Pro-Inflammatory S100A8 and S100A9 Proteins: Self-Assembly into Multifunctional Native and Amyloid Complexes

S100A8 and S100A9 are EF-hand Ca(2+) binding proteins belonging to the S100 family. They are abundant in cytosol of phagocytes and play critical roles in numerous cellular processes such as motility and danger signaling by interacting and modulating the activity of target proteins. S100A8 and S100A9 expression levels increased in many types of cancer, neurodegenerative disorders, inflammatory and autoimmune diseases and they are implicated in the numerous disease pathologies. The Ca(2+) and Zn(2+)-binding properties of S100A8/A9 have a pivotal influence on their conformation and oligomerization state, including self-assembly into homo- and heterodimers, tetramers and larger oligomers. Here we review how the unique chemical and conformational properties of individual proteins and their structural plasticity at the quaternary level account for S100A8/A9 functional diversity. Additional functional diversification occurs via non-covalent assembly into oligomeric and fibrillar amyloid complexes discovered in the aging prostate and reproduced in vitro. This process is also regulated by Ca(2+)and Zn(2+)-binding and effectively competes with the formation of the native complexes. High intrinsic amyloid-forming capacity of S100A8/A9 proteins may lead to their amyloid depositions in numerous ailments characterized by their elevated expression patterns and have additional pathological significance requiring further thorough investigation.     

Directed evolution for the development of conformation-specific affinity reagents using yeast display

Yeast display is a powerful tool for increasing the affinity and thermal stability of scFv antibodies through directed evolution. Mammalian calmodulin (CaM) is a highly conserved signaling protein that undergoes structural changes upon Ca(2+) binding. In an attempt to generate conformation-specific antibodies for proteomic applications, a selection against CaM was undertaken. Flow cytometry-based screening strategies to isolate easily scFv recognizing CaM in either the Ca(2+)-bound (Ca(2+)-CaM) or Ca(2+)-free (apo-CaM) states are presented. Both full-length scFv and single-domain VH only clones were isolated. One scFv clone having very high affinity (K(d) = 0.8 nM) and specificity (>1000-fold) for Ca(2+)-CaM was obtained from de novo selections. Subsequent directed evolution allowed the development of antibodies with higher affinity (K(d) = 1 nM) and specificity (>300-fold) for apo-CaM from a parental single-domain clone with both a modest affinity and specificity for that particular isoform. CaM-binding activity was unexpectedly lost upon conversion of both conformation-specific clones into soluble fragments. However, these results demonstrate that conformation-specific antibodies can be quickly and easily isolated by directed evolution using the yeast display platform.     

Fragment docking to S100 proteins reveals a wide diversity of weak interaction sites

The S100 protein family is a highly conserved group of Ca(2+)-binding proteins that belong to the EF-hand type and are considered potential drug targets. In the present study we focused our attention on two members of the family: S100A13 and S100B; the former is involved in the nonclassical protein release of two proangiogenic polypeptides FGF-1 and IL-1alpha that are involved in inflammatory processes, whereas S100B is known to interact with the C-terminal domain of the intracellular tumor suppressor p53 and promote cancer development. We screened, using waterLOGSY NMR experiments, 430 molecules of a generic fragment library and we identified different hits for each protein. The subset of fragments interacting with S100B has very few members in common with the subset interacting with S100A13. From the (15)N-HSQC NMR spectra of the proteins in the presence of those hits the chemical shift differences Deltadelta(HN) were calculated, and the main regions of surface interaction were identified. A relatively large variety of interaction regions for various ligands were identified for the two proteins, including known or suggested protein-protein interaction sites.     

