In Search for Low-Molecular-Weight Ligands of Human Serum Albumin That Affect Its Affinity for Monomeric Amyloid ß Peptide.

An imbalance between production and excretion of amyloid ß peptide (Aß) in the brain tissues of Alzheimer's disease (AD) patients leads to Aß accumulation and the formation of noxious Aß oligomers/plaques. A promising approach to AD prevention is the reduction of free Aß levels by directed enhancement of Aß binding to its natural depot, human serum albumin (HSA). We previously demonstrated the ability of specific low-molecular-weight ligands (LMWLs) in HSA to improve its affinity for Aß. Here we develop this approach through a bioinformatic search for the clinically approved AD-related LMWLs in HSA, followed by classification of the candidates according to the predicted location of their binding sites on the HSA surface, ranking of the candidates, and selective experimental validation of their impact on HSA affinity for Aß. The top 100 candidate LMWLs were classified into five clusters. The specific representatives of the different clusters exhibit dramatically different behavior, with 3- to 13-fold changes in equilibrium dissociation constants for the HSA-Aß40 interaction: prednisone favors HSA-Aß interaction, mefenamic acid shows the opposite effect, and levothyroxine exhibits bidirectional effects. Overall, the LMWLs in HSA chosen here provide a basis for drug repurposing for AD prevention, and for the search of medications promoting AD progression.

Ibuprofen Favors Binding of Amyloid-ß Peptide to Its Depot, Serum Albumin.

The deposition of amyloid-ß peptide (Aß) in the brain is a critical event in the progression of Alzheimer's disease (AD). This Aß deposition could be prevented by directed enhancement of Aß binding to its natural depot, human serum albumin (HSA). Previously, we revealed that specific endogenous ligands of HSA improve its affinity to monomeric Aß. We show here that an exogenous HSA ligand, ibuprofen (IBU), exerts the analogous effect. Plasmon resonance spectroscopy data evidence that a therapeutic IBU level increases HSA affinity to monomeric Aß40/Aß42 by a factor of 3-5. Using thioflavin T fluorescence assay and transmission electron microcopy, we show that IBU favors the suppression of Aß40 fibrillation by HSA. Molecular docking data indicate partial overlap between the IBU/Aß40-binding sites of HSA. The revealed enhancement of the HSA-Aß interaction by IBU and the strengthened inhibition of Aß fibrillation by HSA in the presence of IBU could contribute to the neuroprotective effects of the latter, previously observed in mouse and human studies of AD.

Interferon-ß Activity Is Affected by S100B Protein.

Interferon-ß (IFN-ß) is a pleiotropic cytokine secreted in response to various pathological conditions and is clinically used for therapy of multiple sclerosis. Its application for treatment of cancer, infections and pulmonary diseases is limited by incomplete understanding of regulatory mechanisms of its functioning. Recently, we reported that IFN-ß activity is affected by interactions with S100A1, S100A4, S100A6, and S100P proteins, which are members of the S100 protein family of multifunctional Ca2+-binding proteins possessing cytokine-like activities (Int J Mol Sci. 2020;21(24):9473). Here we show that IFN-ß interacts with one more representative of the S100 protein family, the S100B protein, involved in numerous oncological and neurological diseases. The use of chemical crosslinking, intrinsic fluorescence, and surface plasmon resonance spectroscopy revealed IFN-ß binding to Ca2+-loaded dimeric and monomeric forms of the S100B protein. Calcium depletion blocks the S100B-IFN-ß interaction. S100B monomerization increases its affinity to IFN-ß by 2.7 orders of magnitude (equilibrium dissociation constant of the complex reaches 47 pM). Crystal violet assay demonstrated that combined application of IFN-ß and S100B (5-25 nM) eliminates their inhibitory effects on MCF-7 cell viability. Bioinformatics analysis showed that the direct modulation of IFN-ß activity by the S100B protein described here could be relevant to progression of multiple oncological and neurological diseases.

