ABSTRACT
First-line treatment of multiple myeloma, a prevalent blood cancer lacking a cure, using anti-CD38 daratumumab antibody and lenalidomide is often inadequate due to relapse and severe side effects. To enhance drug safety and efficacy, an antibody-drug conjugate, TE-1146, comprising six lenalidomide drug molecules site-specifically conjugated to a reconfigured daratumumab to deliver cytotoxic lenalidomide to tumor cells is developed. TE-1146 is prepared using the HighDAR platform, which employs i) a maleimide-containing "multi-arm linker" to conjugate multiple drug molecules creating a drug bundle, and ii) a designed peptide with a Zn2+-binding cysteine at the C-termini of a reconfigured daratumumab for site-specific drug bundle conjugation. It is shown that TE-1146 remains intact and effectively enters CD38-expressing tumor cells, releasing lenalidomide, leading to enhanced cell-killing effects compared to lenalidomide/daratumumab alone or their combination. This reveals the remarkable potency of lenalidomide once internalized by myeloma cells. TE-1146 precisely delivers lenalidomide to target CD38-overexpressing tumor cells. In contrast, lenalidomide without daratumumab cannot easily enter cells, whereas daratumumab without lenalidomide relies on Fc-dependent effector functions to kill tumor cells.
Subject(s)
Antibodies, Monoclonal , Immunoconjugates , Lenalidomide , Multiple Myeloma , Multiple Myeloma/drug therapy , Humans , Immunoconjugates/pharmacology , Immunoconjugates/chemistry , Lenalidomide/pharmacology , Lenalidomide/therapeutic use , Antibodies, Monoclonal/pharmacology , Cell Line, Tumor , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Mice , Animals , Disease Models, AnimalABSTRACT
Antibodies conjugated with diagnostic/therapeutic radionuclides are attractive options for inoperable cancers lacking accurate imaging methods and effective therapeutics, such as pancreatic cancer. Hence, we have produced an antibody radionuclide conjugate termed TE-1132 comprising a α-CA19-9 scFv-Fc that is site-specifically conjugated at each C-terminus to 3 DOTA chelators via a cysteine-containing peptide linker. The smaller scFv-Fc size facilitates diffusivity within solid tumors, whereas the chelator-to-antibody ratio of six enabled 177Lu-radiolabeled TE-1132 to exhibit high radioactivity up to 520 MBq/nmol. In mice bearing BxPC3 tumors, immuno-SPECT/CT imaging of [111In]In-TE-1132 and the biodistribution of [177Lu]Lu-TE-1132 showed selective tumor accumulation. Single and multiple doses of [177Lu]Lu-TE-1132 effectively inhibited the BxPC3 tumor growth and prolonged the survival of mice with no irreversible body weight loss or hematopoietic damage. The adequate pharmacokinetic parameters, prominent tumor accumulation, and efficacy with good safety in mice encourage the further investigation of theranostic TE-1132 for treating pancreatic cancer.
Subject(s)
Immunoconjugates , Pancreatic Neoplasms , Mice , Animals , Chelating Agents , CA-19-9 Antigen , Tissue Distribution , Pancreatic Neoplasms/diagnostic imaging , Pancreatic Neoplasms/drug therapy , Radiopharmaceuticals/pharmacokinetics , Cell Line, Tumor , Lutetium , Pancreatic NeoplasmsABSTRACT
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+.
Subject(s)
Calcium , Strontium , Strontium/chemistry , Ligands , Calcium/chemistry , Binding Sites , SolventsABSTRACT
The primary cilium, a microtubule-based sensory organelle, undergoes cycles of assembly and disassembly that govern the cell cycle progression critical to cell proliferation and differentiation. Although cilia assembly has been studied extensively, the molecular mechanisms underlying cilia disassembly are less well understood. Here, we uncover a γ-tubulin ring complex (γ-TuRC)-dependent pathway that promotes cilia disassembly and thereby prevents cilia formation. We further demonstrate that Kif2A, a kinesin motor that bears microtubule-depolymerizing activity, is recruited to the cilium basal body in a γ-TuRC-dependent manner. Our mechanistic analyses show that γ-TuRC specifically recruits Kif2A via the GCP2 subunit and its binding partner Mzt2. Hence, despite the long-standing view that γ-TuRC acts mainly as a microtubule template, we illustrate that its functional heterogeneity at the basal body facilitates both microtubule nucleation and Kif2A recruitment-mediated regulation of ciliogenesis, ensuring cell cycle progression.
