Molecular Insights into the Calcium Binding in Troponin C through a Molecular Dynamics Study.

Calcium-binding proteins play critical roles in various biological processes such as signal transduction, cell growth, and transcription factor regulation. Ion binding and target binding of Ca2+-binding proteins are highly related. Therefore, understanding the ion binding mechanism will benefit the relevant inhibitor design toward the Ca2+-binding proteins. The EF-hand is the typical ion binding motif in Ca2+-binding proteins. Previous studies indicate that the ion binding affinity of the EF-hand increases with the peptide length, but this mechanism has not been fully understood. Herein, using molecular dynamics simulations, thermodynamic integration calculations, and molecular mechanics Poisson-Boltzmann surface area analysis, we systematically investigated four Ca2+-binding peptides containing the EF-hand loop in site III of rabbit skeletal troponin C. These four peptides have 13, 21, 26, and 34 residues. Our simulations reproduced the observed trend that the ion binding affinity increases with the peptide length. Our results implied that the E-helix motif preceding the EF-hand loop, likely the Phe99 residue in particular, plays a significant role in this regulation. The E-helix has a significant impact on the backbone and side-chain conformations of the Asp103 residue, rigidifying important hydrogen bonds in the EF-hand and decreasing the solvent exposure of the Ca2+ ion, hence leading to more favorable Ca2+ binding in longer peptides. The present study provides molecular insights into the ion binding in the EF-hand and establishes an important step toward elucidating the responses of Ca2+-binding proteins toward the ion and target availability.

Eliminating the First Inactive State and Stabilizing the Active State of the Cardiac Regulatory System Alters Behavior in Solution and in Ordered Systems.

Calcium binding to troponin C (TnC) is insufficient for full activation of myosin ATPase activity by actin-tropomyosin-troponin. Previous attempts to investigate full activation utilized ATP-free myosin or chemically modified myosin to stabilize the active state of regulated actin. We utilized the Δ14-TnT and the A8V-TnC mutants to stabilize the activated state at saturating Ca2+ and to eliminate one of the inactive states at low Ca2+. The observed effects differed in solution studies and in the more ordered in vitro motility assay and in skinned cardiac muscle preparations. At saturating Ca2+, full activation with Δ14-TnT·A8V-TnC decreased the apparent KM for actin-activated ATPase activity compared to bare actin filaments. Rates of in vitro motility increased at both high and low Ca2+ with Δ14-TnT; the maximum shortening speed at high Ca2+ increased 1.8-fold. Cardiac muscle preparations exhibited increased Ca2+ sensitivity and large increases in resting force with either Δ14-TnT or Δ14-TnT·A8V-TnC. We also observed a significant increase in the maximal rate of tension redevelopment. The results of full activation with Ca2+ and Δ14-TnT·A8V-TnC confirmed and extended several earlier observations using other means of reaching full activation. Furthermore, at low Ca2+, elimination of the first inactive state led to partial activation. This work also confirms, in three distinct experimental systems, that troponin is able to stabilize the active state of actin-tropomyosin-troponin without the need for high-affinity myosin binding. The results are relevant to the reason for two inactive states and for the role of force producing myosin in regulation.

Characterization of the L29Q Hypertrophic Cardiomyopathy Mutation in Cardiac Troponin C by Paramagnetic Relaxation Enhancement Nuclear Magnetic Resonance.

The key events in regulating muscle contraction involve the troponin (Tn) heterotrimeric protein complex in which the binding to and release of Ca2+ from the highly conserved troponin C (TnC) subunit trigger a series of structural changes within Tn, and the other thin filament proteins, to result in contraction. In the heart, the control of contraction and relaxation events can be altered by many single-point mutations that may result in cardiomyopathy and sometimes sudden cardiac death. Here we have examined the structural effects of one hypertrophic cardiomyopathy mutation, L29Q, on Ca2+-induced structural transitions within whole TnC. This mutation is of particular interest as several physiological and structural studies have indicated that the response of TnC to Ca2+ binding is altered in the presence of the L29Q mutation, but the structural nature of these changes continues to be debated. In addition, little is known about the effect of this mutation in the Ca2+ free state. Here we have used paramagnetic relaxation enhancement nuclear magnetic resonance (PRE-NMR) to assess the structural effects arising from the L29Q mutation. PRE-NMR distances obtained from a nitroxide spin-label at Cys84 showed that the L29Q mutation perturbs the structure of the TnC N-domain in the presence and absence of Ca2+, with a more "open" TnC N-domain observed in the apo form. In addition, binding of Ca2+ to the TnC-L29Q construct triggers a change in the orientation between the two domains of TnC. Together, these structural perturbations, revealed by PRE-NMR, provide insight into the pathogenesis of this mutation.

