Tubulin polyglutamylation differentially regulates microtubule-interacting proteins.

Tubulin posttranslational modifications have been predicted to control cytoskeletal functions by coordinating the molecular interactions between microtubules and their associating proteins. A prominent tubulin modification in neurons is polyglutamylation, the deregulation of which causes neurodegeneration. Yet, the underlying molecular mechanisms have remained elusive. Here, using in-vitro reconstitution, we determine how polyglutamylation generated by the two predominant neuronal polyglutamylases, TTLL1 and TTLL7, specifically modulates the activities of three major microtubule interactors: the microtubule-associated protein Tau, the microtubule-severing enzyme katanin and the molecular motor kinesin-1. We demonstrate that the unique modification patterns generated by TTLL1 and TTLL7 differentially impact those three effector proteins, thus allowing for their selective regulation. Given that our experiments were performed with brain tubulin from mouse models in which physiological levels and patterns of polyglutamylation were altered by the genetic knockout of the main modifying enzymes, our quantitative measurements provide direct mechanistic insight into how polyglutamylation could selectively control microtubule interactions in neurons.

Calmodulin and S100A1 protein interact with N terminus of TRPM3 channel.

Transient receptor potential melastatin 3 ion channel (TRPM3) belongs to the TRP family of cation-permeable ion channels involved in many important biological functions such as pain transduction, thermosensation, and mechanoregulation. The channel was reported to play an important role in Ca(2+) homeostasis, but its gating mechanisms, functions, and regulation are still under research. Utilizing biophysical and biochemical methods, we characterized two independent domains, Ala-35-Lys-124 and His-291-Gly-382, on the TRPM3 N terminus, responsible for interactions with the Ca(2+)-binding proteins calmodulin (CaM) and S100A1. We identified several positively charged residues within these domains as having a crucial impact on CaM/S100A1 binding. The data also suggest that the interaction is calcium-dependent. We also performed competition assays, which suggested that CaM and S100A1 are able to compete for the same binding sites within the TRPM3 N terminus. This is the first time that such an interaction has been shown for TRP family members.

Characterization of calmodulin binding domains in TRPV2 and TRPV5 C-tails.

The transient receptor potential channels TRPV2 and TRPV5 belong to the vanilloid TRP subfamily. TRPV2 is highly similar to TRPV1 and shares many common properties with it. TRPV5 (and also its homolog TRPV6) is a rather distinct member of the TRPV subfamily. It is distant for being strictly Ca(2+)-selective and features quite different properties from the rest of the TRPV subfamily. It is known that TRP channels are regulated by calmodulin in a calcium-dependent manner. In our study we identified a calmodulin binding site on the C-termini of TRPV2 (654-683) and TRPV5 (587-616) corresponding to the consensus CaM binding motif 1-5-10. The R679 and K681 single mutants of TRPV2 caused a 50% decrease in binding affinity and a double mutation of K661/K664 of the same peptide lowered the binding affinity by up to 75%. A double mutation of R606/K607 and triple mutation of R594/R606/R610 in TRPV5 C-terminal peptide resulted in the total loss of binding affinity to calmodulin. These results demonstrate that the TRPV2 C-tail and TRPV5 C-tail contain calmodulin binding sites and that the basic residues are strongly involved in TRP channel binding to calmodulin.

ATP and magnesium drive conformational changes of the Na+/K+-ATPase cytoplasmic headpiece.

Conformational changes of the Na(+)/K(+)-ATPase isolated large cytoplasmic segment connecting transmembrane helices M4 and M5 (C45) induced by the interaction with enzyme ligands (i.e. Mg(2+) and/or ATP) were investigated by means of the intrinsic tryptophan fluorescence measurement and molecular dynamic simulations. Our data revealed that this model system consisting of only two domains retained the ability to adopt open or closed conformation, i.e. behavior, which is expected from the crystal structures of relative Ca(2+)-ATPase from sarco(endo)plasmic reticulum for the corresponding part of the entire enzyme. Our data revealed that the C45 is found in the closed conformation in the absence of any ligand, in the presence of Mg(2+) only, or in the simultaneous presence of Mg(2+) and ATP. Binding of the ATP alone (i.e. in the absence of Mg(2+)) induced open conformation of the C45. The fact that the transmembrane part of the enzyme was absent in our experiments suggested that the observed conformational changes are consequences only of the interaction with ATP or Mg(2+) and may not be related to the transported cations binding/release, as generally believed. Our data are consistent with the model, where ATP binding to the low-affinity site induces conformational change of the cytoplasmic part of the enzyme, traditionally attributed to E2-->E1 transition, and subsequent Mg(2+) binding to the enzyme-ATP complex induces in turn conformational change traditionally attributed to E1-->E2 transition.

Mitochondria-adaptor TRAK1 promotes kinesin-1 driven transport in crowded environments.

