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1.  Structural and functional consequences of the cardiac troponin C L48Q Ca2+-sensitizing mutation 
Biochemistry  2012;51(22):4473-4487.
Calcium binding to the regulatory domain of cardiac troponin C (cNTnC) causes a conformational change that exposes a hydrophobic surface to which troponin I (cTnI) binds, prompting a series of protein-protein interactions that culminate in muscle contraction. A number of cTnC variants that alter the Ca2+-sensitivity of the thin filament have been linked to disease. Tikunova and Davis have engineered a series of cNTnC mutations that altered Ca2+ binding properties and studied the effects on the Ca2+ sensitivity of the thin filament and contraction [Tikunova and Davis (2004) J Biol Chem279, 35341–35352]. One of the mutations they engineered, the L48Q variant, resulted in a pronounced increase in cNTnC Ca2+ binding affinity and Ca2+ sensitivity of cardiac muscle force development. In this work, we sought structural and mechanistic explanations for the increased Ca2+ sensitivity of contraction for the L48Q cNTnC variant, using an array of biophysical techniques. We found that the L48Q mutation enhanced binding of both Ca2+ and cTnI to cTnC. NMR chemical shift and relaxation data provided evidence that the cNTnC hydrophobic core is more exposed with the L48Q variant. Molecular dynamics simulations suggest that the mutation disrupts a network of crucial hydrophobic interactions so that the closed form of cNTnC is destabilized. The findings emphasize the importance of cNTnC's conformation in the regulation of contraction and suggest that mutations in cNTnC that alter myofilament Ca2+ sensitivity can do so by modulating Ca2+ and cTnI binding.
doi:10.1021/bi3003007
PMCID: PMC3437384  PMID: 22591429
Troponin C; Troponin I; Calcium binding; Fluorescence spectroscopy; Molecular dynamic simulation; NMR spectroscopy; Isothermal Titration Calorimetry
2.  Structure of trans-resveratrol in complex with the cardiac regulatory protein troponin C 
Biochemistry  2011;50(8):1309-1320.
Cardiac troponin – a heterotrimeric protein complex that regulates heart contraction – represents an attractive target for the development of drugs to treat heart disease. Cardiovascular diseases are one of the chief causes of morbidity and mortality worldwide. In France, however, the death rate from heart disease is remarkably low relative to fat consumption. This so called “French paradox” has been attributed to the high consumption of wine in France; and the antioxidant trans-resveratrol is thought to be the primary basis for wine’s cardioprotective nature. It has been demonstrated that trans-resveratrol increases the myofilament Ca2+-sensitivity of guinea-pig myocytes (Liew, R., Stagg, M.A., MacLeod, K.T., and Collins, P., (2005) Eur. J. Pharmacol. 519, 1–8.), however, the specific mode of its action is unknown. In this study, the structure of trans-resveratrol free and bound to the calcium-binding protein, troponin C, was determined by NMR spectroscopy. The results indicate that trans-resveratrol undergoes a minor conformational change upon binding to the hydrophobic pocket of the C-domain of troponin C. The location occupied by trans-resveratrol coincides with the binding site of troponin I – troponin C’s natural binding partner. This has been seen for other troponin C-targeting inotropes and implicates the modulation of the troponin C-troponin I interaction as a possible mechanism of action for trans-resveratrol.
doi:10.1021/bi101985j
PMCID: PMC3043152  PMID: 21226534
Trans-resveratrol; troponin C; NMR spectroscopy; cardiac muscle; contraction; Ca2+-sensitizer
3.  Solution Structure of a DNA Duplex Containing the Potent Anti-Poxvirus Agent Cidofovir 
Cidofovir (1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine, CDV) is a potent inhibitor of orthopoxvirus DNA replication. Prior studies have shown that when CDV is incorporated into a growing primer strand, it can inhibit both the 3′-to-5′ exonuclease and the 5′-to-3′ chain extension activities of vaccinia virus DNA polymerase. This drug can also be incorporated into DNA, creating a significant impediment to trans-lesion DNA synthesis in a manner resembling DNA damage. CDV and deoxycytidine share a common nucleobase but CDV lacks the deoxyribose sugar. The acyclic phosphonate bears a hydroxyl moiety that is equivalent to the 3′-hydroxyl of dCMP and permits CDV incorporation into duplex DNA. To study the structural consequences of inserting CDV into DNA, we have used 1H NMR to solve the solution structures of a dodecamer DNA duplex containing a CDV molecule at position 7 and of a control DNA duplex. The overall structures of both DNA duplexes were found to be very similar. We observed a decrease of intensity (>50%) for the imino protons neighboring the CDV (G6, T8) and the cognate base G18, and a large chemical shift change for G18. This indicates higher proton exchange rates for this region, which was confirmed using NMR monitored melting experiments. DNA duplex melting experiments monitored by circular dichroism revealed a lower Tm for the CDV DNA duplex (46°C) compared to the control (58°C) in 0.2 M salt. Our results suggest that the CDV drug is well accommodated and stable within the dodecamer DNA duplex, but the stability of the complex is less than the control suggesting increased dynamics around the CDV.