Reversible Covalent Reaction of Levosimendan with Cardiac Troponin C in Vitro and in Situ.

The development of calcium sensitizers for the treatment of systolic heart failure presents difficulties, including judging the optimal efficacy and the specificity to target cardiac muscle. The thin filament is an attractive target because cardiac troponin C (cTnC) is the site of calcium binding and the trigger for subsequent contraction. One widely studied calcium sensitizer is levosimendan. We have recently shown that when a covalent cTnC-levosimendan analogue is exchanged into cardiac muscle cells, they become constitutively active, demonstrating the potency of a covalent complex. We have also demonstrated that levosimendan reacts in vitro to form a reversible covalent thioimidate bond specifically with cysteine 84, unique to cTnC. In this study, we use mass spectrometry to show that the in vitro mechanism of action of levosimendan is consistent with an allosteric, reversible covalent inhibitor; to determine whether the presence of the cTnI switch peptide or changes in either Ca2+ concentration or pH modify the reaction kinetics; and to determine whether the reaction can occur with cTnC in situ in cardiac myofibrils. Using the derived kinetic rate constants, we predict the degree of covalently modified cTnC in vivo under the conditions studied. We observe that covalent bond formation would be highest under the acidotic conditions resulting from ischemia and discuss whether the predicted level could be sufficient to have therapeutic value. Irrespective of the in vivo mechanism of action for levosimendan, our results provide a rationale and basis for the development of reversible covalent drugs to target the failing heart.

Successful Identification of Cardiac Troponin Calcium Sensitizers Using a Combination of Virtual Screening and ROC Analysis of Known Troponin C Binders.

Calcium-dependent cardiac muscle contraction is regulated by the protein complex troponin. Calcium binds to the N-terminal domain of troponin C (cNTnC) which initiates the process of contraction. Heart failure is a consequence of a disruption of this process. With the prevalence of this condition, a strong need exists to find novel compounds to increase the calcium sensitivity of cNTnC. Desirable are small chemical molecules that bind to the interface between cTnC and the cTnI switch peptide and exhibit calcium sensitizing properties by possibly stabilizing cTnC in an open conformation. To identify novel drug candidates, we employed a structure-based drug discovery protocol that incorporated the use of a relaxed complex scheme (RCS). In preparation for the virtual screening, cNTnC conformations were identified based on their ability to correctly predict known cNTnC binders using a receiver operating characteristics analysis. Following a virtual screen of the National Cancer Institute's Developmental Therapeutic Program database, a small number of molecules were experimentally tested using stopped-flow kinetics and steady-state fluorescence titrations. We identified two novel compounds, 3-(4-methoxyphenyl)-6,7-chromanediol (NSC600285) and 3-(4-methylphenyl)-7,8-chromanediol (NSC611817), that show increased calcium sensitivity of cTnC in the presence of the regulatory domain of cTnI. The effects of NSC600285 and NSC611817 on the calcium dissociation rate was stronger than that of the known calcium sensitizer bepridil. Thus, we identified a 3-phenylchromane group as a possible key pharmacophore in the sensitization of cardiac muscle contraction. Building on this finding is of interest to researchers working on development of drugs for calcium sensitization.

The concerted movement of the switch region of Troponin I in cardiac muscle thin filaments as tracked by conventional and pulsed (DEER) EPR.