Intracellular trafficking of organelles, driven by kinesin-1 stepping along microtubules, underpins essential cellular processes. In absence of other proteins on the microtubule surface, kinesin-1 performs micron-long runs. Under crowding conditions, however, kinesin-1 motility is drastically impeded. It is thus unclear how kinesin-1 acts as an efficient transporter in intracellular environments. Here, we demonstrate that TRAK1 (Milton), an adaptor protein essential for mitochondrial trafficking, activates kinesin-1 and increases robustness of kinesin-1 stepping on crowded microtubule surfaces. Interaction with TRAK1 i) facilitates kinesin-1 navigation around obstacles, ii) increases the probability of kinesin-1 passing through cohesive islands of tau and iii) increases the run length of kinesin-1 in cell lysate. We explain the enhanced motility by the observed direct interaction of TRAK1 with microtubules, providing an additional anchor for the kinesin-1-TRAK1 complex. Furthermore, TRAK1 enables mitochondrial transport in vitro. We propose adaptor-mediated tethering as a mechanism regulating kinesin-1 motility in various cellular environments.

Changes in electrostatic surface potential of Na+/K+-ATPase cytoplasmic headpiece induced by cytoplasmic ligand(s) binding.

A set of single-tryptophan mutants of the Na(+)/K(+)-ATPase isolated, large cytoplasmic loop connecting transmembrane helices M4 and M5 (C45) was prepared to monitor effects of the natural cytoplasmic ligands (i.e., Mg(2+) and/or ATP) binding. We introduced a novel method for the monitoring of the changes in the electrostatic surface potential (ESP) induced by ligand binding, using the quenching of the intrinsic tryptophan fluorescence by acrylamide or iodide. This approach opens a new way to understanding the interactions within the proteins. Our experiments revealed that the C45 conformation in the presence of the ATP (without magnesium) substantially differed from the conformation in the presence of Mg(2+) or MgATP or in the absence of any ligand not only in the sense of geometry but also in the sense of the ESP. Notably, the set of ESP-sensitive residues was different from the set of geometry-sensitive residues. Moreover, our data indicate that the effect of the ligand binding is not restricted only to the close environment of the binding site and that the information is in fact transmitted also to the distal parts of the molecule. This property could be important for the communication between the cytoplasmic headpiece and the cation binding sites located within the transmembrane domain.

NMR determination of pKa values of indoloquinoline alkaloids.

Malaria is one of the most serious global health problems. Isolating new therapeutic agents with potential antimalarial activity from natural sources or preparing such agents either semisynthetically or synthetically is one strategy for solving the problem of resistance constantly evolving to the drugs currently in use. For alkaloids, the acid-base dissociation constant, pK(a), is an important characteristic, thought to be associated with biological activity. In this contribution, pK(a) values for several indoloquinoline alkaloids were determined by using (1)H NMR spectroscopy in a mixture of solvents. The data were recalculated for water solutions using the correction factors reported previously. The structural dependence of the pK(a) values for cryptolepine and its isomers neocryptolepine, isocryptolepine and isoneocryptolepine as well as some substituted neocryptolepine derivatives is discussed.

Ionic interactions are essential for TRPV1 C-terminus binding to calmodulin.

Calmodulin (CaM) is known to play an important role in the regulation of TRP channels activity. Although it has been reported that CaM binds to the C-terminus of TRPV1 (TRPV1-CT), no classic CaM-binding motif was found in this region. In this work, we explored this unusual TRPV1 CaM-binding motif in detail and found that five residues from a putative CaM-binding motif are important for TRPV1-CT's binding to CaM, with arginine R785 being the most essential residue. The homology modelling suggests that a CaM-binding motif of TRPV1-CT forms an alpha helix that docks into the central cavity of CaM.

The role of proline residues in the structure and function of human MT2 melatonin receptor.

Melatonin functions as an essential regulator of various physiological processes in all vertebrate species. In mammals, two G protein-coupled melatonin receptors (GPCR) mediate some melatonin's actions: MT1 and MT2. Transmembrane domains (TM) of most GPCRs contain a set of highly conserved proline residues that presumably play important structural and functional roles. As TM segments of MT2 receptor display several interesting differences in expression of specific proline residues compared to other rhodopsin-like receptors (rGPCRs), we investigated the role of proline residues in the structure and function of this receptor. All prolines in TM segments of MT2 receptor were individually replaced with alanine and/or glycine. In addition, the unusual NAxxY motif located in TM7 was mutated to generate highly conserved NPxxY motif found in the majority of rGPCR proteins. Following transient expression in CHO-K1 cells, binding properties of the mutant receptors and their ability to transduce signals were analyzed using (125)I-mel- and [(35)S]GTPgammaS-binding assays, respectively. The impact of the performed mutations on the receptor structure was assessed by molecular dynamic simulations of MT2 receptors embedded in the fully hydrated phospholipid bilayer. Our results indicate that residues P174, P212 and P266 are important for the ligand binding and/or signaling of the human MT2 receptor. We also show that changes within the unusual NAxxY sequence in the TM7 (mutations A305P and A305V) produce defective MT2 receptors indicating an important role of this motif in the function of melatonin receptors.