Structural Aspects and Prediction of Calmodulin-Binding Proteins

Calmodulin (CaM) is an important intracellular protein that binds Ca²⁺ and functions as a critical second messenger involved in numerous biological activities through extensive interactions with proteins and peptides. CaM’s ability to adapt to binding targets with different structures is related to the flexible central helix separating the N- and C-terminal lobes, which allows for conformational changes between extended and collapsed forms of the protein. CaM-binding targets are most often identified using prediction algorithms that utilize sequence and structural data to predict regions of peptides and proteins that can interact with CaM. In this review, we provide an overview of different CaM-binding proteins, the motifs through which they interact with CaM, and shared properties that make them good binding partners for CaM. Additionally, we discuss the historical and current methods for predicting CaM binding, and the similarities and differences between these methods and their relative success at prediction. As new CaM-binding proteins are identified and classified, we will gain a broader understanding of the biological processes regulated through changes in Ca²⁺ concentration through interactions with CaM.     

Molecular modeling of single polypeptide chain of calcium-binding protein p26olf from dimeric S100B()

P26olf from olfactory tissue of frog, which may be involvedin olfactory transduction or adaptation, is a Ca²⁺-binding proteinwith 217 amino acids. The p26olf molecule contains two homologousparts consisting of the N-terminal half with amino acids 1–109and the C-terminal half with amino acids 110–217. Eachhalf resembles S100 protein with about 100 amino acids and containstwo helix–loop–helix Ca²⁺-binding structural motifsknown as EF-hands: a normal EF-hand at the C-terminus and apseudo EF-hand at the N-terminus. Multiple alignment of thetwo S100-like domains of p26olf with 18 S100 proteins indicatedthat the C-terminal putative EF-hand of each domain containsa four-residue insertion when compared with the typical EF-handmotifs in the S100 protein, while the N-terminal EF-hand ishomologous to its pseudo EF-hand. We constructed a three-dimensionalmodel of the p26olf molecule based on results of the multiplealignment and NMR structures of dimeric S100B(ßß)in the Ca²⁺-free state. The predicted structure of the p26olfsingle polypeptide chain satisfactorily adopts a folding patternremarkably similar to dimeric S100B(ßß). Each domainof p26olf consists of a unicornate-type four-helix bundle andthey interact with each other in an antiparallel manner formingan X-type four-helix bundle between the two domains. The twoS100-like domains of p26olf are linked by a loop with no sterichindrance, suggesting that this loop might play an importantrole in the function of p26olf. The circular dichroism spectraldata support the predicted structure of p26olf and indicatethat Ca²⁺-dependent conformational changes occur. Since theC-terminal putative EF-hand of each domain fully keeps the helix–loop–helixmotif having a longer Ca²⁺-binding loop, regardless of the four-residueinsertion, we propose that it is a new, novel EF-hand, althoughit is unclear whether this EF-hand binds Ca²⁺. P26olf is a newmember of the S100 protein family.     

Further Evidence of the Melatonin Calmodulin Interaction: Effect on CaMKII Activity

Melatonin (MEL) is a pleiotropic indolamine that reaches multiple intracellular targets. Among these, MEL binds to calmodulin (CaM) with high affinity. In presence of Ca²⁺, CaM binds to CaM-dependent kinase II (CaMKII). The Ca²⁺-CaM/CaMKII pathway regulates a myriad of brain functions in different cellular compartments. Evidence showing the regulation of this cellular pathway by MEL is scarce. Thus, our main objective was to study the interaction of MEL with CaM and its effects on CaMKII activity in two microenvironments (aqueous and lipidic) naturally occurring within the cell. In addition, colocalization of MEL with CaM in vivo was explored in mice brain hippocampus. In vitro CaM-MEL interaction and the structural conformations of CaM in the presence of this indoleamine were assessed through electrophoretic mobility and isoelectric point. The functional consequence of this interaction was evaluated by measuring CaMKII activity. Ca²⁺-CaM-MEL increased the activity of CaMKII in aqueous buffer but reduced the kinase activity in lipid buffer. Importantly, MEL colocalizes in vivo with Ca²⁺-CaM in the hippocampus. Our evidence suggests that MEL regulates the key cellular Ca²⁺-CaM/CaMKII pathway and might explain why physiological MEL concentrations reduce CaMKII activity in some experimental conditions, while in others it drives biological processes through activation of this kinase.     