Subject(s)
Microtubule-Associated Proteins , Tubulin , Tubulin/metabolism , Microtubule-Associated Proteins/metabolism , Cilia/metabolism , Microtubule-Organizing Center/metabolism , Microtubules/metabolismABSTRACT
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.
Subject(s)
Lithium , Protein Kinase C-alpha , Binding Sites , Calcium/metabolism , Cations , Humans , Protein Kinase C-alpha/metabolism , Static ElectricityABSTRACT
Opening of two-pore domain K+ channels (K2Ps) is regulated by various external cues, such as pH, membrane tension, or temperature, which allosterically modulate the selectivity filter (SF) gate. However, how these cues cause conformational changes in the SF of some K2P channels remains unclear. Herein, we investigate the mechanisms by which extracellular pH affects gating in an alkaline-activated K2P channel, TALK1, using electrophysiology and molecular dynamics (MD) simulations. We show that R233, located at the N-terminal end of transmembrane segment 4, is the primary pHo sensor. This residue distally regulates the orientation of the carbonyl group at the S1 potassium-binding site through an interacting network composed of residues on transmembrane segment 4, the pore helix domain 1, and the SF. Moreover, in the presence of divalent cations, we found the acidic pH-activated R233E mutant recapitulates the network interactions of protonated R233. Intriguingly, our data further suggested stochastic coupling between R233 and the SF gate, which can be described by an allosteric gating model. We propose that this allosteric model could predict the hybrid pH sensitivity in heterodimeric channels with alkaline-activated and acidic-activated K2P subunits.
Subject(s)
Ion Channel Gating , Potassium Channels, Tandem Pore Domain , Hydrogen-Ion Concentration , Ion Channel Gating/physiology , Molecular Dynamics Simulation , Potassium Channels, Tandem Pore Domain/metabolismABSTRACT
One strategy to counter viruses that persistently cause outbreaks is to design molecules that can specifically inhibit an essential multifunctional viral protease. Herein, we present such a strategy using well-established methods to first identify a region present only in viral (but not human) proteases and find peptides that can bind specifically to this "unique" region by maximizing the protease-peptide binding free energy iteratively using single-point mutations starting with the substrate peptide. We applied this strategy to discover pseudosubstrate peptide inhibitors for the multifunctional 2A protease of enterovirus 71 (EV71), a key causative pathogen for hand-foot-and-mouth disease affecting young children, along with coxsackievirus A16. Four peptide candidates predicted to bind EV71 2A protease more tightly than the natural substrate were experimentally validated and found to inhibit protease activity. Furthermore, the crystal structure of the best pseudosubstrate peptide bound to the EV71 2A protease was determined to provide a molecular basis for the observed inhibition. Since the 2A proteases of EV71 and coxsackievirus A16 share nearly identical sequences and structures, our pseudosubstrate peptide inhibitor may prove useful in inhibiting the two key pathogens of hand-foot-and-mouth disease.
ABSTRACT
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.
Subject(s)
Calcium , Signal Transduction , Binding Sites , Cations , Cations, DivalentABSTRACT
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.
ABSTRACT
[This corrects the article DOI: 10.1039/D0SC02646H.].
