Daniel Tietze

The experimental power of FR900359 to study Gq-regulated biological processes

Despite the discovery of heterotrimeric αβγ G proteins ∼25 years ago, their selective perturbation by cell-permeable inhibitors remains a fundamental challenge. Here we report that the plant-derived depsipeptide FR900359 (FR) is ideally suited to this task. Using a multifaceted approach we systematically characterize FR as a selective inhibitor of Gq/11/14 over all other mammalian Gα isoforms and elaborate its molecular mechanism of action. We also use FR to investigate whether inhibition of Gq proteins is an effective post-receptor strategy to target oncogenic signalling, using melanoma as a model system. FR suppresses many of the hallmark features that are central to the malignancy of melanoma cells, thereby providing new opportunities for therapeutic intervention. Just as pertussis toxin is used extensively to probe and inhibit the signalling of Gi/o proteins, we anticipate that FR will at least be its equivalent for investigating the biological relevance of Gq.

Braces for the peptide backbone: insights into structure-activity relationships of protease inhibitor mimics with locked amide conformations.

The architecture of protein macromolecules fundamentallydepends on the sequential arrangement of peptide backbonebonds in defined conformations. Among the three torsionangles(f,y,andw)presentateachaminoacid,itistheamidebond (w) which is intrinsically hindered as a result of itspartial double-bond character and it is thus more or lessrestricted to either a trans or a cis conformation (Figure 1).

Drug-induced conformational and dynamical changes of the S31N mutant of the influenza M2 proton channel investigated by solid-state NMR.

The M2 protein of influenza A viruses forms a tetrameric proton channel that is targeted by the amantadine class of antiviral drugs. A S31N mutation in the transmembrane (TM) domain of the protein has caused widespread amantadine resistance in most of the currently circulating flu viruses. Recently, a new family of compounds based on amantadine- and aryl-substituted isoxazole were discovered to inhibit the S31N channel activity and reduce replication of S31N-harboring viruses. We now use solid-state NMR spectroscopy to investigate the effects of one of these isoxazole compounds, WJ352, on the conformation of the S31N TM segment and the dynamics of the proton-selective residue, His37. Chemical shift perturbations show that WJ352 changes the conformational equilibrium of multiple TM residues, with the maximal perturbation occurring at the crucial Asn31. (13)C-(2)H distance measurements and (1)H-(1)H NOE cross peaks indicate that the adamantane moiety of the drug is bound in the spacious pore between Asn31 and Gly34 while the phenyl tail is located near Val27. Thus, the polar amine points to the channel exterior rather than to His37, in contrast to amantadine and rimantadine in the wild-type channel, suggesting that the drug is significantly stabilized by hydrophobic interactions between the adamantane and the TM peptide. (15)N and (13)C chemical shifts indicate that at low pH, His37 undergoes fast exchange among the τ tautomer, the π tautomer, and the cationic state due to proton transfer with water. The exchange rate is higher than the wild-type channel, consistent with the larger single-channel conductance of the mutant. Drug binding at acidic pH largely suppresses this exchange, reverting the histidines to a similar charge distribution as that of the high-pH closed state.

New Insight into the Mode of Action of Nickel Superoxide Dismutase by Investigating Metallopeptide Substrate Models

For the first time, the existence of a substrate adduct of a nickel superoxide dismutase (NiSOD) model, based on the first nine residues from the N terminus of the active form of Streptomyces coelicolor NiSOD, has been proven and the adduct has been isolated. This adduct is based on the cyanide anion (CN(-)), as a substrate analogue of the superoxide anion (O(2)(*-)), and the nickel metallopeptide H-HCDLPCGVY-NH(2)-Ni. Spectroscopic studies, including IR, UV/Vis, and liquid- and solid-state NMR spectroscopy, show a single nickel-bound cyanide anion, which is embedded in the metallopeptide structure. This complex sheds new light on the question of whether the mode of action of the NiSOD enzyme is an inner- or outer-sphere mechanism. Whereas discussion was previously biased in favor of an outer-sphere electron-transfer mechanism due to the fact that binding of cyanide or azide moieties to the nickel active site had never been observed, our results are a clear indication in favor of the inner-sphere electron-transfer mechanism for the disproportionation of the O(2)(*-) ion, whereby the substrate is attached to the Ni atom in the active site of the NiSOD.