The absence of a crystal structure of the calcium free state of the cardiac isoform of the troponin complex has hindered our understanding of how the simple binding of Ca2+ triggers conformational changes in troponin which are then propagated to enable muscle contraction. Here we have used continuous wave (CW) and Double Electron-Electron Resonance (DEER) pulsed EPR spectroscopy to measure distances between TnI and TnC to track the movement of the functionally important regulatory 'switch' region of cardiac Tn. Spin labels were placed on the switch region of Troponin I and distances measured to Troponin C. Under conditions of high Ca2+, the interspin distances for one set (TnI151/TnC84) were 'short' (9-10Å) with narrow distance distribution widths (3-8Å) indicating the close interaction of the switch region with the N-lobe of TnC. Additional spin populations representative of longer interspin distances were detected by DEER. These longer distance populations, which were ∼16-19Å longer than the short distance populations, possessed notably broader distance distribution widths (14-29Å). Upon Ca2+ removal, the interspin population shifted toward the longer distances, indicating the release of the switch region from TnC and an overall increase in disorder for this region. Together, our results suggest that under conditions of low Ca2+, the close proximity of the TnI switch region to TnC in the cardiac isoform is necessary for promoting the interaction between the regulatory switch helix with the N-lobe of cardiac Troponin C, which, unlike the skeletal isoform, is largely in a closed conformation.

Clinically Divergent Mutation Effects on the Structure and Function of the Human Cardiac Tropomyosin Overlap.

The progression of genetically inherited cardiomyopathies from an altered protein structure to clinical presentation of disease is not well understood. One of the main roadblocks to mechanistic insight remains a lack of high-resolution structural information about multiprotein complexes within the cardiac sarcomere. One example is the tropomyosin (Tm) overlap region of the thin filament that is crucial for the function of the cardiac sarcomere. To address this central question, we devised coupled experimental and computational modalities to characterize the baseline function and structure of the Tm overlap, as well as the effects of mutations causing divergent patterns of ventricular remodeling on both structure and function. Because the Tm overlap contributes to the cooperativity of myofilament activation, we hypothesized that mutations that enhance the interactions between overlap proteins result in more cooperativity, and conversely, those that weaken interaction between these elements lower cooperativity. Our results suggest that the Tm overlap region is affected differentially by dilated cardiomyopathy-associated Tm D230N and hypertrophic cardiomyopathy-associated human cardiac troponin T (cTnT) R92L. The Tm D230N mutation compacts the Tm overlap region, increasing the cooperativity of the Tm filament, contributing to a dilated cardiomyopathy phenotype. The cTnT R92L mutation causes weakened interactions closer to the N-terminal end of the overlap, resulting in decreased cooperativity. These studies demonstrate that mutations with differential phenotypes exert opposite effects on the Tm-Tn overlap, and that these effects can be directly correlated to a molecular level understanding of the structure and dynamics of the component proteins.

Troponin C Mutations Partially Stabilize the Active State of Regulated Actin and Fully Stabilize the Active State When Paired with Δ14 TnT.

Striated muscle contraction is regulated by the actin-associated proteins tropomyosin and troponin. The extent of activation of myosin ATPase activity is lowest in the absence of both Ca2+ and activating cross-bridges (i.e., S1-ADP or rigor S1). Binding of activating species of myosin to actin at a saturating Ca2+ concentration stabilizes the most active state (M state) of the actin-tropomyosin-troponin complex (regulated actin). Ca2+ binding alone produces partial stabilization of the active state. The extent of stabilization at a saturating Ca2+ concentration depends on the isoform of the troponin subunits, the phosphorylation state of troponin, and, in the case of cardiac muscle, the presence of hypertrophic cardiomyopathy-producing mutants of troponin T and troponin I. Cardiac dysfunction is also associated with mutations of troponin C (TnC). Troponin C mutants A8V, C84Y, and D145E increase the Ca2+ sensitivity of ATPase activity. We show that these mutants change the distribution of regulated actin states. The A8V and C84Y TnC mutants decreased the inactive B state distribution slightly at low Ca2+ concentrations, but the D145E mutants had no effect on that state. All TnC mutants increased the level of the active M state compared to that of the wild type, at a saturating Ca2+ concentration. Troponin complexes that contained two mutations that stabilize the active M state, A8V TnC and Δ14 TnT, appeared to be completely in the active state in the presence of only Ca2+. Because Ca2+ gives full activation, in this situation, troponin must be capable of positioning tropomyosin in the active M state without the need for rigor myosin binding.

Enhanced troponin I binding explains the functional changes produced by the hypertrophic cardiomyopathy mutation A8V of cardiac troponin C.