ABSTRACT
Monophosphate, an essential component of nucleic acids, as well as cell membranes and signaling molecules, is often bound to metal cations. Despite the biological importance of monophosphate-containing cell-signaling or lipid molecules, their propensity to bind the two most abundant cellular dications, Mg2+ and Ca2+, in a particular mode (inner/outer shell, mono/bidentate) is not well understood. Whether they prefer binding to Mg2+ than to Ca2+ and if they can outcompete the carboxylates of excitatory Asp/Glu and inhibitory gamma-aminobutyric acid (GABA) neurotransmitters in binding to Mg2+/Ca2+ remain unclear. To address these questions, we modeled cyclic adenosine/guanosine monophosphate (cAMP/cGMP), nucleoside 2',3'-cyclic phosphate, phosphatidylinositol (PI), phosphatidylserine (PS), and phosphatidylethanolamine (PEA) and determined their most stable metal-binding modes, including those of Asp/Glu and GABA, as well as their selectivity for Mg2+/Ca2+ using density functional theory combined with the polarizable continuum model. The results obtained, which are consistent with the available experimental findings, reveal that the structurally and functionally diverse monophosphate-containing ligands studied prefer monodentate coordination of Mg2+ because of the greater strain encountered upon bidentate coordination, whereas the larger Ca2+ imposes less strain upon bidentate binding and has reduced/no preference for monodentate coordination. We further show that in a low-dielectric environment, negatively charged monophosphate-containing ligands favor the better charge-accepting dication, that is, Mg2+ rather than Ca2+. By promoting Mg2+ over Ca2+ binding, signaling monophosphates (cAMP/cGMP) do not entrap cellular Ca2+ and interfere with signal transduction processes employing Ca2+ as a second messenger. In regions with high glutamate cytoplasmic concentration, glutamate may sequester Mg2+ bound to isolated five-/six-membered ring phosphates, PI, or neutral PEA, but not anionic phospholipids constituting the inner leaflet of the cell membrane.
Subject(s)
Calcium/chemistry , Cyclic AMP/chemistry , Cyclic GMP/chemistry , Lipids/chemistry , Magnesium/chemistry , Models, Molecular , Molecular Structure , ThermodynamicsABSTRACT
Whole-cell biosensors are useful for monitoring heavy metal toxicity in public health and ecosystems, but their development has been hindered by intrinsic trade-offs between sensitivity and specificity. Here, we demonstrated an effective engineering solution by building a sensitive, specific, and high-response biosensor for carcinogenic cadmium ions. We genetically programmed the metal transport system of Escherichia coli to enrich intracellular cadmium ions and deprive interfering metal species. We then selected 16 cadmium-sensing transcription factors from the GenBank database and tested their reactivity to 14 metal ions in the engineered E. coli using the expression of the green fluorescent protein as the readout. The resulting cadmium biosensor was highly specific and showed a detection limit of 3 nM, a linear increase in fluorescent intensities from 0 to 200 nM, and a maximal 777-fold signal change. Using this whole-cell biosensor, a smartphone, and low-tech equipment, we developed a simple assay capable of measuring cadmium ions at the same concentration range in irrigation water and human urine. This method is user-friendly and cost-effective, making it affordable to screen large amounts of samples for cadmium toxicity in agriculture and medicine. Moreover, our work highlights natural gene repositories as a treasure chest for bioengineering.
Subject(s)
Biosensing Techniques , Cadmium , Ecosystem , Escherichia coli/genetics , Humans , MetalsABSTRACT
We present a near-term treatment strategy to tackle pandemic outbreaks of coronaviruses with no specific drugs/vaccines by combining evolutionary and physical principles to identify conserved viral domains containing druggable Zn-sites that can be targeted by clinically safe Zn-ejecting compounds. By applying this strategy to SARS-CoV-2 polyprotein-1ab, we predicted multiple labile Zn-sites in papain-like cysteine protease (PLpro), nsp10 transcription factor, and nsp13 helicase. These are attractive drug targets because they are highly conserved among coronaviruses and play vital structural/catalytic roles in viral proteins indispensable for virus replication. We show that five Zn-ejectors can release Zn2+ from PLpro and nsp10, and clinically-safe disulfiram and ebselen can not only covalently bind to the Zn-bound cysteines in both proteins, but also inhibit PLpro protease. We propose combining disulfiram/ebselen with broad-spectrum antivirals/drugs to target different conserved domains acting at various stages of the virus life cycle to synergistically inhibit SARS-CoV-2 replication and reduce the emergence of drug resistance.
ABSTRACT
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 Mg2+ 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 Mg2+and Ca2+ 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 Mg2+ and Ca2+. We show that the thermodynamic outcome of the competition between Mg2+ and Ca2+ 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, Mg2+. 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 Mg2+ compared to Ca2+, enables Mg2+ to outcompete Ca2+ in binding to these highly charged anions. This suggests an explanation for why nature has chosen Mg2+ but not Ca2+ to perform most of the essential tasks associated with biological triphosphates.
