Philipp O. Tsvetkov

Binding of ATP to Heat Shock Protein 90 EVIDENCE FOR AN ATP-BINDING SITE IN THE C-TERMINAL DOMAIN

The presence of a nucleotide binding site on hsp90 was very controversial until x-ray structure of the hsp90 N-terminal domain, showing a nonconventional nucleotide binding site, appeared. A recent study suggested that the hsp90 C-terminal domain also binds ATP (Marcu, M. G., Chadli, A., Bouhouche, I., Catelli, M. G., and Neckers, L. M. (2000) J. Biol. Chem. 275, 37181–37186). In this paper, the interactions of ATP with native hsp90 and its recombinant N-terminal (positions 1–221) and C-terminal (positions 446–728) domains were studied by isothermal titration calorimetry, scanning differential calorimetry, and fluorescence spectroscopy. Results clearly demonstrate that hsp90 possesses a second ATP-binding site located on the C-terminal part of the protein. The association constant between this domain of hsp90 and ATP-Mg and a comparison with the binding constant on the full-length protein are reported for the first time. Secondary structure prediction revealed motifs compatible with a Rossmann fold in the C-terminal part of hsp90. It is proposed that this potential Rossmann fold may constitute the C-terminal ATP-binding site. This work also suggests allosteric interaction between N- and C-terminal domains of hsp90.

Isomerization of the Asp7 Residue Results in Zinc-Induced Oligomerization of Alzheimer’s Disease Amyloid β(1–16) Peptide

Alzheimer’s disease (AD)—a fatal neurodegenerative disorder that primarily affects the elderly—is pathophysiologically characterized by the extracellular deposition of a 40/42-aminoacid-long protein, referred to as amyloid-b peptide (Ab), in the brains of AD victims. Although the molecular mechanism of AD onset is unknown, the transformation of Ab from its native monomer conformation via soluble dimers and higher oligomers into insoluble fibrillar b-sheet aggregates, which finally accumulate into the amyloid plaques, is believed to be a key event in AD pathogenesis. One plausible hypothesis suggests that the amyloid neuropathology of AD depends on zinc ions released during neurotransmission, and so it is assumed that binding of zinc to Ab might play an important role in initiating pathogenic amyloid deposition, as well as some additional still unidentified proteinaceous factors. The Ab molecules isolated from AD brain lesions have numerous endogenous post-translational modifications (PTMs), which should profoundly affect both the Ab conformation and its oligomeric state and make up a pool of potential pathogenic agents in AD. The most abundant PTM of Ab is isomerization of the Asp7 residue; this results in the formation of an lisoAsp7 isoform (isoaspartate). This nonenzymatic modification occurs spontaneously in polypeptides through an intramolecular rearrangement of aspartate or asparagine residues and is generally regarded as a degradation reaction that occurs in vivo during tissue ageing. In the case of isomerized Ab (isoAb) it is still unclear whether the isoaspartyl residues are the cause or the result of the pathological accumulation of Ab. Nevertheless, recent in vitro experimental evidence indicates that isoAb might potentially be involved in the onset of AD. To investigate the role of the Asp7 isomerization in zinc-induced oligomerization of Ab we have studied the thermodynamics of zinc binding and the oligomeric states of two synthetic model peptides that correspond to region 1–16 in Ab and in isoAb : Ab16 and isoAb16, respectively. Earlier, this region was identified as the zinc-binding domain of Ab, which binds Zn with 1:1 stoichiometry and a 6 mm dissociation constant. Both Ab16 and its complex with Zn 2+ were found to be monomeric under physiological conditions for at least six months over a wide concentration range, and so were used as monomer reference standards throughout this work. The isoAb16 was also shown to possess zinc-binding ability; [11] however, the properties of the Zn–isoAb16 complex have not been studied previously. To compare Zn binding to Ab16 and to isoAb16 (in 50 mm Tris buffer at pH 7.3), isothermal titration calorimetry (ITC) was used. The thermodynamic data demonstrate that Ab16 binds one zinc ion with an association constant of 1.7ACHTUNGTRENNUNG( 0.4)A10m 1 (Figure 1), which corresponds to previously published da ACHTUNGTRENNUNGta.