Development of a Functional cis‐Prolyl Bond Biomimetic and Mechanistic Implications for Nickel Superoxide Dismutase

During recent years several peptide-based Ni superoxide dismutase (NiSOD) models have been developed. These NiSOD models show an important structural difference compared to the native NiSOD enzyme, which could cause a completely different mechanism of superoxide dismutation. In the native enzyme the peptide bond between Leu4 and Pro5 is cis-configured, while the NiSOD models exhibit a trans-configured peptide bond between these two residues. To shed light on how the configuration of this single peptide bond influences the activity of the NiSOD model peptides, a new cis-prolyl bond surrogate was developed. As surrogate we chose a leucine/alanine-based disubstituted 1,2,3-triazole, which was incorporated into the NiSOD model peptide replacing residues Leu4 and Pro5. The yielded 1,5-disubstituted triazole nickel peptide exhibited high SOD activity, which was approximately the same activity as its parent trans-configured analogue. Hence, the conformation of the prolyl peptide bond apparently has of minor importance for the catalytic activity of the metallopeptides as postulated in literature. Furthermore, it is shown that the triazole metallopeptide is forming a stable cyanide adduct as a substrate analogue model complex.

Structurally Diverse μ‐Conotoxin PIIIA Isomers Block Sodium Channel NaV1.4

Certain VGSC subtypes (NaV1.3, 1.7, 1.8, and 1.9) are expressed in the peripheral nervous system and mediate the transmission of signals leading to the sensation of different kinds of pain, such as nociception (NaV1.8), acute inflammatory (NaV1.7), and neuropathic (NaV1.3) pain. [2] Therefore, VGSCs are potential targets for novel analgesics, ideally those with strong channel specificity. Among sodium channel antagonists, m- and mO-conotoxins from the venoms of marine cone snails have attracted considerable attention because of their analgesic potency. [2a, 3] m-Conotoxins are 14to 26-mer peptides with six cysteine residues (Supporting Information, Table S1). [4] They inhibit muscle and/or neuronal VGSCs by occluding the ion channel pore. [5] A specific cysteine framework, that is, CCXnCXnCXnCC, confers conformational restriction to their three-dimensional structure upon formation of three disulfide bonds. It is generally accepted that the native fold of the toxins carries the disulfide connectivities Cys1–Cys4, Cys2–Cys5, and Cys3–Cys6 (numbered in the order of occurrence in the amino acid sequence). [3, 5b] However, three-dimensional structures are available only for a limited subset of m-conotoxins, that is, PIIIA, [6]

Mechanisms of Dipolar Ortho/Para-H2O Conversion in Ice

Abstract In this paper a possible explanation for an unexpected ortho/para-water ratio in the gas clouds of comets is given. The description is based on the quantum-mechanical density matrix formalism and the spin temperature concept. Only the nuclear spin system is treated quantum-mechanically. Employing the model of a four spin system, created by two nearest neighbour water molecules, spin eigenstates and their dynamics under the influence of their mutual dipolar interactions are studied. It is shown that a fast conversion between ortho- and para-states occurs on a msec time scale, caused by the intermolecular homonuclear magnetic dipolar interaction. Moreover the spin eigenstates of water in an ice crystal are determined by magnetic dipolar interactions and are not given by normal ortho- and para-H2O states of gaseous water. As a result of this the spin temperature of gaseous water evaporated from ice depends strongly on its evaporation history and the ortho/para-ratio of water molecules are only an indirect measure of the temperature of ice crystals from where they descend. This result could explain the unexpected experimentally observed ortho/para-ratios in the clouds of comets.

The influenza m2 cytoplasmic tail changes the proton-exchange equilibria and the backbone conformation of the transmembrane histidine residue to facilitate proton conduction.

The influenza M2 protein forms an acid-activated tetrameric proton channel important for the virus lifecycle. Residue His37 in the transmembrane domain is responsible for channel activation and proton selectivity. While the structure and dynamics of His37 have been well studied in TM peptide constructs, it has not been investigated in the presence of the full cytoplasmic domain, which increases the proton conductivity by 2-fold compared to the TM peptide. We report here (13)C and (15)N chemical shifts of His37 in the cytoplasmic-containing M2(21-97) and show that cationic histidines are already present at neutral pH, in contrast to the TM peptide, indicating that the cytoplasmic domain shifts the protonation equilibria. Quantification of the imidazole (15)N intensities yielded two resolved proton dissociation constants (pKas) of 7.1 and 5.4, which differ from the TM result but resemble the M2(18-60) result, suggesting cooperative proton binding. The average His37 pKa is higher for M2(21-97) than for the shorter constructs. We attribute this higher pKa to direct and indirect effects of the cytoplasmic domain, which is rich in acidic residues. 2D (13)C-(13)C correlation spectra reveal seven His37 Cα-Cβ cross peaks at different pH, some of which are unique to the cytoplasmic-containing M2 and correspond to more ideal α-helical conformations. Based on the pH at which these chemical shifts appear and their side chain structures, we assign these conformations to His37 in differently charged tetramers. Thus, the cytoplasmic domain facilitates proton conduction through the transmembrane pore by modifying the His37-water proton exchange equilibria and the His37 backbone conformational distribution.