Calcium-Bound S100P Protein Is a Promiscuous Binding Partner of the Four-Helical Cytokines.

S100 proteins are multifunctional calcium-binding proteins of vertebrates that act intracellularly, extracellularly, or both, and are engaged in the progression of many socially significant diseases. Their extracellular action is typically mediated by the recognition of specific receptor proteins. Recent studies indicate the ability of some S100 proteins to affect cytokine signaling through direct interaction with cytokines. S100P was shown to be the S100 protein most actively involved in interactions with some four-helical cytokines. To assess the selectivity of the S100P protein binding to four-helical cytokines, we have probed the interaction of Ca2+-bound recombinant human S100P with a panel of 32 four-helical human cytokines covering all structural families of this fold, using surface plasmon resonance spectroscopy. A total of 22 cytokines from all families of four-helical cytokines are S100P binders with the equilibrium dissociation constants, Kd, ranging from 1 nM to 3 µM (below the Kd value for the S100P complex with the V domain of its conventional receptor, receptor for advanced glycation end products, RAGE). Molecular docking and mutagenesis studies revealed the presence in the S100P molecule of a cytokine-binding site, which overlaps with the RAGE-binding site. Since S100 binding to four-helical cytokines inhibits their signaling in some cases, the revealed ability of the S100P protein to interact with ca. 71% of the four-helical cytokines indicates that S100P may serve as a poorly selective inhibitor of their action.

Specific S100 Proteins Bind Tumor Necrosis Factor and Inhibit Its Activity.

Tumor necrosis factor (TNF) inhibitors (anti-TNFs) represent a cornerstone of the treatment of various immune-mediated inflammatory diseases and are among the most commercially successful therapeutic agents. Knowledge of TNF binding partners is critical for identification of the factors able to affect clinical efficacy of the anti-TNFs. Here, we report that among eighteen representatives of the multifunctional S100 protein family, only S100A11, S100A12 and S100A13 interact with the soluble form of TNF (sTNF) in vitro. The lowest equilibrium dissociation constants (Kd) for the complexes with monomeric sTNF determined using surface plasmon resonance spectroscopy range from 2 nM to 28 nM. The apparent Kd values for the complexes of multimeric sTNF with S100A11/A12 estimated from fluorimetric titrations are 0.1-0.3 µM. S100A12/A13 suppress the cytotoxic activity of sTNF against Huh-7 cells, as evidenced by the MTT assay. Structural modeling indicates that the sTNF-S100 interactions may interfere with the sTNF recognition by the therapeutic anti-TNFs. Bioinformatics analysis reveals dysregulation of TNF and S100A11/A12/A13 in numerous disorders. Overall, we have shown a novel potential regulatory role of the extracellular forms of specific S100 proteins that may affect the efficacy of anti-TNF treatment in various diseases.

Serotonin Promotes Serum Albumin Interaction with the Monomeric Amyloid ß Peptide.

Prevention of amyloid ß peptide (Aß) deposition via facilitation of Aß binding to its natural depot, human serum albumin (HSA), is a promising approach to preclude Alzheimer's disease (AD) onset and progression. Previously, we demonstrated the ability of natural HSA ligands, fatty acids, to improve the affinity of this protein to monomeric Aß by a factor of 3 (BBRC, 510(2), 248-253). Using plasmon resonance spectroscopy, we show here that another HSA ligand related to AD pathogenesis, serotonin (SRO), increases the affinity of the Aß monomer to HSA by a factor of 7/17 for Aß40/Aß42, respectively. Meanwhile, the structurally homologous SRO precursor, tryptophan (TRP), does not affect HSA's affinity to monomeric Aß, despite slowdown of the association and dissociation processes. Crosslinking with glutaraldehyde and dynamic light scattering experiments reveal that, compared with the TRP-induced effects, SRO binding causes more marked changes in the quaternary structure of HSA. Furthermore, molecular docking reveals distinct structural differences between SRO/TRP complexes with HSA. The disintegration of the serotonergic system during AD pathogenesis may contribute to Aß release from HSA in the central nervous system due to impairment of the SRO-mediated Aß trapping by HSA.