Evaluation of the Calmodulin‐SOX9 Interaction by “Magnetic Fishing” Coupled to Mass Spectrometry

Disruption of calmodulin (CaM)‐based protein interactions has been touted as a potential means for modulating several disease pathways. Among these is SOX9, which is a DNA binding protein that is involved in chrondrocyte differentiation and regulation of the hormones that control sexual development. In this work, we employed a “magnetic fishing”/mass spectrometry assay in conjunction with intrinsic fluorescence to examine the interaction of CaM with the CaM‐binding domain of SOX9 (SOX‐CAL), and to assess the modulation of this interaction by known anti‐CaM compounds. Our data show that there is a high affinity interaction between CaM and SOX‐CAL (27±9 nM ), and that SOX‐CAL bound to the same location as the well‐known CaM antagonist melittin; unexpectedly, we also found that addition of CaM‐binding small molecules initially produced increased SOX‐CAL binding, indicative of binding to both the well‐known high‐affinity CaM binding site and a second, lower‐affinity binding site.     

Decreased Interactions between Calmodulin and a Mutant Huntingtin Model Might Reduce the Cytotoxic Level of Intracellular Ca2+: A Molecular Dynamics Study

Mutant huntingtin (m-HTT) proteins and calmodulin (CaM) co-localize in the cerebral cortex with significant effects on the intracellular calcium levels by altering the specific calcium-mediated signals. Furthermore, the mutant huntingtin proteins show great affinity for CaM that can lead to a further stabilization of the mutant huntingtin aggregates. In this context, the present study focuses on describing the interactions between CaM and two huntingtin mutants from a biophysical point of view, by using classical Molecular Dynamics techniques. The huntingtin models consist of a wild-type structure, one mutant with 45 glutamine residues and the second mutant with nine additional key-point mutations from glutamine residues into proline residues (9P(EM) model). Our docking scores and binding free energy calculations show higher binding affinities of all HTT models for the C-lobe end of the CaM protein. In terms of dynamic evolution, the 9P(EM) model triggered great structural changes into the CaM protein’s structure and shows the highest fluctuation rates due to its structural transitions at the helical level from α-helices to turns and random coils. Moreover, our proposed 9P(EM) model suggests much lower interaction energies when compared to the 45Qs-HTT mutant model, this finding being in good agreement with the 9P(EM)’s antagonistic effect hypothesis on highly toxic protein–protein interactions.     

Calcium-induced membrane microdomains trigger plant phospholipase D activity

Plant alpha-type phospholipase D proteins are calcium-dependent, lipolytic enzymes. The morphology of the aggregates of their phospholipid substrate fundamentally defines the interaction between the enzyme and the surface. Here we demonstrate that the Ca(2+)-induced generation of membrane microdomains dramatically activates alpha-type phospholipase D from white cabbage. 500-fold stimulation was observed upon incorporation of 10 mol % 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA) into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles in the presence of Ca(2+) ions. Enhanced association of PLDalpha2 with phospholipid surfaces containing anionic components was indicated by lag phase analysis and film balance measurements. Differential scanning calorimetry showed that the POPA-specific activation correlates with the phase behavior of the POPC/POPA vesicles in the presence of Ca(2+) ions. We conclude from the results that the Ca(2+)-induced formation of POPA microdomains is the crucial parameter that facilitates the binding of PLD to the phospholipid surface and suggest that this effect serves as a cellular switch for controlling PLD activity.     

A molecular mechanism for toxin block in N-type calcium channels

A series of highly toxic snail venoms, the omega-conotoxins, have been shown to bind selectively, and often irreversibly to the N-type voltage- gated calcium channel alpha-1 subunit. The most potent of these is known as omega-conotoxin GVIA from the species Conus geographus, a marine snail that has been responsible for a number of human fatalities. Using theoretical techniques we present a plausible binding model of the conotoxin to a loop region of the channel. Our model of the toxin binding region also contains a possible EF-hand motif and we suggest that this Ca2+ binding domain lies on the ion permeation pathway, a possible Ca2+ recruitment site.      

