Highly specific interaction of monomeric S100P protein with interferon beta.

S100 proteins are EF-hand calcium-binding proteins of vertebrates exerting numerous intra- and extracellular actions and involved into multiple diseases. Some of S100 proteins serve as extracellular damage signals via interaction with receptors. Although several S100 proteins directly bind specific cytokines, this phenomenon remains underexplored. Using chemical crosslinking, intrinsic fluorescence and surface plasmon resonance spectroscopies, we show that S100P protein interacts with interferon beta (IFN-ß) depending on calcium level and oligomeric state of S100P. Dimeric Ca2+-loaded S100P binds IFN-ß with equilibrium dissociation constants, Kd, of 0.05-0.6 µM. S100P monomerization favors this interaction decreasing Kd values down to 0.3-2 nM. Calcium depletion drastically lowers S100P affinity to IFN-ß. Other related EF-hand proteins studied (calmodulin, α-parvalbumin and S100G) do not bind IFN-ß, thereby confirming selectivity of the S100P - IFN-ß interaction. Crystal violet assay reveals that the S100P binding suppresses IFN-ß cytotoxicity to MCF-7 breast cancer cells. Since several cancers (breast, colon, lung, liver, etc.) exhibit dysregulated functioning of S100P and IFN-ß, their interaction could be relevant to the cancer progression and directed therapeutic interventions.

Monomeric state of S100P protein: Experimental and molecular dynamics study.

S100 proteins constitute a large subfamily of the EF-hand superfamily of calcium binding proteins. They possess one classical EF-hand Ca2+-binding domain and an atypical EF-hand domain. Most of the S100 proteins form stable symmetric homodimers. An analysis of literature data on S100 proteins showed that their physiological concentrations could be much lower than dissociation constants of their dimeric forms. It means that just monomeric forms of these proteins are important for their functioning. In the present work, thermal denaturation of apo-S100P protein monitored by intrinsic tyrosine fluorescence has been studied at various protein concentrations within the region from 0.04-10 µM. A transition from the dimeric to monomeric form results in a decrease in protein thermal stability shifting the mid-transition temperature from 85 to 75 °C. Monomeric S100P immobilized on the surface of a sensor chip of a surface plasmon resonance instrument forms calcium dependent 1 to 1 complexes with human interleukin-11 (equilibrium dissociation constant 1.2 nM). In contrast, immobilized interleukin-11 binds two molecules of dimeric S100P with dissociation constants of 32 nM and 288 nM. Since effective dissociation constant of dimeric S100P protein is very low (0.5 µM as evaluated from our data) the sensitivity of the existing physical methods does not allow carrying out a detailed study of S100P monomer properties. For this reason, we have used molecular dynamics methods to evaluate structural changes in S100P upon its transition from the dimeric to monomeric state. 80-ns molecular dynamics simulations of kinetics of formation of S100P, S100B and S100A11 monomers from the corresponding dimers have been carried out. It was found that during the transition from the homo-dimer to monomer form, the three S100 monomer structures undergo the following changes: (1) the helices in the four-helix bundles within each monomer rotate in order to shield the exposed non-polar residues; (2) almost all lost contacts at the dimer interface are substituted with equivalent and newly formed interactions inside each monomer, and new stabilizing interactions are formed; and (3) all monomers recreate functional hydrophobic cores. The results of the present study show that both dimeric and monomeric forms of S100 proteins can be functional.

Interleukin-11 binds specific EF-hand proteins via their conserved structural motifs.