Higher affinity for TnI explains how troponin C (TnC) carrying a causative hypertrophic cardiomyopathy mutation, TnC(A8V), sensitizes muscle cells to Ca(2+). Muscle fibers reconstituted with TnC(A8V) require ∼2.3-fold less [Ca(2+)] to achieve 50% maximum-tension compared to fibers reconstituted with wild-type TnC (TnC(WT)). Binding measurements rule out a significant change in N-terminus Ca(2+)-affinity of isolated TnC(A8V), and TnC(A8V) binds the switch-peptide of troponin-I (TnI(sp)) ∼1.6-fold more strongly than TnC(WT); thus we model the TnC-TnI(sp) interaction as competing with the TnI-actin interaction. Tension data are well-fit by a model constrained to conditions in which the affinity of TnC(A8V) for TnI(sp) is 1.5-1.7-fold higher than that of TnC(WT) at all [Ca(2+)]. Mean ATPase rates of reconstituted cardiac myofibrils is greater for TnC(A8V) than TnC(WT) at all [Ca(2+)], with statistically significant differences in the means at higher [Ca(2+)]. To probe TnC-TnI interaction in low Ca(2+), displacement of bis-ANS from TnI was monitored as a function of TnC. Whereas Ca(2+)-TnC(WT) displaces significantly more bis-ANS than Mg(2+)-TnC(WT), Ca(2+)-TnC(A8V) displaces probe equivalently to Mg(2+)-TnC(A8V) and Ca(2+)-TnC(WT), consistent with stronger Ca(2+)-independent TnC(A8V)-TnI(sp). A Matlab program for computing theoretical activation is reported. Our work suggests that contractility is constantly above normal in hearts made hypertrophic by TnC(A8V).

The functional significance of the last 5 residues of the C-terminus of cardiac troponin I.

The C-terminal region of cardiac troponin I (cTnI) is known to be important in cardiac function, as removal of the last 17 C-terminal residues of human cTnI has been associated with myocardial stunning. To investigate the C-terminal region of cTnI, three C-terminal deletion mutations in human cTnI were generated: Δ1 (deletion of residue 210), Δ3 (deletion of residues 208-210), and Δ5 (deletion of residues 206-210). Mammalian two-hybrid studies showed that the interactions between cTnI mutants and cardiac troponin C (cTnC) or cardiac troponin T (cTnT) were impaired in Δ3 and Δ5 mutants when compared to wild-type cTnI. Troponin complexes containing 2-[4'-(iodoacetamido) anilino] naphthalene-6-sulfonic acid (IAANS) labeled cTnC showed that the troponin complex containing cTnI Δ5 had a small increase in Ca(2+) affinity (P < 0.05); while the cTnI Δ1- and Δ3 troponin complexes showed no difference in Ca(2+) affinity when compared to wild-type troponin. In vitro motility assays showed that all truncation mutants had increased Ca(2+) dependent motility relative to wild-type cTnI. These results suggest that the last 5 C-terminal residues of cTnI influence the binding of cTnI with cTnC and cTnT and affect the Ca(2+) dependence of filament sliding, and demonstrate the importance of this region of cTnI.

A peptide hydrogel derived from a fragment of human cardiac troponin C.

The human cardiac troponin C peptide fragment H-V(9)EQLTEEQKNEFKAAFDIFVLGA(31)-OH, which covers helix-A in the native protein, self-assembles into ß-sheet fibrils in solution. These fibrils further entangle to give a hydrogel. This peptide may therefore serve as a template for development of novel biomaterials.

The structural and functional effects of the familial hypertrophic cardiomyopathy-linked cardiac troponin C mutation, L29Q.

Familial hypertrophic cardiomyopathy (FHC) is characterized by severe abnormal cardiac muscle growth. The traditional view of disease progression in FHC is that an increase in the Ca(2+)-sensitivity of cardiac muscle contraction ultimately leads to pathogenic myocardial remodeling, though recent studies suggest this may be an oversimplification. For example, FHC may be developed through altered signaling that prevents downstream regulation of contraction. The mutation L29Q, found in the Ca(2+)-binding regulatory protein in heart muscle, cardiac troponin C (cTnC), has been linked to cardiac hypertrophy. However, reports on the functional effects of this mutation are conflicting, and our goal was to combine in vitro and in situ structural and functional data to elucidate its mechanism of action. We used nuclear magnetic resonance and circular dichroism to solve the structure and characterize the backbone dynamics and stability of the regulatory domain of cTnC with the L29Q mutation. The overall structure and dynamics of cTnC were unperturbed, although a slight rearrangement of site 1, an increase in backbone flexibility, and a small decrease in protein stability were observed. The structure and function of cTnC was also assessed in demembranated ventricular trabeculae using fluorescence for in situ structure. L29Q reduced the cooperativity of the Ca(2+)-dependent structural change in cTnC in trabeculae under basal conditions and abolished the effect of force-generating myosin cross-bridges on this structural change. These effects could contribute to the pathogenesis of this mutation.