Zinc-induced dimerization of the amyloid-β metal-binding domain 1–16 is mediated by residues 11–14

Analysis of complex formation between amyloid-β fragments using surface plasmon resonance biosensing and electrospray mass spectrometry reveals that region 11-14 mediates zinc-induced dimerization of amyloid-β and may serve as a potential drug target for preventing development and progression of Alzheimers disease.

Apocalmodulin binds to the myosin light chain kinase calmodulin target site.

The interaction of a 20-residue-long peptide derived from the calmodulin-binding domain of the smooth muscle myosin light chain kinase with calcium-free calmodulin (apocalmodulin) was studied using a combination of isothermal titration calorimetry and differential scanning calorimetry. We showed that: (i) a significant binding between apocalmodulin and the target peptide (RS20) exists in the absence of salt (K a = 106 m −1), (ii) the peptide interacts with the C-terminal lobe of calmodulin and adopts a partly helical conformation, and (iii) the presence of salt weakens the affinity of the peptide for apocalmodulin, emphasizing the importance of electrostatic interactions in the complex. Based on these results and taking into account the work of Bayley et al. (Bayley, P. M., Findlay, W.A., and Martin, S. R. (1996) Protein Sci. 5, 1215–1228), we suggest a physiological role for apocalmodulin.

Stathmin/Op18 is a novel mediator of vinblastine activity.

MINT‐6603918: tubulin beta (uniprotkb:Q9H4B7), tubulin alpha (uniprotkb:Q71U36) and stathmin (uniprotkb:Q71U36) physically interact (MI:0218) by cosedimentation (MI:0027) MINT‐6603930: tubulin alpha (uniprotkb:Q71U36) physically interacts (MI:0218) with tubulin beta (uniprotkb:Q9H4B7) and stathmin (uniprotkb:P16949) by isothermal titration calorimetry (MI:0065)

Disordering of human telomeric G-quadruplex with novel antiproliferative anthrathiophenedione.

Linear heteroareneanthracenediones have been shown to interfere with DNA functions, thereby causing death of human tumor cells and their drug resistant counterparts. Here we report the interaction of our novel antiproliferative agent 4,11-bis[(2-{[acetimido]amino}ethyl)amino]anthra[2,3-b]thiophene-5,10-dione with telomeric DNA structures studied by isothermal titration calorimetry, circular dichroism and UV absorption spectroscopy. New compound demonstrated a high affinity (Kass∼106 M−1) for human telomeric antiparallel quadruplex d(TTAGGG)4 and duplex d(TTAGGG)4∶d(CCCTAA)4. Importantly, a ∼100-fold higher affinity was determined for the ligand binding to an unordered oligonucleotide d(TTAGGG TTAGAG TTAGGG TTAGGG unable to form quadruplex structures. Moreover, in the presence of Na+ the compound caused dramatic conformational perturbation of the telomeric G-quadruplex, namely, almost complete disordering of G-quartets. Disorganization of a portion of G-quartets in the presence of K+ was also detected. Molecular dynamics simulations were performed to illustrate how the binding of one molecule of the ligand might disrupt the G-quartet adjacent to the diagonal loop of telomeric G-quadruplex. Our results provide evidence for a non-trivial mode of alteration of G-quadruplex structure by tentative antiproliferative drugs.

New insights into tau-microtubules interaction revealed by isothermal titration calorimetry.