doi:10.1021/ja109823e
PMCID: PMC3051402  PMID: 21280608
4.  A structural and functional perspective into the mechanism of Ca2+-sensitizers that target the cardiac troponin complex 
The Ca2+ dependant interaction between troponin I (cTnI) and troponin C (cTnC) triggers contraction in heart muscle. Heart failure is characterized by a decrease in cardiac output, and compounds that increase the sensitivity of cardiac muscle to Ca2+ have therapeutic potential. The Ca2+-sensitizer, levosimendan, targets cTnC; however, detailed understanding of its mechanism has been obscured by its instability. In order to understand how this class of positive inotropes function, we investigated the mode of action of two fluorine containing novel analogues of levosimendan; 2’ ,4’-difluoro(1,1’-biphenyl)-4-yloxy acetic acid (dfbp-o) and 2’ ,4’-difluoro(1,1’-biphenyl)-4-yl acetic acid (dfbp). The affinities of dfbp and dfbp-o for the regulatory domain of cTnC were measured in the absence and presence of cTnI by NMR spectroscopy, and dfbp-o was found to bind more strongly than dfbp. Dfbp-o also increased the affinity of cTnI for cTnC. Dfbp-o increased the Ca2+-sensitivity of demembranated cardiac trabeculae in a manner similar to levosimendan. The high resolution NMR solution structure of the cTnC-cTnI-dfbp-o ternary complex showed that dfbp-o bound at the hydrophobic interface formed by cTnC and cTnI making critical interactions with residues such as Arg147 of cTnI. In the absence of cTnI, docking localized dfbp-o to the same position in the hydrophobic groove of cTnC. The structural and functional data reveal that the levosimendan class of Ca2+-sensitizers work by binding to the regulatory domain of cTnC and stabilizing the pivotal cTnC-cTnI regulatory unit via a network of hydrophobic and electrostatic interactions, in contrast to the destabilizing effects of antagonists such as W7 at the same interface.
doi:10.1016/j.yjmcc.2010.08.019
PMCID: PMC2975748  PMID: 20801130
Levosimendan; troponin C; troponin I; Ca2+-sensitizer; NMR spectroscopy
5.  The carboxyl terminal segment of apolipoprotein A-V undergoes a lipid-induced conformational change 
Biochemistry  2010;49(23):4821-4826.
Apolipoprotein (apo) A-V is a 343 residue, multi-domain protein that plays an important role in regulation of plasma triglyceride homeostasis. Primary sequence analysis revealed a unique tetra-proline sequence (Pro293 – Pro296) near the carboxyl terminus of the protein. A peptide corresponding to the 48 residue segment beyond the tetra-proline motif was generated from a recombinant apoA-V precursor wherein Pro295 was replaced by Met. Cyanogen bromide cleavage of the precursor protein, followed by negative affinity chromatography, yielded a purified peptide. Nondenaturing polyacrylamide gel electrophoresis verified that apoA-V(296-343) solubilizes phospholipid vesicles, forming a relatively heterogeneous population of reconstituted high density lipoprotein with Stoke's diameters > 17 nm. At the same time, apoA-V(296-343) failed to bind a spherical lipoprotein substrate in vitro. Far UV circular dichroism spectroscopy revealed the peptide is unstructured in buffer yet adopts significant α-helical secondary structure in the presence of the lipid mimetic solvent, trifluoroethanol (TFE; 50% v/v). Heteronuclear multidemensional NMR spectroscopy experiments were conducted with uniformly 15N and 15N/13C labeled peptide in 50 % TFE. Peptide backbone assignment and secondary structure prediction using TALOS+ reveals the peptide adopts α-helix secondary structure from residues 309 – 334. In TFE, apoA-V(296-343) adopts an extended amphipathic α-helix, consistent with a role in lipoprotein binding as a component of full-length apoA-V.