Coarse-Grained Modeling of Peptide Docking Associated with Large Conformation Transitions of the Binding Protein: Troponin I Fragment-Troponin C System.

Most of the current docking procedures are focused on fine conformational adjustments of assembled complexes and fail to reproduce large-scale protein motion. In this paper, we test a new modeling approach developed to address this problem. CABS-dock is a versatile and efficient tool for modeling the structure, dynamics and interactions of protein complexes. The docking protocol employs a coarse-grained representation of proteins, a simplified model of interactions and advanced protocols for conformational sampling. CABS-dock is one of the very few tools that allow unrestrained docking with large conformational freedom of the receptor. In an example application we modeled the process of complex assembly between two proteins: Troponin C (TnC) and the N-terminal helix of Troponin I (TnI N-helix), which occurs in vivo during muscle contraction. Docking simulations illustrated how the TnC molecule undergoes significant conformational transition on complex formation, a phenomenon that can be modeled only when protein flexibility is properly accounted for. This way our procedure opens up a new possibility for studying mechanisms of protein complex assembly, which may be a supporting tool for rational drug design.

In situ time-resolved FRET reveals effects of sarcomere length on cardiac thin-filament activation.

During cardiac thin-filament activation, the N-domain of cardiac troponin C (N-cTnC) binds to Ca(2+) and interacts with the actomyosin inhibitory troponin I (cTnI). The interaction between N-cTnC and cTnI stabilizes the Ca(2+)-induced opening of N-cTnC and is presumed to also destabilize cTnI-actin interactions that work together with steric effects of tropomyosin to inhibit force generation. Recently, our in situ steady-state FRET measurements based on N-cTnC opening suggested that at long sarcomere length, strongly bound cross-bridges indirectly stabilize this Ca(2+)-sensitizing N-cTnC-cTnI interaction through structural effects on tropomyosin and cTnI. However, the method previously used was unable to determine whether N-cTnC opening depends on sarcomere length. In this study, we used time-resolved FRET to monitor the effects of cross-bridge state and sarcomere length on the Ca(2+)-dependent conformational behavior of N-cTnC in skinned cardiac muscle fibers. FRET donor (AEDANS) and acceptor (DDPM)-labeled double-cysteine mutant cTnC(T13C/N51C)AEDANS-DDPM was incorporated into skinned muscle fibers to monitor N-cTnC opening. To study the structural effects of sarcomere length on N-cTnC, we monitored N-cTnC opening at relaxing and saturating levels of Ca(2+) and 1.80 and 2.2-µm sarcomere length. Mg(2+)-ADP and orthovanadate were used to examine the structural effects of noncycling strong-binding and weak-binding cross-bridges, respectively. We found that the stabilizing effect of strongly bound cross-bridges on N-cTnC opening (which we interpret as transmitted through related changes in cTnI and tropomyosin) become diminished by decreases in sarcomere length. Additionally, orthovanadate blunted the effect of sarcomere length on N-cTnC conformational behavior such that weak-binding cross-bridges had no effect on N-cTnC opening at any tested [Ca(2+)] or sarcomere length. Based on our findings, we conclude that the observed sarcomere length-dependent positive feedback regulation is a key determinant in the length-dependent Ca(2+) sensitivity of myofilament activation and consequently the mechanism underlying the Frank-Starling law of the heart.

Molecular and functional consequences of mutations in the central helix of cardiac troponin C.