Disulfide Dimerization of Neuronal Calcium Sensor-1: Implications for Zinc and Redox Signaling.

Neuronal calcium sensor-1 (NCS-1) is a four-EF-hand ubiquitous signaling protein modulating neuronal function and survival, which participates in neurodegeneration and carcinogenesis. NCS-1 recognizes specific sites on cellular membranes and regulates numerous targets, including G-protein coupled receptors and their kinases (GRKs). Here, with the use of cellular models and various biophysical and computational techniques, we demonstrate that NCS-1 is a redox-sensitive protein, which responds to oxidizing conditions by the formation of disulfide dimer (dNCS-1), involving its single, highly conservative cysteine C38. The dimer content is unaffected by the elevation of intracellular calcium levels but increases to 10-30% at high free zinc concentrations (characteristic of oxidative stress), which is accompanied by accumulation of the protein in punctual clusters in the perinuclear area. The formation of dNCS-1 represents a specific Zn2+-promoted process, requiring proper folding of the protein and occurring at redox potential values approaching apoptotic levels. The dimer binds Ca2+ only in one EF-hand per monomer, thereby representing a unique state, with decreased α-helicity and thermal stability, increased surface hydrophobicity, and markedly improved inhibitory activity against GRK1 due to 20-fold higher affinity towards the enzyme. Furthermore, dNCS-1 can coordinate zinc and, according to molecular modeling, has an asymmetrical structure and increased conformational flexibility of the subunits, which may underlie their enhanced target-binding properties. In HEK293 cells, dNCS-1 can be reduced by the thioredoxin system, otherwise accumulating as protein aggregates, which are degraded by the proteasome. Interestingly, NCS-1 silencing diminishes the susceptibility of Y79 cancer cells to oxidative stress-induced apoptosis, suggesting that NCS-1 may mediate redox-regulated pathways governing cell death/survival in response to oxidative conditions.

Interferon Beta Activity Is Modulated via Binding of Specific S100 Proteins.

Interferon-ß (IFN-ß) is a pleiotropic cytokine used for therapy of multiple sclerosis, which is also effective in suppression of viral and bacterial infections and cancer. Recently, we reported a highly specific interaction between IFN-ß and S100P lowering IFN-ß cytotoxicity to cancer cells (Int J Biol Macromol. 2020; 143: 633-639). S100P is a member of large family of multifunctional Ca2+-binding proteins with cytokine-like activities. To probe selectivity of IFN-ß-S100 interaction with respect to S100 proteins, we used surface plasmon resonance spectroscopy, chemical crosslinking, and crystal violet assay. Among the thirteen S100 proteins studied S100A1, S100A4, and S100A6 proteins exhibit strictly Ca2+-dependent binding to IFN-ß with equilibrium dissociation constants, Kd, of 0.04-1.5 µM for their Ca2+-bound homodimeric forms. Calcium depletion abolishes the S100-IFN-ß interactions. Monomerization of S100A1/A4/A6 decreases Kd values down to 0.11-1.0 nM. Interferon-α is unable of binding to the S100 proteins studied. S100A1/A4 proteins inhibit IFN-ß-induced suppression of MCF-7 cells viability. The revealed direct influence of specific S100 proteins on IFN-ß activity uncovers a novel regulatory role of particular S100 proteins, and opens up novel approaches to enhancement of therapeutic efficacy of IFN-ß.

Building kit for metal cation binding sites in proteins.