Interleukin-11 (IL-11) is a hematopoietic cytokine engaged in numerous biological processes and validated as a target for treatment of various cancers. IL-11 contains intrinsically disordered regions that might recognize multiple targets. Recently we found that aside from IL-11RA and gp130 receptors, IL-11 interacts with calcium sensor protein S100P. Strict calcium dependence of this interaction suggests a possibility of IL-11 interaction with other calcium sensor proteins. Here we probed specificity of IL-11 to calcium-binding proteins of various types: calcium sensors of the EF-hand family (calmodulin, S100B and neuronal calcium sensors: recoverin, NCS-1, GCAP-1, GCAP-2), calcium buffers of the EF-hand family (S100G, oncomodulin), and a non-EF-hand calcium buffer (α-lactalbumin). A specific subset of the calcium sensor proteins (calmodulin, S100B, NCS-1, GCAP-1/2) exhibits metal-dependent binding of IL-11 with dissociation constants of 1-19 µM. These proteins share several amino acid residues belonging to conservative structural motifs of the EF-hand proteins, 'black' and 'gray' clusters. Replacements of the respective S100P residues by alanine drastically decrease its affinity to IL-11, suggesting their involvement into the association process. Secondary structure and accessibility of the hinge region of the EF-hand proteins studied are predicted to control specificity and selectivity of their binding to IL-11. The IL-11 interaction with the EF-hand proteins is expected to occur under numerous pathological conditions, accompanied by disintegration of plasma membrane and efflux of cellular components into the extracellular milieu.

Negative modulation of signal transduction via interleukin splice variation.

Interleukin 6 (IL-6) belongs to a large group of secreted proteins called cytokines functioning to mediate and regulate immunity, inflammation, and hematopoiesis with direct effects on cell proliferation, apoptosis, and differentiation. Along with the IL-6 protein, two of its splice variants, IL-6delta2 and IL-6delta4, were reported to be transcribed or expressed in vivo in human, and the mRNAs of IL-6delta3 and IL-6delta5 had been observed in mouse. While the existence of different splice variants of IL-6 has been shown, very little is known on how the structural modifications of IL-6 resulting from the formation of the different splice variants may alter cytokine functions. We have analyzed the potential effects splicing would have on interactions with the cell surface receptor complex. We (1) constructed three-dimensional structures of the IL-6 splice variants, IL-6delta2, IL-6delta3, and IL-6delta4, with the assumption that an interleukin splice variant as a folded protein should retain a functional hydrophobic core; (2) reconstructed the ternary structural complexes consisting of the modeled IL-6 splice variants, the IL-6 receptor molecule (IL-6R) and the dimeric signal-transducing protein, gp130, and (3) analyzed all complexes and made comparisons with the X-ray structure of the wild-type IL-6 complex. We identified three separate sites on IL-6 where interactions are made with IL-6R and with each of the two copies of gp130. The structural consequences of losing an exon lead to a unique pattern of lost interaction with different components of the receptor complex. Thus, in IL-6 and its splice variants, the exons appear to have compartmentalized roles contributing to the combined function of the cytokine. The modeled interactions suggest that splice variants could act as antagonists, and that IL-6delta2, missing the signal peptide, would be a cytoplasmic protein and be released and interact with nearby cell-surface receptors when cells are damaged. We argue that in the case of IL-6, helix E may act as a "silent secondary structure," which only has an active role when it substitutes for a part of the hydrophobic core, for example, replacing helix A in IL-6delta2.

Novel CalphaNN structural motif for protein recognition of phosphate ions.

Phosphate is one of the most frequently exploited chemical moieties in nature, present in a wide range of naturally occurring and critically important small molecules. Several phosphate group recognition motifs have been found for a few narrow groups of proteins, but for many protein families and folds the mode of phosphate recognition remains unclear. Here, we have analyzed the structures of all fold-representative protein-ligand complexes listed in the FSSP database, regardless of whether the bound ligand included a phosphate group. Based on a phosphate-binding motif that we identified in pyridoxal phosphate binding proteins, we have identified a new anion-binding structural motif, CalphaNN, common to 104 fold-representative protein structures that belong to 62 different folds, of which 86% of the fold-representative structures (51 folds) bind phosphate or lone sulfate ions. This motif leads to a precise mode for phosphate group recognition forming a structure where atoms of the phosphate group occupy the most favorable stabilizing positions. The anion-binding CalphaNN motif is based only on main-chain atoms from three adjacent residues, has a conservative betaalphaalpha or betaalphabeta geometry, and recognizes the free phosphate (sulfate) ion as well as one or more phosphate groups in nucleotides and in a variety of cofactors. Moreover, the CalphaNN motif is positioned in functionally important regions of protein structures and often residues of the motif directly participate in the function of the protein.

"Acceptor-donor-acceptor" motifs recognize the Watson-Crick, Hoogsteen and Sugar "donor-acceptor-donor" edges of adenine and adenosine-containing ligands.