The objective of this work was to investigate the role of acidic residues within the exposed middle segment of the central helix of cTnC in (1) cTnC-cTnI interactions, (2) Ca(2+) binding and exchange with the regulatory N-domain of cTnC in increasingly complex biochemical systems, and (3) ability of the cTn complex to regulate actomyosin ATPase. In order to achieve this objective, we introduced the D87A/D88A and E94A/E95A/E96A mutations into the central helix of cTnC. The D87A/D88A and E94A/E95A/E96A mutations decreased affinity of cTnC for the regulatory region of cTnI. The Ca(2+) sensitivity of the regulatory N-domain of isolated cTnC was decreased by the D87A/D88A, but not E94A/E95A/E96A mutation. However, both the D87A/D88A and E94A/E95A/E96A mutations desensitized the cTn complex and reconstituted thin filaments to Ca(2+). Decreases in the Ca(2+) sensitivity of the cTn complex and reconstituted thin filaments were, at least in part, due to faster rates of Ca(2+) dissociation. In addition, the D87A/D88A and E94A/E95A/E96A mutations desensitized actomyosin ATPase to Ca(2+), and decreased maximal actomyosin ATPase activity. Thus, our results indicate that conserved acidic residues within the exposed middle segment of the central helix of cTnC are important for the proper regulatory function of the cTn complex.

A computational exploration of the interactions of the green tea polyphenol (-)-Epigallocatechin 3-Gallate with cardiac muscle troponin C.

Thanks to its polyphenols and phytochemicals, green tea is believed to have a number of health benefits, including protecting from heart disease, but its mechanism of action at the molecular level is still not understood. Here we explore, by means of atomistic simulations, how the most abundant of the green tea polyphenols, (-)-Epigallocatechin 3-Gallate (EGCg), interacts with the structural C terminal domain of cardiac muscle troponin C (cCTnC), a calcium binding protein that plays an important role in heart contractions. We find that EGCg favourably binds to the hydrophobic cleft of cCTnC consistently with solution NMR experiments. It also binds to cCTnC in the presence of the anchoring region of troponin I (cTnI(34-71)) at the interface between the E and H helices. This appears to affect the strength of the interaction between cCTnC and cTnI(34-71) and also counter-acts the effects of the Gly159Asp mutation, related to dilated cardiomyopathy. Our simulations support the picture that EGCg interacting with the C terminal domain of troponin C may help in regulating the calcium signalling either through competitive binding with the anchoring domain of cTnI or by affecting the interaction between cCTnC and cTnI(34-71).

Structural basis for the in situ Ca(2+) sensitization of cardiac troponin C by positive feedback from force-generating myosin cross-bridges.

The in situ structural coupling between the cardiac troponin (cTn) Ca(2+)-sensitive regulatory switch (CRS) and strong myosin cross-bridges was investigated using Förster resonance energy transfer (FRET). The double cysteine mutant cTnC(T13C/N51C) was fluorescently labeled with the FRET pair 5-(iodoacetamidoethyl)aminonaphthelene-1-sulfonic acid (IAEDENS) and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM) and then incorporated into detergent skinned left ventricular papillary fiber bundles. Ca(2+) titrations of cTnC(T13C/N51C)AEDENS/DDPM-reconstituted fibers showed that the Ca(2+)-dependence of the opening of the N-domain of cTnC (N-cTnC) statistically matched the force-Ca(2+) relationship. N-cTnC opening still occurred steeply during Ca(2+) titrations in the presence of 1mM vanadate, but the maximal extent of ensemble-averaged N-cTnC opening and the Ca(2+)-sensitivity of the CRS were significantly reduced. At nanomolar, resting Ca(2+) levels, treatment with ADP·Mg in the absence of ATP caused a partial opening of N-cTnC. During subsequent Ca(2+) titrations in the presence of ADP·Mg and absence of ATP, further N-cTnC opening was stimulated as the CRS responded to Ca(2+) with increased Ca(2+)-sensitivity and reduced steepness. These findings supported our hypothesis here that strong cross-bridge interactions with the cardiac thin filament exert a Ca(2+)-sensitizing effect on the CRS by stabilizing the interaction between the exposed hydrophobic patch of N-cTnC and the switch region of cTnI.

Computational analysis of protein-protein interfaces involving an alpha helix: insights for terphenyl-like molecules binding.