Starting with conformations of calcium-binding sites in parvalbumin and integrin (representative structures of EF-hand and calcium blade zones, respectively) we introduce four new different local Ca2+-recognition units in proteins: a one-residue unit type I (ORI); a three-residue unit type I (TRI); a one-residue unit type II (ORII) and a three-residue unit type II (TRII). Based on the amount and nature of variable atoms, the type I and II units theoretically can have four and twelve variants, respectively. Analysis of known "Ca2+-bound functional niches" in proteins revealed presence of almost all possible variants of Ca2+-recognition units in actual structures. Parvalbumin, integrin alpha-IIb and sixteen other proteins with different Ca2+-bound functional niches contain various consecutively joined combinations of OR(I/II) and TR(I/II) units. Such a OR(I/II)+TR(I/II) joint unit forms a tripeptide, which uses three main-chain atoms for metal binding: nitrogenn (Donor), oxygenn (Acceptor) and nitrogenn+2 (Donor). Thus, taken together, the described ORI, TRI, ORII and TRII units can serve as elementary blocks to construct more complex calcium recognizing substructures in a variety of calcium binding sites of unrelated proteins.

Novel calcium recognition constructions in proteins: Calcium blade and EF-hand zone.

Metal ions can regulate various cell processes being first, second or third messengers, and some of them, especially transition metal ions, take part in catalysis in many enzymes. As an intracellular ion, Ca2+ is involved in many cellular functions from fertilization and contraction, cell differentiation and proliferation, to apoptosis and cancer. Here, we have identified and described two novel calcium recognition environments in proteins: the calcium blade zone and the EF-hand zone, common to 12 and 8 different protein families, respectively. Each of the two environments contains three distinct structural elements: (a) the well-known characteristic Dx[DN]xDG motif; (b) an adjacent structurally identical segment, which binds metal ion in the same way between the calcium blade zone and the EF-hand zone; and (c) the following structurally variable segment, which distinguishes the calcium blade zone from the EF-hand zone. Both zones have sequence insertions between the last residue of the zone and calcium-binding residues in positions V or VI. The long insertion often connects the active and the calcium-binding sites in proteins. Using the structurally identical segments as an anchor, we were able to construct the classical calmodulin type EF-hand calcium-binding site out of two different calcium-binding motifs from two unrelated proteins.

Modulation of linoleic acid-binding properties of human serum albumin by divalent metal cations.

Human serum albumin (HSA) is an abundant multiligand carrier protein, linked to progression of Alzheimer's disease (AD). Blood HSA serves as a depot of amyloid ß (Aß) peptide. Aß peptide-buffering properties of HSA depend on interaction with its ligands. Some of the ligands, namely, linoleic acid (LA), zinc and copper ions are involved into AD progression. To clarify the interplay between LA and metal ion binding to HSA, the dependence of LA binding to HSA on Zn2+, Cu2+, Mg2+ and Ca2+ levels and structural consequences of these interactions have been explored. Seven LA molecules are bound per HSA molecule in the absence of the metal ions. Zn2+ binding to HSA causes a loss of one bound LA molecule, while the other metals studied exert an opposite effect (1-2 extra LA molecules are bound). In most cases, the observed effects are not related to the metal-induced changes in HSA quaternary structure. However, the Zn2+-induced decline in LA capacity of HSA could be due to accumulation of multimeric HSA forms. Opposite to Ca2+/Mg2+-binding, Zn2+ or Cu2+ association with HSA induces marked changes in its hydrophobic surface. Overall, the divalent metal ions modulate LA capacity and affinity of HSA to a different extent. LA- and Ca2+-binding to HSA synergistically support each other. Zn2+ and Cu2+ induce more pronounced changes in hydrophobic surface and quaternary structure of HSA and its LA capacity. A misbalanced metabolism of these ions in AD could modify interactions of HSA with LA, other fatty acids and hydrophobic substances, associated with AD.

Regulatory function of the C-terminal segment of guanylate cyclase-activating protein 2.