Covalent bonding of azoles to quaternary protoberberine alkaloids.

Adducts of the quaternary protoberberine alkaloids (QPA) berberine, palmatine, and coptisine were prepared with nucleophiles derived from pyrrole, pyrazole, imidazole, and 1,2,4-triazole. The products, 8-substituted 7,8-dihydroprotoberberines, were identified by mass spectrometry and 1D and 2D NMR spectroscopy, including (1)H--(15)N shift correlations at natural abundance. In addition, two adducts of QPA with chloroform and methanethiolate were characterized by using NMR data. Single-crystal X-ray structures of 8-pyrrolyl-7,8-dihydroberberine, 8-pyrazolyl-7,8-dihydroberberine, and 8-imidazolyl-7,8-dihydroberberine are also presented.

Quaternary protoberberine alkaloids.

This contribution reviews some general aspects of the quaternary iminium protoberberine alkaloids. The alkaloids represent a very extensive group of secondary metabolites with diverse structures, distribution in nature, and biological effects. The quaternary protoberberine alkaloids (QPA), derived from the 5,6-dihydrodibenzo[a,g]quinolizinium system, belong to a large class of isoquinoline alkaloids. Following a general introduction, the plant sources of QPA, their biosynthesis, and procedures for their isolation are discussed. Analytical methods and spectral data are summarized with emphasis on NMR spectroscopy. The reactivity of QPA is characterized by the sensitivity of the iminium bond CN(+) to nucleophilic attack. The addition of various nucleophiles to the protoberberine skeleton is discussed. An extended discussion of the principal chemical reactivity is included since this governs interactions with biological targets. Quaternary protoberberine alkaloids and some related compounds exhibit considerable biological activities. Recently reported structural studies indicate that the QPA interact with nucleic acids predominantly as intercalators or minor groove binders. Currently, investigations in many laboratories worldwide are focused on the antibacterial and antimalarial activity, cytotoxicity, and potential genotoxicity of QPA.

Ca(2+) binding protein S100A1 competes with calmodulin and PIP2 for binding site on the C-terminus of the TPRV1 receptor.

Transient receptor potential vanilloid 1 ion channel (TRPV1) belongs to the TRP family of ion channels. These channels play a role in many important biological processes such as thermosensation and pain transduction. The TRPV1 channel was reported to be also involved in nociception. Ca(2+) ions are described to participate in the regulation of TRP channels through the interaction with Ca(2+)-binding proteins, such as calmodulin or S100A1. Calmodulin is involved in the Ca(2+)-dependent regulation of TRPV1 via its binding to the TRPV1 C-terminal region. However, the role of the Ca(2+)-binding protein S100A1 in the process of TRP channel regulation remains elusive. Here we characterized a region on the TRPV1 C-terminus responsible for the interaction with S100A1 using biochemical and biophysical tools. We found that this region overlaps with previously identified calmodulin and PIP2 binding sites and that S100A1 competes with calmodulin and PIP2 for this binding site. We identified several positively charged residues within this region, which have crucial impact on S100A1 binding, and we show that the reported S100A1-TRPV1 interaction is calcium-dependent. Taken together, our data suggest a mechanism for the mutual regulation of PIP2 and the Ca(2+)-binding proteins S100A1 and calmodulin to TRPV1.

Characterization of the S100A1 protein binding site on TRPC6 C-terminus.

The transient receptor potential (TRP) protein superfamily consists of seven major groups, among them the "canonical TRP" family. The TRPC proteins are calcium-permeable nonselective cation channels activated after the emptying of intracellular calcium stores and appear to be gated by various types of messengers. The TRPC6 channel has been shown to be expressed in various tissues and cells, where it modulates the calcium level in response to external signals. Calcium binding proteins such as Calmodulin or the family of S100A proteins are regulators of TRPC channels. Here we characterized the overlapping integrative binding site for S100A1 at the C-tail of TRPC6, which is also able to accomodate various ligands such as Calmodulin and phosphatidyl-inositol-(4,5)-bisphosphate. Several positively charged amino acid residues (Arg852, Lys856, Lys859, Arg860 and Arg864) were determined by fluorescence anisotropy measurements for their participation in the calcium-dependent binding of S100A1 to the C terminus of TRPC6. The triple mutation Arg852/Lys859/Arg860 exhibited significant disruption of the binding of S100A1 to TRPC6. This indicates a unique involvement of these three basic residues in the integrative overlapping binding site for S100A1 on the C tail of TRPC6.