Subject(s)
Adenosine Diphosphate/chemistry , Adenosine Triphosphate/chemistry , Calcium , Guanosine Diphosphate/chemistry , Guanosine Triphosphate/chemistry , Magnesium/chemistry , Binding Sites , Calcium/chemistry , Databases, Protein , Density Functional Theory , ThermodynamicsABSTRACT
To facilitate proper mitotic cell partitioning, the Golgi disassembles by suppressing vesicle fusion. However, the underlying mechanism has not been characterized previously. Here, we report a Ran pathway-independent attenuation mechanism that allows Importin-α (a nuclear transport factor) to suppress the vesicle fusion mediated by p115 (a vesicular tethering factor) and is required for mitotic Golgi disassembly. We demonstrate that Importin-α directly competes with p115 for interaction with the Golgi protein GM130. This interaction, promoted by a phosphate moiety on GM130, is independent of Importin-ß and Ran. A GM130 K34A mutant, in which the Importin-α-GM130 interaction is specifically disrupted, exhibited abundant Golgi puncta during metaphase. Importantly, a mutant showing enhanced p115-GM130 interaction presented proliferative defects and G2/M arrest, demonstrating that Importin-α-GM130 binding modulates the Golgi disassembly that governs mitotic progression. Our findings illuminate that the Ran and kinase-phosphatase pathways regulate multiple aspects of mitosis coordinated by Importin-α (e.g. spindle assembly, Golgi disassembly).
Subject(s)
Autoantigens/metabolism , Golgi Apparatus/metabolism , Golgi Matrix Proteins/metabolism , Membrane Proteins/metabolism , Metaphase/physiology , Vesicular Transport Proteins/metabolism , alpha Karyopherins/metabolism , Autoantigens/genetics , Crystallography, X-Ray , G2 Phase Cell Cycle Checkpoints , HEK293 Cells , Humans , Membrane Fusion , Membrane Proteins/genetics , Mitosis/physiology , Phosphorylation , Protein Binding , beta Karyopherins/metabolism , ran GTP-Binding Protein/metabolismABSTRACT
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.
Subject(s)
Brain/cytology , Lithium/chemistry , Lithium/therapeutic use , Signal Transduction/drug effects , Animals , Brain/drug effects , Brain/metabolism , Brain/pathology , Humans , Lithium/pharmacologyABSTRACT
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 Zn2+ bound to ligands commonly found in both structural and catalytic Zn-sites, namely, Zn-CC(C/H)x (x = D, E, or H2O) 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) H2O sites.
Subject(s)
Models, Molecular , Proteins/chemistry , Proteins/metabolism , Zinc/metabolism , Binding Sites , Ligands , Protein ConformationABSTRACT
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
Abiogenic lead (Pb2+), 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 Pb2+ is known to be a potent competitor of Ca2+ in protein binding sites, why/how Pb2+ can compete with Ca2+ in proteins remains unclear, raising multiple outstanding questions, including the following: (1) What are the physicochemical factors governing the competition between Pb2+ and Ca2+? (2) Which Ca2+-binding sites in terms of the structure, composition, overall charge, flexibility, and solvent exposure are the most likely targets for Pb2+ attack? Using density functional theory combined with polarizable continuum model calculations, we address these questions by studying the thermodynamic outcome of the competition between Pb2+ and Ca2+ in various model Ca2+-binding sites, including those modeling voltage-gated calcium channel selectivity filters and EF-hand and non-EF-hand Ca2+-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 Pb2+ and Ca2+: If the Ca2+-binding site is relatively rigid and crowded with protein ligands, then Pb2+, upon binding, preserves the native metal-binding site geometry and at low concentrations, can act as an activator of the host protein. If the Ca2+-binding site is flexible and consists of only a few protein ligands, then Pb2+ can displace Ca2+ and deform the native metal-binding site geometry, resulting in protein malfunction.
Subject(s)
Calcium-Binding Proteins/chemistry , Calcium/metabolism , Density Functional Theory , Lead/chemistry , Binding Sites , Ligands , ThermodynamicsABSTRACT
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-shellsecond-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.