Tryptophan fluorescence of calmodulin binding domain peptides interacting with calmodulin containing unnatural methionine analogues

The interactions between the abundant methionine residues ofthe calcium regulatory protein calmodulin (CaM) and severalof its binding targets were probed using fluorescence spectroscopy.Tryptophan steady-state fluorescence from peptides encompassingthe CaM-binding domains of the target proteins myosin lightchain kinase (MLCK), cyclic nucleotide phosphodiesterase (PDE)and caldesmon site A and B (CaD A, CaD B), and the model peptidemelittin showed Ca²⁺-dependent blue-shifts in their maximumemission wavelength when complexed with wild-type CaM. Blue-shiftswere also observed for complexes in which the CaM methionineresidues were replaced by selenomethionine, norleucine and ethionine,and when a quadruple methionine to leucine C-terminal mutantof CaM was studied. Quenching of the tryptophan fluorescenceintensity was observed with selenomethionine, but not with norleucineor ethionine substituted protein. Fluorescence quenching studieswith added potassium iodide (KI) demonstrate that the non-nativeproteins limit the solvent accessibility of the Trp in the MLCKpeptide to levels close to that of the wild-type CaM–MLCKinteraction. Our results show that the methionine residues fromCaM are highly sensitive to the target peptide in question,confirming the importance of their role in binding interactions.In addition, we provide evidence that the nature of bindingin the CaM–CaD B complex is unique compared with the othercomplexes studied, as the Trp residue of this peptide remainspartially solvent exposed upon binding to CaM.     

A Designed Enzyme Promotes Selective Post‐translational Acylation

A computationally designed, allosterically regulated catalyst (CaM M144H) produced by substituting a single residue in calmodulin, a non‐enzymatic protein, is capable of efficient and site selective post‐translational acylation of lysines in peptides with highly diverse sequences. Calmodulin′s binding partners are involved in regulating a large number of cellular processes; this new chemical‐biology tool will help to identify them and provide structural insight into their interactions with calmodulin.     

Structural Basis for the Functional Diversity of Centrins: A Focus on Calcium Sensing Properties and Target Recognition

Centrins are a family of small, EF hand-containing proteins that are found in all eukaryotes and are often complexed with centrosome-related structures. Since their discovery, centrins have attracted increasing interest due to their multiple, diverse cellular functions. Centrins are similar to calmodulin (CaM) in size, structure and domain organization, although in contrast to CaM, the majority of centrins possess at least one calcium (Ca²⁺) binding site that is non-functional, thus displaying large variance in Ca²⁺ sensing abilities that could support their functional versatility. In this review, we summarize current knowledge on centrins from both biophysical and structural perspectives with an emphasis on centrin-target interactions. In-depth analysis of the Ca²⁺ sensing properties of centrins and structures of centrins complexed with target proteins can provide useful insight into the mechanisms of the different functions of centrins and how these proteins contribute to the complexity of the Ca²⁺ signaling cascade. Moreover, it can help to better understand the functional redundancy of centrin isoforms and centrin-binding proteins.     

Effects of mutations in the calcium-binding sites of recoverin on its calcium affinity: evidence for successive filling of the calcium binding sites

A molecule of the photoreceptor Ca²⁺-binding protein recoverincontains four potential EF-hand Ca²⁺-binding sites, of whichonly two, the second and the third, are capable of binding calciumions. We have studied the effects of substitutions in the second,third and fourth EF-hand sites of recoverin on its Ca²⁺-bindingproperties and some other characteristics, using intrinsic fluorescence,circular dichroism spectroscopy and differential scanning microcalorimetry.The interaction of the two operating binding sites of wild-typerecoverin with calcium increases the protein's thermal stability,but makes the environment around the tryptophan residues moreflexible. The amino acid substitution in the EF-hand 3 (E121Q)totally abolishes the high calcium affinity of recoverin, whilethe mutation in the EF-hand 2 (E85Q) causes only a moderatedecrease in calcium binding. Based on this evidence, we suggestthat the binding of calcium ions to recoverin is a sequentialprocess with the EF-hand 3 being filled first. Estimation ofCa²⁺-binding constants according to the sequential binding schemegave the values 3.7 x 10⁶ and 3.1 x 10⁵ M^–1 for thirdand second EF-hands, respectively. The substitutions in theEF-hand 2 or 3 (or in both the sites simultaneously) do notdisturb significantly either tertiary or secondary structureof the apo-protein. Amino acid substitutions, which have beendesigned to restore the calcium affinity of the EF-hand 4 (G160D,K161E, K162N, D165G and K166Q), increase the calcium capacityand affinity of recoverin but also perturb the protein structureand decrease the thermostability of its apo-form.     