doi:10.1021/bi1005859
PMCID: PMC2893581  PMID: 20469899
6.  Solution Structure of the Regulatory Domain of Human Cardiac Troponin C in Complex with the Switch Region of Cardiac Troponin I and W7: The Basis of W7 as an Inhibitor of Cardiac Muscle Contraction 
The solution structure of Ca2+-bound regulatory domain of cardiac troponin C (cNTnC) in complex with the switch region of troponin I (cTnI147-163) and the calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfinamide (W7), has been determined by NMR spectroscopy. The structure reveals that the W7 naphthalene ring interacts with the terminal methyl groups of M47, M60, and M81 as well as aliphatic and aromatic side-chains of several other residues in the hydrophobic pocket of cNTnC. The H3 ring proton of W7 also contacts the methyl groups of I148 and M153 of cTnI147-163. The N-(6-aminohexyl) tail interacts primarily with the methyl groups of V64 and M81, which are located on the C- and D-helices of cNTnC. Compared to the structure of the cNTnC•Ca2+•W7 complex (Hoffman, R. M. B. and Sykes, B. D. (2009) Biochemistry 48, 5541-5552), the tail of W7 reorients slightly towards the surface of cNTnC while the ring remains in the hydrophobic pocket. The positively charged -NH3+ group from the tail of W7 repels the positively charged R147 of cTnI147-163. As a result, the N-terminus of the peptide moves away from cNTnC and the helical content of cTnI147-163 is diminished, when compared to the structure of cNTnC•Ca2+•cTnI147-163 (Li, M. X., Spyracopoulos, L., and Sykes B. D. (1999) Biochemistry 38, 8289-8298). Thus the ternary structure cNTnC•Ca2+•W7•cTnI147-163 reported in this study offers an explanation for the ∼13-fold affinity reduction of cTnI147-163 for cNTnC•Ca2+ in the presence of W7, and provides a structural basis for the inhibitory effect of W7 in cardiac muscle contraction. This generates molecular insight into structural features that are useful for the design of cTnC-specific Ca2+-desensitizing drugs.
doi:10.1016/j.yjmcc.2010.01.016
PMCID: PMC2854253  PMID: 20116385
troponin; structure; drugs; mechanism; inhibition
7.  Structure of the Inhibitor W7 Bound to the Regulatory Domain of Cardiac Troponin C† 
Biochemistry  2009;48(24):5541-5552.
The calmodulin antagonist W7 binds to troponin C in the presence of Ca2+ and inhibits striated muscle contraction. This study integrates multiple data into the structure of the regulatory domain of human cardiac troponin C (cNTnC) bound to Ca2+ and W7. The protein−W7 interface is defined through a three-dimensional {1H,13C}-edited-{1H,12C}-detected NOESY NMR experiment, and other aspects of the structure are modeled as perturbations to previously known coordinates and restraints. The structure determination protocol optimizes the protein−W7 contacts prior to the introduction of protein−W7 steric interactions or conformational changes in the protein. The structure determination protocol gives families of conformers that all have an optimal docking as assessed by satisfaction of the target function. The structure supports the previously proposed troponin I blocking mechanism for the activity of W7 in striated muscle and suggests a role for the flexible tail of W7 in stabilization of the bound state. This clarifies the structure−activity relationships of W7 and implicates an electrostatically mediated component of activity in common analogues of W7, including the antipsychotic trifluoroperazine and the cardiotonic levosimendan.
doi:10.1021/bi9001826
PMCID: PMC2697600  PMID: 19419198
8.  A glycolytic burst drives glucose induction of global histone acetylation by picNuA4 and SAGA 
Nucleic Acids Research  2009;37(12):3969-3980.
Little is known about what enzyme complexes or mechanisms control global lysine acetylation in the amino-terminal tails of the histones. Here, we show that glucose induces overall acetylation of H3 K9, 18, 27 and H4 K5, 8, 12 in quiescent yeast cells mainly by stimulating two KATs, Gcn5 and Esa1. Genetic and pharmacological perturbation of carbon metabolism, combined with 1H-NMR metabolic profiling, revealed that glucose induction of KAT activity directly depends on increased glucose catabolism. Glucose-inducible Esa1 and Gcn5 activities predominantly reside in the picNuA4 and SAGA complexes, respectively, and act on chromatin by an untargeted mechanism. We conclude that direct metabolic regulation of globally acting KATs can be a potent driving force for reconfiguration of overall histone acetylation in response to a physiological cue.