Nucleotides are among the most extensively exploited chemical moieties in nature and, as a part of a handful of different protein ligands, nucleotides play key roles in energy transduction, enzymatic catalysis and regulation of protein function. We have previously reported that in many proteins with different folds and functions a distinctive adenine-binding motif is involved in the recognition of the Watson-Crick edge of adenine. Here, we show that many proteins do have clear structural motifs that recognize adenosine (and some other nucleotides and nucleotide analogs) not only through the Watson-Crick edge, but also through the sugar and Hoogsteen edges. Each of the three edges of adenosine has a donor-acceptor-donor (DAD) pattern that is often recognized by proteins via a complementary acceptor-donor-acceptor (ADA) motif, whereby three distinct hydrogen bonds are formed: two conventional N-H...O and N-H...N hydrogen bonds, and one weak C-H...O hydrogen bond. The local conformation of the adenine-binding loop is betabetabeta or betabetaalpha and reflects the mode of nucleotide binding. Additionally, we report 21 proteins from five different folds that simultaneously recognize both the sugar edge and the Watson-Crick edge of adenine. In these proteins a unique beta-loop-beta supersecondary structure grasps an adenine-containing ligand between two identical adenine-binding motifs as part of the betaalphabeta-loop-beta fold.

Phosphate group binding "cup" of PLP-dependent and non-PLP-dependent enzymes: leitmotif and variations.

Pyridoxal-5'-phosphate (PLP) is widely used by many enzymes in reactions where amino acids are interconverted. Whereas the role of the pyridoxal ring in catalysis is well understood, the functional role of the single phosphate group in PLP has been less studied. Here we construct unambiguous connection diagrams that describe the interactions among the three non-ester phosphate oxygen atoms of PLP and surrounding atoms from the protein binding site and from water molecules, the so-called phosphate group binding "cup". These diagrams provide a simple means to identify common recognition motifs for the phosphate group in both similar and different protein folds. Diagrams were constructed and compared in the cases of five newly determined structures of PLP-dependent transferases (fold type I enzymes) and, additionally, two non-PLP protein complexes (indole-3-glycerol phosphate synthase (IGPS) with bound indole-3-glycerol phosphate (IGP) and old yellow enzyme (OYE) with bound flavin mononucleotide (FMN)). A detailed comparison of the diagrams shows that three positions out of ten in the structure of the phosphate group binding "cup" contain invariant atoms, while seven others are occupied by conserved atom types. This level of similarity was also observed in the fold type III (TIM beta/alpha-barrel) enzymes that bind three different ligands: PLP, IGP and FMN.

Functional attributes of the phosphate group binding cup of pyridoxal phosphate-dependent enzymes.

Twenty-four structures of pyridoxal-5'-phosphate (PLP)-dependent enzymes that represent five different folds are shown to share a common recognition pattern for the phosphate group of their PLP-ligands. All atoms that interact with the phosphate group of PLP in these proteins are organized within a two-layer structure so that the first interacting layer contains from five to seven atoms and parallel with this is a second layer containing from three to seven interacting atoms. In order to identify features of the phosphate-binding site common to PLP-dependent enzymes, a simple procedure is described that assigns relative positions to all interacting atoms unambiguously, such that the networks of interactions for different proteins can be compared. On the basis of these diagrams for 24 enzyme-cofactor complexes, a detailed comparison of the two-layer structures of PLP-dependent enzymes, with both similar and different folds, was made. A majority of the structurally defined PLP-dependent proteins use the same atom types in analogous "key" positions to bind their PLP-ligands. In some instances, proteins use water molecules when a key position is unoccupied. A similar two-layer recognition pattern extends to protein recognition of at least one other, non-PLP ligand, glucosamine 6-phosphate. We refer to this three-dimensional recognition pattern as the phosphate-binding cup. In general, the phosphate-binding cup provides a very stable anchoring point for PLP. When numerous water molecules occur within the cup, however, then the phosphate group of PLP participates directly in the enzymatic reactions with inorganic phosphate replacing the water molecules of the cup. With glucosamine-6-phosphate synthase, the water molecules of the phosphate-binding cup facilitate the entry of substrate and the exit of product.