Short-Term Blockade of Pro-Inflammatory Alarmin S100A9 Favorably Modulates Left Ventricle Proteome and Related Signaling Pathways Involved in Post-Myocardial Infarction Recovery

Prognosis after myocardial infarction (MI) varies greatly depending on the extent of damaged area and the management of biological processes during recovery. Reportedly, the inhibition of the pro-inflammatory S100A9 reduces myocardial damage after MI. We hypothesize that a S100A9 blockade induces changes of major signaling pathways implicated in post-MI healing. Mass spectrometry-based proteomics and gene analyses of infarcted mice left ventricle were performed. The S100A9 blocker (ABR-23890) was given for 3 days after coronary ligation. At 3 and 7 days post-MI, ventricle samples were analyzed versus control and Sham-operated mice. Blockade of S100A9 modulated the expressed proteins involved in five biological processes: leukocyte cell–cell adhesion, regulation of the muscle cell apoptotic process, regulation of the intrinsic apoptotic signaling pathway, sarcomere organization and cardiac muscle hypertrophy. The blocker induced regulation of 36 proteins interacting with or targeted by the cellular tumor antigen p53, prevented myocardial compensatory hypertrophy, and reduced cardiac markers of post-ischemic stress. The blockade effect was prominent at day 7 post-MI when the quantitative features of the ventricle proteome were closer to controls. Blockade of S100A9 restores key biological processes altered post-MI. These processes could be valuable new pharmacological targets for the treatment of ischemic heart. Mass spectrometry data are available via ProteomeXchange with identifier PXD033683.     

Is competition between Li+ and Mg2+ the underlying theme in the proposed mechanisms for the pharmacological action of lithium salts in bipolar disorder?

Lithium salts have been in use for the treatment of bipolar disorder for more than 50 years, but their pharmacological mode of action remains a matter of conjecture. Li(+) and Mg(2+) share many physicochemical properties. Not surprisingly, many reported cellular targets for Li(+) action involve Mg(2+)-activated enzymes, which are inhibited by Li(+). In this Account, we describe results from our and other laboratories that suggest that a competition mechanism between Li(+) and Mg(2+) ions for Mg(2+)-binding sites in cellular components is the underlying theme in putative mechanisms of Li(+) action.     

Three-dimensional pharmacophore design and biochemical screening identifies substituted 1,2,4-triazoles as inhibitors of the annexin A2-S100A10 protein interaction

Protein interactions are increasingly appreciated as targets in small‐molecule drug discovery. The interaction between the adapter protein S100A10 and its binding partner annexin A2 is a potentially important drug target. To obtain small‐molecule starting points for inhibitors of this interaction, a three‐dimensional pharmacophore model was constructed from the X‐ray crystal structure of the complex between S100A10 and annexin A2. The pharmacophore model represents the favourable hydrophobic and hydrogen bond interactions between the two partners, as well as spatial and receptor site constraints (excluded volume spheres). Using this pharmacophore model, UNITY flex searches were carried out on a 3D library of 0.7 million commercially available compounds. This resulted in 568 hit compounds. Subsequently, GOLD docking studies were performed on these hits, and a set of 190 compounds were purchased and tested biochemically for inhibition of the protein interaction. Three compounds of similar chemical structure were identified as genuine inhibitors of the binding of annexin A2 to S100A10. The binding modes predicted by GOLD were in good agreement with their UNITY‐generated conformations. We synthesised a series of analogues revealing areas critical for binding. Thus computational predictions and biochemical screening can be used successfully to derive novel chemical classes of protein–protein interaction blockers.     