doi:10.1093/nar/gkp270
PMCID: PMC2709565  PMID: 19406923
9.  Interaction of cardiac troponin with cardiotonic drugs 
Over the forty years since its discovery, many studies have focused on understanding the role of troponin as a myofilament based molecular switch in regulating the Ca2+-dependent activation of striated muscle contraction. Recently, studies have explored the role of cardiac troponin as a target for cardiotonic agents. These drugs are clinically useful for treating heart failure, a condition in which the heart is no longer able to pump enough blood to other organs. These agents act via a mechanism that modulates the Ca2+-sensitivity of troponin; such a mode of action is therapeutically desirable because intracellular Ca2+ concentration is not perturbed, preserving the regulation of other Ca2+-based signaling pathways. This review describes molecular details of the interaction of cardiac troponin with a variety of cardiotonic drugs. We present recent structural work that has identified the docking sites of several cardiotonic drugs in the troponin C - troponin I interface and discuss their relevance in the design of troponin based drugs for the treatment of heart disease.
doi:10.1016/j.bbrc.2007.12.108
PMCID: PMC2349097  PMID: 18162171
cardiac muscle contraction; myofilament; troponin; cardiotonic drugs; calcium sensitizers
10.  Defining the Binding Site of Levosimendan and Its Analogues in a Regulatory Cardiac Troponin C−Troponin I Complex† 
Biochemistry  2008;47(28):7485-7495.
The interaction of Cardiac Troponin C (cTnC) and Cardiac Troponin I (cTnI) plays a critical role in transmitting the Ca2+ signal to the other myofilament proteins in the activation of cardiac muscle contraction. As such, the cTnC−cTnI interface is a logical target for cardiotonic agents such as levosimendan that can modulate the Ca2+ sensitivity of the myofilaments. Evidence indicates that drug candidates may exert their effects by targeting a site formed by binding of the switch region of cTnI to the regulatory N domain of cTnC (cNTnC). In this study, we utilized two-dimensional 1H−15N HSQC NMR spectroscopy to monitor the binding of levosimendan and its analogues, CMDP, AMDP, CI-930, imazodan, and MPDP, to cNTnC·Ca2+ in complex with two versions of the switch region of cTnI (cTnI147−163 and cTnI144−163). Levosimendan, CMDP, AMDP, and CI-930 were found to bind to both cNTnC·Ca2+·cTnI147−163 and cNTnC·Ca2+·cTnI144−163 complexes. These compounds contain a methyl group that is absent in MPDP or imazodan. Thus, the methyl group is one of the pharmacophores responsible for the action of these pyridazinone drugs on cTnC. Furthermore, the results showed that the cNTnC·Ca2+·cTnI144−163 complex presents a higher-affinity binding site for these compounds than the cNTnC·Ca2+·cTnI147−163 complex. This is consistent with our observation that the affinity of cTnI144−163 for cNTnC·Ca2+ is ∼10-fold stronger than that of cTnI147−163, likely a result of electrostatic forces between the N-terminal RRV extension in cTnI144−163 and the acidic residues in the C and D helices of cNTnC. These results will help in the delineation of the mode of action of levosimendan on the important functional unit of cardiac troponin that constitutes the regulatory domain of cTnC and the switch region of cTnI.
doi:10.1021/bi800438k
PMCID: PMC2652250  PMID: 18570382
11.  DNA Binding: a Novel Function of Pseudomonas aeruginosa Type IV Pili 
Journal of Bacteriology  2005;187(4):1455-1464.
The opportunistic pathogen Pseudomonas aeruginosa produces multifunctional, polar, filamentous appendages termed type IV pili. Type IV pili are involved in colonization during infection, twitching motility, biofilm formation, bacteriophage infection, and natural transformation. Electrostatic surface analysis of modeled pilus fibers generated from P. aeruginosa strain PAK, K122-4, and KB-7 pilin monomers suggested that a solvent-exposed band of positive charge may be a common feature of all type IV pili. Several functions of type IV pili, including natural transformation and biofilm formation, involve DNA. We investigated the ability of P. aeruginosa type IV pili to bind DNA. Purified PAK, K122-4, and KB-7 pili were observed to bind both bacterial plasmid and salmon sperm DNA in a concentration-dependent and saturable manner. PAK pili had the highest affinity for DNA, followed by K122-4 and KB-7 pili. DNA binding involved backbone interactions and preferential binding to pyrimidine residues even though there was no evidence of sequence-specific binding. Pilus-mediated DNA binding was a function of the intact pilus and thus required elements present in the quaternary structure. However, binding also involved the pilus tip as tip-specific, but not base-specific, antibodies inhibited DNA binding. The conservation of a Thr residue in all type IV pilin monomers examined to date, along with the electrostatic data, implies that DNA binding is a conserved function of type IV pili. Pilus-mediated DNA binding could be important for biofilm formation both in vivo during an infection and ex vivo on abiotic surfaces.
