+ binding are sequential processes with the CD site being occupied first. Spectrofluorimetric Sr2+ titration of the Ca2+-loaded α-PA revealed presence of secondary Sr2+ binding site(s) with an apparent equilibrium association constant of 4 × 105 M-1. Fourier-transform infrared spectroscopy data evidence that Ca2+/Sr2+-loaded forms of α-PA exhibit similar states of their COO- groups. Near-UV circular dichroism (CD) data show that Ca2+/Sr2+ binding to α-PA induce similar changes in symmetry of microenvironment of its Phe residues. Far-UV CD experiments reveal that Ca2+/Sr2+ binding are accompanied by nearly identical changes in secondary structure of α-PA. Meanwhile, scanning calorimetry measurements show markedly lower Sr2+-induced increase in stability of tertiary structure of α-PA, compared to the Ca2+-induced effect. Theoretical modeling using Density Functional Theory computations with Polarizable Continuum Model calculations confirms that Ca2+-binding sites of α-PA are well protected against exchange of Ca2+ for Sr2+ regardless of coordination number of Sr2+, solvent exposure or rigidity of sites. The latter appears to be a key determinant of the Ca2+/Sr2+ selectivity. Overall, despite lowered affinity of α-PA to Sr2+, the latter competes with Ca2+ for the same EF-hands and induces similar structural rearrangements. The presence of a secondary Sr2+ binding site(s) could be a factor contributing to Sr2+ impact on the functional activity of proteins.