Calcium Regulates S100A12 Zinc Sequestration by Limiting Structural Variations

Antimicrobial proteins such as S100A12 and S100A8/A9 are highly expressed and secreted by neutrophils during infection and participate in human immune response by sequestering transition metals. At neutral pH, S100A12 sequesters Zn²⁺ with nanomolar affinity, which is further enhanced upon calcium binding. We investigated the pH dependence of human S100A12 zinc sequestration by using Co²⁺ as a surrogate. Apo-S100A12 exhibits strong Co²⁺ binding between pH 7.0 and 10.0 that progressively diminishes as the pH is decreased to 5.3. Ca²⁺-S100A12 can retain nanomolar Co²⁺ binding up to pH 5.7. NMR spectroscopic measurements revealed that calcium binding does not alter the side-chain protonation of the Co²⁺/Zn²⁺ binding histidine residues. Instead, the calcium-mediated modulation is achieved by restraining pH-dependent conformational changes to EF loop 1, which contains Co²⁺/Zn²⁺ binding Asp25. This calcium-induced enhancement of Co²⁺/Zn²⁺ binding might assist in the promotion of antimicrobial activities in humans by S100 proteins during neutrophil activation under subneutral pH conditions.     

An Integrated Computational and Experimental Approach to Gaining Selectivity for MMP‐2 within the Gelatinase Subfamily

Looking for water‐soluble inhibitors of matrix metalloproteinase‐2 (MMP‐2 or gelatinase A), we have previously reported compound 1 , a potent MMP‐2 inhibitor with a promising selectivity over the structurally homologous MMP‐9 (gelatinase B). Here we report the results of Molecular Dynamics (MD) simulations for both gelatinases (MMP‐2 and MMP‐9), and for the corresponding MMP/ 1 complexes, in an attempt to shed light on the observed selectivity between the two enzymes. These studies indicated a higher plasticity of MMP‐2 at the S1′ pocket and suggested an induced‐fit effect at the “back door” of this pocket. On the basis of these observations, we designed 11 a – d to aid further discrimination between MMP‐2 and MMP‐9. Those compounds displayed notably lower inhibitory activities against MMP‐9; in particular, 11 b proved to be over 100 times more active against MMP‐2 than against MMP‐9. MD simulations of the MMP/ 11 b complexes and thermodynamic integration calculations provided structural insight and relative binding energies consistent with the experimentally observed activity data. These findings demonstrate that structural differences in the S1′ pocket bottom permit an improvement in selectivity in the inhibition of MMP‐2 over that of MMP‐9; this is of great relevance for future structure‐based drug design because MMP‐2 is a validated target for cancer therapy, whereas MMP‐9 plays both detrimental and protective roles in cancer. This study also supports the need to consider the dynamics of the S1′ pocket in order to achieve selectivity in the inhibition of MMPs.     

Role of 14-3-3ζ in Platelet Glycoprotein Ibα-von Willebrand Factor Interaction-Induced Signaling

The interaction of platelet glycoprotein (GP) Ib-IX with von Willebrand factor (VWF) exposed at the injured vessel wall or atherosclerotic plaque rupture initiates platelet transient adhesion to the injured vessel wall, which triggers intracellular signaling cascades leading to platelet activation and thrombus formation. 14-3-3ζ has been verified to regulate the VWF binding function of GPIb-IX by interacting with the cytoplasmic domains of GPIb-IX. However, the data regarding the role of 14-3-3ζ in GPIb-IX-VWF interaction-induced signaling still remain controversial. In the present study, the data indicate that the S609A mutation replacing Ser(609) of GPIbα with alanine (S609A) significantly prevented the association of 14-3-3ζ with GPIbα before and after the VWF binding to GPIbα. GPIb-IX-VWF interaction-induced activations of Src family kinases and protein kinase C were clearly reduced in S609A mutation. Furthermore, S609A mutation significantly inhibited GPIb-IX-VWF interaction-induced elevation of cytoplasmic Ca(2+) levels in flow cytometry analysis. Taken together, these data indicate that the association of 14-3-3ζ with the cytoplasmic domain of GPIbα plays an important role in GPIb-IX-VWF interaction-induced signaling.