doi:10.1128/JB.187.4.1455-1464.2005
PMCID: PMC545619  PMID: 15687210
12.  VADAR: a web server for quantitative evaluation of protein structure quality 
Nucleic Acids Research  2003;31(13):3316-3319.
VADAR (Volume Area Dihedral Angle Reporter) is a comprehensive web server for quantitative protein structure evaluation. It accepts Protein Data Bank (PDB) formatted files or PDB accession numbers as input and calculates, identifies, graphs, reports and/or evaluates a large number (>30) of key structural parameters both for individual residues and for the entire protein. These include excluded volume, accessible surface area, backbone and side chain dihedral angles, secondary structure, hydrogen bonding partners, hydrogen bond energies, steric quality, solvation free energy as well as local and overall fold quality. These derived parameters can be used to rapidly identify both general and residue-specific problems within newly determined protein structures. The VADAR web server is freely accessible at http://redpoll.pharmacy.ualberta.ca/vadar.
PMCID: PMC168972  PMID: 12824316
13.  Metal ion-dependent, reversible, protein filament formation by designed beta-roll polypeptides 
Background
A right-handed, calcium-dependent β-roll structure found in secreted proteases and repeat-in-toxin proteins was used as a template for the design of minimal, soluble, monomeric polypeptides that would fold in the presence of Ca2+. Two polypeptides were synthesised to contain two and four metal-binding sites, respectively, and exploit stacked tryptophan pairs to stabilise the fold and report on the conformational state of the polypeptide.
Results
Initial analysis of the two polypeptides in the presence of calcium suggested the polypeptides were disordered. The addition of lanthanum to these peptides caused aggregation. Upon further study by right angle light scattering and electron microscopy, the aggregates were identified as ordered protein filaments that required lanthanum to polymerize. These filaments could be disassembled by the addition of a chelating agent. A simple head-to-tail model is proposed for filament formation that explains the metal ion-dependency. The model is supported by the capping of one of the polypeptides with biotin, which disrupts filament formation and provides the ability to control the average length of the filaments.
Conclusion
Metal ion-dependent, reversible protein filament formation is demonstrated for two designed polypeptides. The polypeptides form filaments that are approximately 3 nm in diameter and several hundred nm in length. They are not amyloid-like in nature as demonstrated by their behaviour in the presence of congo red and thioflavin T. A capping strategy allows for the control of filament length and for potential applications including the "decoration" of a protein filament with various functional moieties.
doi:10.1186/1472-6807-7-63
PMCID: PMC2174480  PMID: 17908326
14.  HMDB: the Human Metabolome Database 
Nucleic Acids Research  2007;35(Database issue):D521-D526.
The Human Metabolome Database (HMDB) is currently the most complete and comprehensive curated collection of human metabolite and human metabolism data in the world. It contains records for more than 2180 endogenous metabolites with information gathered from thousands of books, journal articles and electronic databases. In addition to its comprehensive literature-derived data, the HMDB also contains an extensive collection of experimental metabolite concentration data compiled from hundreds of mass spectra (MS) and Nuclear Magnetic resonance (NMR) metabolomic analyses performed on urine, blood and cerebrospinal fluid samples. This is further supplemented with thousands of NMR and MS spectra collected on purified, reference metabolites. Each metabolite entry in the HMDB contains an average of 90 separate data fields including a comprehensive compound description, names and synonyms, structural information, physico-chemical data, reference NMR and MS spectra, biofluid concentrations, disease associations, pathway information, enzyme data, gene sequence data, SNP and mutation data as well as extensive links to images, references and other public databases. Extensive searching, relational querying and data browsing tools are also provided. The HMDB is designed to address the broad needs of biochemists, clinical chemists, physicians, medical geneticists, nutritionists and members of the metabolomics community. The HMDB is available at:
doi:10.1093/nar/gkl923
PMCID: PMC1899095  PMID: 17202168

Results 1-14 (14)