Revealing the Position of the Substrate in Nickel Superoxide Dismutase: A Model Study

Reactive oxygen species (ROS) are a major factor in the development of several types of cancer, inflammation, and related diseases. These ROS are not only cytotoxic but also involved in cell signaling. [1] The protection from ROS is of vital importance for biological organisms. For aerobic organisms, superoxide dismutases (SODs) play the major role in protecting cells from ROS, which are generated by the reduction of molecular oxygen by reactive metabolites of the respiratory chain. [2] Because of their biological and medical importance, SODs are a subject of intense research, which yielded more than 2000 publications in the first six months of 2010. While this research has led to detailed knowledge about their biological function and enzyme kinetics, the precise mode of action of these enzymes is still not known and two different mechanisms were proposed. [3] A major reason for this lack of knowledge is the high catalytic rate constants of superoxide degradation (O2C ) by SODs. SODs destroy the superoxide anion radical by converting it into hydrogen peroxide and oxygen with a rate near the diffusion limit (kcat > 2�1 0 9 m 1 s 1 ). [4] Thus all transients involved in their action are too short lived to be amenable for a spectroscopic characterization. For this reason model systems of SODs were developed. Herein we show that the investigation of a model system of the nickel superoxide dismutase (NiSOD) is able to shed light into the mode of action of this enzyme and makes it possible to decide between the proposed mechanisms. In particular we are able to reveal not only the mode of binding of the substrate to the enzyme also the presence of functional water molecules in the active site of the enzyme. Three independent classes of SODs are known. They contain either a dinuclear (Cu, Zn) or a mononuclear (Fe, Mn, Ni) cofactor. [1b, 5] NiSOD, as a mononuclear nickel-containing metalloenzyme, cycles between Ni II and Ni III during catalysis. [3a, 4b, 6] NiSOD was first found in 1996 in Streptomyces. [5a] Crystallographic and spectroscopic studies give an impression of the structure of the whole enzyme and the geometry of its active site with a single covalently bound nickel ion. The nickel ion is embedded within the so-called nickel-hook formed by the first six amino acids of the N-terminus of the active form of S. coelicolor NiSOD (Scheme 1). [3a, 4b, 6, 7]

Solid-State NMR Investigation of the Conformation, Proton Conduction, and Hydration of the Influenza B Virus M2 Transmembrane Proton Channel

Together with the influenza A virus, influenza B virus causes seasonal flu epidemics. The M2 protein of influenza B (BM2) forms a tetrameric proton-conducting channel that is important for the virus lifecycle. BM2 shares little sequence homology with AM2, except for a conserved HxxxW motif in the transmembrane (TM) domain. Unlike AM2, no antiviral drugs have been developed to block the BM2 channel. To elucidate the proton-conduction mechanism of BM2 and to facilitate the development of BM2 inhibitors, we have employed solid-state NMR spectroscopy to investigate the conformation, dynamics, and hydration of the BM2 TM domain in lipid bilayers. BM2 adopts an α-helical conformation in lipid membranes. At physiological temperature and low pH, the proton-selective residue, His19, shows relatively narrow (15)N chemical exchange peaks for the imidazole nitrogens, indicating fast proton shuttling that interconverts cationic and neutral histidines. Importantly, pH-dependent (15)N chemical shifts indicate that His19 retains the neutral population to much lower pH than His37 in AM2, indicating larger acid-dissociation constants or lower pKas. We attribute these dynamical and equilibrium differences to the presence of a second titratable histidine, His27, which may increase the proton-dissociation rate of His19. Two-dimensional (1)H-(13)C correlation spectra probing water (1)H polarization transfer to the peptide indicates that the BM2 channel becomes much more hydrated at low pH than at high pH, particularly at Ser12, indicating that the pore-facing serine residues in BM2 mediate proton relay to the proton-selective histidine.