Neuronal responses to Ca2+-signals are provided by EF-hand-type neuronal Ca2+-sensor (NCS) proteins, which have similar core domains containing Ca2+-binding and target-recognizing sites. NCS proteins vary in functional specificity, probably depending on the structure and conformation of their non-conserved C-terminal segments. Here, we investigated the role of the C-terminal segment in guanylate cyclase activating protein-2, GCAP2, an NCS protein controlling the Ca2+-dependent regulation of photoreceptor guanylate cyclases. We obtained two chimeric proteins by exchanging C-terminal segments between GCAP2 and its photoreceptor homolog recoverin, a Ca2+-sensor controlling rhodopsin kinase (RK) activity. The exchange affected neither the structural integrity of GCAP2 and recoverin nor the Ca2+-sensitivity of GCAP2. Intrinsic fluorescence, circular dichroism, biochemical studies and hydrophobic dye probing revealed Ca2+-dependent conformational transition of the C-terminal segment of GCAP2 occurring in the molecular environment of both proteins. In Ca2+-GCAP2, the C-terminal segment was constrained and its replacement provided the protein with approximately two-fold inhibitory activity towards RK, suggesting that the segment contributes to specific target recognition by interfering with RK-binding. Upon Ca2+-release, it became less constrained and more available for phosphorylation by cyclic nucleotide-dependent protein kinase. The transition from the Ca2+-bound to the apo-state exposed hydrophobic sites in GCAP2, and was associated with its activating function without affecting its dimerization. The released C-terminal segment participated further in photoreceptor membrane binding making it sensitive to phosphorylation. Thus, the C-terminal segment in GCAP2 confers target selectivity, facilitates membrane binding and provides sensitivity of the membrane localization of the protein to phosphorylation by signaling kinases.

Expression, Purification, and Characterization of Interleukin-11 Orthologues.

Interleukin-11 (IL-11) is a multifunctional cytokine implicated in several normal and pathological processes. The decoding of IL-11 function and development of IL-11-targeted drugs dictate the use of laboratory animals and need of the better understanding of species specificity of IL-11 signaling. Here, we present a method for the recombinant interleukin-11 (rIL-11) production from the important model animals, mouse and macaque. The purified mouse and macaque rIL-11 interact with extracellular domain of human IL-11 receptor subunit α and activate STAT3 signaling in HEK293 cells co-expressing human IL-11 receptors with efficacies resembling those of human rIL-11. Hence, the evolutionary divergence does not impair IL-11 signaling. Furthermore, compared to human rIL-11 its macaque orthologue is 8-fold more effective STAT3 activator, which favors its use for treatment of thrombocytopenia as a potent substitute for human rIL-11. Compared to IL-6, IL-11 signaling exhibits lower species specificity, likely due to less conserved intrinsic disorder propensity within IL-6 orthologues. The developed express method for preparation of functionally active macaque/mouse rIL-11 samples is suited for exploration of the molecular mechanisms underlying IL-11 action and for development of the drug candidates for therapy of oncologic/hematologic/inflammatory diseases related to IL-11 signaling.

High-affinity interaction between interleukin-11 and S100P protein.

Interleukin-11 (IL-11) and S100P are oncoproteins co-expressed in numerous cancers, which might favor their interaction during oncogenesis. We have explored the possibility of this interaction by surface plasmon resonance spectroscopy, intrinsic fluorescence, and chemical crosslinking. Recombinant forms of IL-11 and S100P interact with each other under physiological level of calcium ions. IL-11 molecule has at least two S100P-binding sites with dissociation constants of 32 nM and 288 nM, which is 5-13-fold lower than its affinity to extracellular domain of IL-11 receptor subunit α. S100P does not alter IL-11-induced STAT3 activation in HEK293 cells co-expressing IL-11 receptors, but could affect other tumorigenic signaling pathways. The highly specific IL-11 - S100P interaction occurring under physiologically relevant conditions should be taken into consideration upon development of the antineoplastics inhibiting IL-11 signaling.

Effects of osmolytes on protein-solvent interactions in crowded environment: Analyzing the effect of TMAO on proteins in crowded solutions.