PtdIns(4,5)P2 interacts with CaM binding domains on TRPM3 N-terminus.

TRPM3 has been reported to play an important role in Ca(2+) homeostasis, but its gating mechanisms and regulation via Ca(2+) are unknown. Ca(2+) binding proteins such as calmodulin (CaM) could be probable modulators of this ion channel. We have shown that this protein binds to two independent domains, A35-K124 and H291-G382 on the TRPM3 N-terminus, which contain conserved hydrophobic as well as positively charged residues in specific positions, and that these residues have a crucial impact on its binding. We also showed that the other Ca(2+) binding protein, S100A1, is able to bind to these regions and that CaM and S100A1 compete for these binding sites on the TRPM3 N-terminus. Moreover, our results suggest that another very important TRP channel activity modulator, PtdIns(4,5)P(2), interacts with the CaM/S100A1 binding sites on the TRPM3 N-terminus with high affinity.

Integrative binding sites within intracellular termini of TRPV1 receptor.

TRPV1 is a nonselective cation channel that integrates wide range of painful stimuli. It has been shown that its activity could be modulated by intracellular ligands PIP2 or calmodulin (CaM). The detailed localization and description of PIP2 interaction sites remain unclear. Here, we used synthesized peptides and purified fusion proteins of intracellular regions of TRPV1 expressed in E.coli in combination with fluorescence anisotropy and surface plasmon resonance measurements to characterize the PIP2 binding to TRPV1. We characterized one PIP2 binding site in TRPV1 N-terminal region, residues F189-V221, and two independent PIP2 binding sites in C-terminus: residues K688-K718 and L777-S820. Moreover we show that two regions, namely F189-V221 and L777-S820, overlap with previously localized CaM binding sites. For all the interactions the equilibrium dissociation constants were estimated. As the structural data regarding C-terminus of TRPV1 are lacking, restraint-based molecular modeling combined with ligand docking was performed providing us with structural insight to the TRPV1/PIP2 binding. Our experimental results are in excellent agreement with our in silico predictions.

The interactions of the C-terminal region of the TRPC6 channel with calmodulin.

The transient receptor potential channel TRPC6 is a non-selective cation channel which modulates the calcium level in eukaryotic cells (including sensory receptor cells) in response to external signals. Calmodulin (CaM) is a ubiquitously expressed Ca(2+) binding protein that is an important mediator of Ca(2+)-dependent regulation of the TRPC6 channel. One CaM binding site was identified within the C-tail of TRPC6. The aim of this study is to map in detail the CaM and inositol (1,4,5)-triphosphate receptor binding (CIRB) domain in the C-terminal region of mouse TRPC6 that is capable of interacting with CaM using in vitro binding assays. Besides the set of positively charged amino acid residues Arg852, Lys856, Arg864, Lys859/Arg860, a hydrophobic Ile857, at the position 1 in 1-5-10 motif, was located and the effect of replacing it with a neutral residue was tested using fluorescence anisotropy measurement. Participation of Ile857 could indicate a strong role of this conserved CaM binding motif.

C-geranyl compounds from Paulownia tomentosa fruits.

Five geranylflavonoids, one prenylated flavonoid, and a simple flavanone were isolated from an ethanolic extract of Paulownia tomentosa fruit. Tomentodiplacol (1), 3'-O-methyl-5'-methoxydiplacol (2), 6-isopentenyl-3'-O-methyltaxifolin (3), and dihydrotricin (4) are reported from a natural source for the first time and 3'-O-methyldiplacone (6) for the first time from the genus Paulownia. The structures of the compounds were determined by mass spectrometry, including HRMS, and by 1D and 2D NMR spectroscopy. The cytotoxicity and DPPH (2,2-diphenyl-1-picrylhydrazyl)-quenching activity of some of these compounds were tested, with diplacone proving to be the best antioxidant, although the most cytotoxic compound.

ATP binding site on the C-terminus of the vanilloid receptor.

Transient receptor potential channel vanilloid receptor subunit 1 (TRPV1) is a thermosensitive cation channel activated by noxious heat as well as a wide range of chemical stimuli. Although ATP by itself does not directly activate TRPV1, it was shown that intracellular ATP increases its activity by directly interacting with the Walker A motif residing on the C-terminus of TRPV1. In order to identify the amino acid residues that are essential for the binding of ATP to the TRPV1 channel, we performed the following point mutations of the Walker A motif: P732A, D733A, G734A, K735A, D736A, and D737A. Employing bulk fluorescence measurements, namely a TNP-ATP competition assay and FITC labelling and quenching experiments, we identified the key role of the K735 residue in the binding of the nucleotide. Experimental data was interpreted according to our molecular modelling simulations.