BACKGROUND: Protein-Protein Interactions (PPIs) are key for many cellular processes. The characterization of PPI interfaces and the prediction of putative ligand binding sites and hot spot residues are essential to design efficient small-molecule modulators of PPI. Terphenyl and its derivatives are small organic molecules known to mimic one face of protein-binding alpha-helical peptides. In this work we focus on several PPIs mediated by alpha-helical peptides. METHOD: We performed computational sequence- and structure-based analyses in order to evaluate several key physicochemical and surface properties of proteins known to interact with alpha-helical peptides and/or terphenyl and its derivatives. RESULTS: Sequence-based analysis revealed low sequence identity between some of the analyzed proteins binding alpha-helical peptides. Structure-based analysis was performed to calculate the volume, the fractal dimension roughness and the hydrophobicity of the binding regions. Besides the overall hydrophobic character of the binding pockets, some specificities were detected. We showed that the hydrophobicity is not uniformly distributed in different alpha-helix binding pockets that can help to identify key hydrophobic hot spots. CONCLUSIONS: The presence of hydrophobic cavities at the protein surface with a more complex shape than the entire protein surface seems to be an important property related to the ability of proteins to bind alpha-helical peptides and low molecular weight mimetics. Characterization of similarities and specificities of PPI binding sites can be helpful for further development of small molecules targeting alpha-helix binding proteins.

The structure of cardiac troponin C regulatory domain with bound Cd2+ reveals a closed conformation and unique ion coordination.

The amino-terminal domain of cardiac troponin C (cNTnC) is an essential Ca(2+) sensor found in cardiomyocytes. It undergoes a conformational change upon Ca(2+) binding and transduces the signal to the rest of the troponin complex to initiate cardiac muscle contraction. Two classical EF-hand motifs (EF1 and EF2) are present in cNTnC. Under physiological conditions, only EF2 binds Ca(2+); EF1 is a vestigial site that has lost its function in binding Ca(2+) owing to amino-acid sequence changes during evolution. Proteins with EF-hand motifs are capable of binding divalent cations other than calcium. Here, the crystal structure of wild-type (WT) human cNTnC in complex with Cd(2+) is presented. The structure of Cd(2+)-bound cNTnC with the disease-related mutation L29Q, as well as a structure with the residue differences D2N, V28I, L29Q and G30D (NIQD), which have been shown to have functional importance in Ca(2+) sensing at lower temperatures in ectothermic species, have also been determined. The structures resemble the overall conformation of NMR structures of Ca(2+)-bound cNTnC, but differ significantly from a previous crystal structure of Cd(2+)-bound cNTnC in complex with deoxycholic acid. The subtle structural changes observed in the region near the mutations may play a role in the increased Ca(2+) affinity. The 1.4 Å resolution WT cNTnC structure, which is the highest resolution structure yet obtained for cardiac troponin C, reveals a Cd(2+) ion coordinated in the canonical pentagonal bipyramidal geometry in EF2 despite three residues in the loop being disordered. A Cd(2+) ion found in the vestigial ion-binding site of EF1 is coordinated in a noncanonical `distorted' octahedral geometry. A comparison of the ion coordination observed within EF-hand-containing proteins for which structures have been solved in the presence of Cd(2+) is presented. A refolded WT cNTnC structure is also presented.

Infrared study of synthetic peptide analogues of the calcium-binding site III of troponin C: The role of helix F of an EF-hand motif.

The EF-hand motif (helix-loop-helix) is a Ca(2+)-binding domain that is common among many intracellular Ca(2+)-binding proteins. We applied Fourier-transform infrared spectroscopy to study the synthetic peptide analogues of site III of rabbit skeletal muscle troponin C (helix E-loop-helix F). The 17-residue peptides corresponding to loop-helix F (DRDADGYIDAEELAEIF), where one residue is substituted by the D-type amino acid, were investigated to disturb the α-helical conformation of helix F systematically. These D-type-substituted peptides showed no band at about 1555 cm(-1) even in the Ca(2+)-loaded state although the native peptide (L-type only) showed a band at about 1555 cm(-1) in the Ca(2+)-loaded state, which is assigned to the side-chain COO(-) group of Glu at the 12th position, serving as the ligand for Ca(2+) in the bidentate coordination mode. Therefore, helix F is vital to the interaction between the Ca(2+) and the side-chain COO(-) group of Glu at the 12th position. Implications of the COO(-) antisymmetric stretch and the amide-I' of the synthetic peptide analogues of the Ca(2+)-binding sites are discussed.