We analyzed the effect of a natural osmolyte, trimethylamine N-oxide (TMAO), on structural properties and conformational stabilities of several proteins under macromolecular crowding conditions by a set of biophysical techniques. We also used the solvent interaction analysis method to look at the peculiarities of the TMAO-protein interactions under crowded conditions. To this end, we analyzed the partitioning of these proteins in TMAO-free and TMAO-containing aqueous two-phase systems (ATPSs). These ATPSs had the same polymer composition of 6.0 wt.% PEG-8000 and 12.0 wt.% dextran-75, and same ionic composition of 0.01 M K/NaPB, pH 7.4. These analyses revealed that there is no direct interaction of TMAO with proteins, suggesting that the TMAO effects on the protein structure in crowded solutions occur via the effects of this osmolyte on solvent properties of aqueous media. The effects of TMAO on protein structure in the presence of polymers were rather complex and protein-specific. Curiously, our study revealed that in highly concentrated polymer solutions, TMAO does not always act to promote further protein folding.

Sequence microheterogeneity of parvalbumin, the major fish allergen.

The microheterogeneity of amino acid sequence observed in various allergens may affect immune response, but incidence of sequence microheterogeneity in allergens and its relation to their allergenicity are unclear. The occurrence of sequence microheterogeneity in major fish allergen, parvalbumin (PA), has been explored using bioinformatics approaches. 44% of 111 species with known PA sequence have PA isoforms. 41% of these species exhibit from 1 to 4 cases of PA sequence microheterogeneity, i.e. unique pairs of PA isoforms with sequence identity above 90%. 29% of 210 PA sequences studied are characterized by microheterogeneity. The occurrence of allergens among them is 2.5-fold higher than among other PAs. The incidence of sequence microheterogeneity within more allergenic ß isoform of PA is 2.0-fold lower than that for its less allergenic α isoform. 39 residues affected by PA microheterogeneity are concentrated in the region of helices A, B, F, while helices D and E are the most conservative region. 44% and 11% of the microheterogeneous substitutions are located in the species-specific and cross-reactive IgE-binding epitopes of PAs, respectively. 45% and 48% of the substitution cases are predicted to cause notable changes in protein disorder propensity and protein stability, respectively. Hence, the increased allergenicity rate among PAs having microheterogeneous isoforms can be related to differences in their IgE-binding caused directly or in an allosteric manner. Overall, sequence microheterogeneity is shown to be inherent to many of PAs and likely contributes to PA allergenicity.

Generic structures of cytotoxic liprotides: nano-sized complexes with oleic acid cores and shells of disordered proteins.

The cytotoxic complex formed between α-lactalbumin and oleic acid (OA) has inspired many studies on protein-fatty acid complexes, but structural insight remains sparse. After having used small-angle X-ray scattering (SAXS) to obtain structural information, we present a new, generic structural model of cytotoxic protein-oleic acid complexes, which we have termed liprotides (lipids and partially denatured proteins). Twelve liprotides formed from seven structurally unrelated proteins and prepared by different procedures all displayed core-shell structures, each with a micellar OA core and a shell consisting of flexible, partially unfolded protein, which stabilizes the OA micelle. The common structure explains similar effects exerted on cells by different liprotides and is consistent with a cargo off-loading of the OA into cell membranes.

The active site of the SGNH hydrolase-like fold proteins: Nucleophile-oxyanion (Nuc-Oxy) and Acid-Base zones.

SGNH hydrolase-like fold proteins are serine proteases with the default Asp-His-Ser catalytic triad. Here, we show that these proteins share two unique conserved structural organizations around the active site: (1) the Nuc-Oxy Zone around the catalytic nucleophile and the oxyanion hole, and (2) the Acid-Base Zone around the catalytic acid and base. The Nuc-Oxy Zone consists of 14 amino acids cross-linked with eight conserved intra- and inter-block hydrogen bonds. The Acid-Base Zone is constructed from a single fragment of the polypeptide chain, which incorporates both the catalytic acid and base, and whose N- and C-terminal residues are linked together by a conserved hydrogen bond. The Nuc-Oxy and Acid-Base Zones are connected by an SHLink, a two-bond conserved interaction from amino acids, adjacent to the catalytic nucleophile and base.