Microtubule dynamic instability is tightly regulated by coordinated action of stabilizing and destabilizing microtubule associated proteins. Among the stabilizing proteins, tau plays a pivotal role in both physiological and pathological processes. Nevertheless, the detailed mechanism of tau-tubulin interaction is still subject to controversy. In this report, we studied for the first time tau binding to tubulin by a direct thermodynamic method in the absence of any tubulin polymerization cofactors that could influence this process. Isothermal titration calorimetry enabled us to evidence two types of tau-tubulin binding modes: one corresponding to a high affinity binding site with a tau:tubulin stoichiometry of 0.2 and the other one to a low affinity binding site with a stoichiometry of 0.8. The same stoichiometries were obtained at all temperatures tested (10-37°C), indicating that the mechanism of interaction does not depend on the type of tubulin polymer triggered upon tau binding. These findings allowed us to get new insights into the topology of tau on microtubules.

Methylated 23S rRNA nucleotide m2G1835 of Escherichia coli ribosome facilitates subunit association

Among 4.5 thousand nucleotides of Escherichia coli ribosome 36 are modified. These nucleotides are clustered in the functional centers of ribosome, particularly on the interface of large and small subunits. Nucleotide m(2)G1835 located on the 50S side of intersubunit bridge cluster B2 is modified by N2-methyltransferase RlmG. By means of isothermal titration calorimetry and Rayleigh light scattering, we have found that methylation of m(2)G1835 specifically enhances association of ribosomal subunits. No defects in fidelity of translation or interaction with translation GTPases could be ascribed to the ribosomes unmethylated at G1835 of the 23S rRNA. Methylation of G1835 was found to provide a significant advantage for bacteria at osmotic and oxidative stress.

Deciphering the molecular mechanisms of anti-tubulin plant derived drugs

Even though commercialized anticancer drugs are now produced by pharmaceutical companies, most of them were originally obtained from natural sources, and more particularly from plants. Indeed, many structurally diverse compounds isolated from plants or marine flora have been purified and synthesized for their anticancer bioactivity. Among these, several molecules belong to the class of anticancer drugs which target the microtubule cytoskeleton, either by stabilizing it or destabilizing it. To characterize the activity of these drugs and to understand in which physiological context they are more likely to be used as therapeutic agents, it is necessary to fully determine their interaction with tubulin. Understanding the molecular basis of their effects on microtubule cytoskeleton is an important step in designing analogs with greater pharmacological activity and with fewer side effects. In addition, knowing the molecular mechanism of action of each drug that is already used in chemotherapy protocols will also help to find strategies to circumvent resistance. By taking examples of known anti-tubulin plant derived drugs, we show how identification of microtubule targeting agents and further characterization of their activity can be achieved combining biophysical and biochemical approaches. We also illustrate how continuing in depth study of molecules with already known primary mechanisms of action can lead to the discovery of new targets or biomarkers which can open new perspectives in anticancer strategies.

Microtubule-associated proteins and tubulin interaction by isothermal titration calorimetry.

Microtubules play an important role in a number of vital cell processes such as cell division, intracellular transport, and cell architecture. The highly dynamic structure of microtubules is tightly regulated by a number of stabilizing and destabilizing microtubule-associated proteins (MAPs), such as tau and stathmin. Because of their importance, tubulin-MAPs interactions have been extensively studied using various methods that provide researchers with complementary but sometimes contradictory thermodynamic data. Isothermal titration calorimetry (ITC) is the only direct thermodynamic method that enables a full thermodynamic characterization (stoichiometry, enthalpy, entropy of binding, and association constant) of the interaction after a single titration experiment. This method has been recently applied to study tubulin-MAPs interactions in order to bring new insights into molecular mechanisms of tubulin regulation. In this chapter, we review the technical specificity of this method and then focus on the use of ITC in the investigation of tubulin-MAPs binding. We describe technical issues which could arise during planning and carrying out the ITC experiments, in particular with fragile proteins such as tubulin. Using examples of stathmin and tau, we demonstrate how ITC can be used to gain major insights into tubulin-MAP interaction.