Recognition of granulocyte-macrophage colony-stimulating factor by specific S100 proteins.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic myelopoietic growth factor and proinflammatory cytokine, clinically used for multiple indications and serving as a promising target for treatment of many disorders, including cancer, multiple sclerosis, rheumatoid arthritis, psoriasis, asthma, COVID-19. We have previously shown that dimeric Ca2+-bound forms of S100A6 and S100P proteins, members of the multifunctional S100 protein family, are specific to GM-CSF. To probe selectivity of these interactions, the affinity of recombinant human GM-CSF to dimeric Ca2+-loaded forms of 18 recombinant human S100 proteins was studied by surface plasmon resonance spectroscopy. Of them, only S100A4 protein specifically binds to GM-CSF with equilibrium dissociation constant, Kd, values of 0.3-2 µM, as confirmed by intrinsic fluorescence and chemical crosslinking data. Calcium removal prevents S100A4 binding to GM-CSF, whereas monomerization of S100A4/A6/P proteins disrupts S100A4/A6 interaction with GM-CSF and induces a slight decrease in S100P affinity for GM-CSF. Structural modelling indicates the presence in the GM-CSF molecule of a conserved S100A4/A6/P-binding site, consisting of the residues from its termini, helices I and III, some of which are involved in the interaction with GM-CSF receptors. The predicted involvement of the 'hinge' region and F89 residue of S100P in GM-CSF recognition was confirmed by mutagenesis. Examination of S100A4/A6/P ability to affect GM-CSF signaling showed that S100A4/A6 inhibit GM-CSF-induced suppression of viability of monocytic THP-1 cells. The ability of the S100 proteins to modulate GM-CSF activity is relevant to progression of various neoplasms and other diseases, according to bioinformatics analysis. The direct regulation of GM-CSF signaling by extracellular forms of the S100 proteins should be taken into account in the clinical use of GM-CSF and development of the therapeutic interventions targeting GM-CSF or its receptors.

Structural characterization of more potent alternatives to HAMLET, a tumoricidal complex of α-lactalbumin and oleic acid.

HAMLET is a complex of human α-lactalbumin (hLA) with oleic acid (OA) that kills various tumor cells and strains of Streptococcus pneumoniae. More potent protein-OA complexes were previously reported for bovine α-lactalbumin (bLA) and ß-lactoglobulin (bLG), and pike parvalbumin (pPA), and here we explore their structural features. The concentration dependencies of the tryptophan fluorescence of hLA, bLA, and bLG complexes with OA reveal their disintegration at protein concentrations below the micromolar level. Chemical cross-linking experiments provide evidence that association with OA shifts the distribution of oligomeric forms of hLA, bLA, bLG, and pPA toward higher-order oligomers. This effect is confirmed for bLA and bLG using the dynamic light scattering method, while pPA is shown to associate with OA vesicles. Like hLA binding, OA binding increases the affinity of bLG for small unilamellar dipalmitoylphosphatidylcholine vesicles, while pPA efficiently binds to the vesicles irrespective of OA binding. The association of OA with bLG and pPA increases their α-helix and cross-ß-sheet content and resistance to enzymatic proteolysis, which is indicative of OA-induced protein structuring. The lack of excess heat sorption during melting of bLG and pPA in complex with OA and the presence of a cooperative thermal transition at the level of their secondary structure suggest that the OA-bound forms of bLG and pPA lack a fixed tertiary structure but exhibit a continuous thermal transition. Overall, despite marked differences, the HAMLET-like complexes that were studied exhibit a common feature: a tendency toward protein oligomerization. Because OA-induced oligomerization has been reported for other proteins, this phenomenon is inherent to many proteins.