3,130 research outputs found
Rethinking Leucine Zipper : ribonuclease activity and structural dynamics of a ubiquitous oligomerization motif
No full textThis dissertation focuses on structural, dynamic and catalytic properties of a Leucine
Zipper (LZ) motif – a family of protein oligomerization domains which belong to the
structural class of coiled coil proteins. LZ possess unique stability owing to high abundance of
leucine residues in the key positions of the oligomerization interface. This allows increased
combinatorial flexibility for the sidechains in coiled coil positions defining oligomerization
specificity, thus making LZ an ideal protein-protein interaction determinant. This potential is
reflected in the omnipresence of LZ within protein signalling pathways. Summarized in the
Chapter I, we review the structure, interaction specificity, folding characteristics and functional
diversity of LZ motifs, revealing the molecular mechanisms underlying LZ-enabled protein
signaling.
Beyond the widely acknowledged role of a protein oligomerization motif, recently it
was shown that LZ motifs from bZIP factors GCN4 and cJun are capable of catalyzing
degradation of RNA. Moreover catalytic RNase activity is conserved within full-length bZIP
factors. This discovery was made in the laboratory of Prof. Bernd Gutte (University of Zurich)
and served as a basis for the structural studies of LZ presented in this thesis. The manuscript
presented as the Chapter II summarizes the results of the initial LZ RNase studies, performed
in collaboration with Christine Deillon and Stefan Hoffman.
Our first structural trials on LZ-GCN4 employing solution NMR led to the discovery of
the x-form – a novel monomeric folding intermediate of LZ that exists in equilibrium with the
classical coiled coil state. Although marginally populated at experimental in vitro conditions,
x-form might represent a considerable fraction of the LZ structural ensemble in vivo, providing
a transient interface for specific recombination of interaction partners within bZIP networks.
Results of these studies are presented as Chapter III of this thesis. Finally, our structural NMR studies of LZ–RNA interactions have shown that the
substrate interacts with the coiled coil (dimeric) conformation, while the x-form is incapable of
binding RNA molecules. This is supported by the fact that the catalytic site is formed at the
interface of two LZ chains, and therefore is only available upon assembly of the coiled coil
dimer. Experimental data show that LZ from GCN4 and cJun differ in the topology and
catalytic properties of the active site, which points to the ability of LZ to provide a general
scaffold for assembly of catalytic sites with different properties. These results are presented in
the Chapter IV of this thesis
Some Cryptanalytic Results on Zipper Hash and Concatenated Hash
No full textAbstract. At SAC 2006, Liskov proposed the zipper hash, a technique for constructing secure (indifferentiable from random oracles) hash func-tions based on weak (invertible) compression functions. Zipper hash is a two pass scheme, which makes it unfit for practical consideration. But, from the theoretical point of view it seemed to be secure, as it had resisted standard attacks for long. Recently, Andreeva et al. gave a forced-suffix herding attack on the zipper hash, and Chen and Jin showed a second preimage attack provided f1 is strong invertible. In this paper, we analyse the construction under the random oracle model as well as when the un-derlying compression functions have some weakness. We show (second) preimage, and herding attacks on an n-bit zipper hash and its relaxed variant with f1 = f2, all of which require less than 2 n online computa-tions. Hoch and Shamir have shown that the concatenated hash offers only n 2 bits security when both the underlying compression functions are strong invertible. We show that the bound is tight even when only one of the underlying compression functions is strong invertible
Zipper Connectors for Flexible Electronic Circuits
No full textDevices that look and function much like conventional zippers on clothing have been proposed as connectors for flexible electronic circuits. Heretofore, flexible electronic circuits have commonly included rigid connectors like those of conventional rigid electronic circuits. The proposed zipper connectors would make it possible to connect and disconnect flexible circuits quickly and easily. Moreover, the flexibility of zipper connectors would make them more (relative to rigid connectors) compatible with flexible circuits, so that the advantages of flexible circuitry could be realized more fully. Like a conventional zipper, a zipper according to the proposal would include teeth anchored on flexible tapes, a slider with a loosely attached clasp, a box at one end of the rows of mating teeth, and stops at the opposite ends. The tapes would be made of a plastic or other dielectric material. On each of the two mating sides of the zipper, metal teeth would alternate with dielectric (plastic) teeth, there being two metal teeth for each plastic one. When the zipper was closed, each metal tooth from one side would be in mechanical and electrical contact with a designated metal tooth from the other side, and these mating metal teeth would be electrically insulated from the next pair of mating metal teeth by an intervening plastic tooth. The metal teeth would be soldered or crimped to copper tabs. Wires or other conductors connected to electronic circuits would be soldered or crimped to the ends of the tabs opposite the teeth
Deregulation of sucrose-controlled translation of a bZIP-type transcription factor results in sucrose accumulation in leaves
No full textSucrose is known to repress the translation of Arabidopsis thaliana AtbZIP11 transcript which encodes a protein belonging to the group of S (S - stands for small) basic region-leucine zipper (bZIP)-type transcription factor. This repression is called sucrose-induced repression of translation (SIRT). It is mediated through the sucrose-controlled upstream open reading frame (SC-uORF) found in the AtbZIP11 transcript. The SIRT is reported for 4 other genes belonging to the group of S bZIP in Arabidopsis. Tobacco tbz17 is phylogenetically closely related to AtbZIP11 and carries a putative SC-uORF in its 5′-leader region. Here we demonstrate that tbz17 exhibits SIRT mediated by its SC-uORF in a manner similar to genes belonging to the S bZIP group of the Arabidopsis genus. Furthermore, constitutive transgenic expression of tbz17 lacking its 5′-leader region containing the SC-uORF leads to production of tobacco plants with thicker leaves composed of enlarged cells with 3–4 times higher sucrose content compared to wild type plants. Our finding provides a novel strategy to generate plants with high sucrose content
A leucine zipper in the N terminus confers membrane association to SLP-65
No full textMembrane recruitment of adaptor proteins is crucial for coupling antigen receptors to downstream signaling events. Despite the essential function of the B cell adaptor SLP-65, the mechanism of its recruitment to the plasma membrane is not yet understood. Here we show that a highly conserved leucine zipper in the SLP-65 N terminus is responsible for membrane association. Alterations in the N terminus abolished SLP-65 membrane localization and activity, both of which were restored by replacement of the N terminus with a myristoylation signal. The N terminus is an autonomous domain that confers specific localization and function when transferred to green fluorescent protein or the adaptor protein SLP-76. Our data elucidate the mechanism of SLP-65 membrane recruitment and suggest that leucine zipper motifs are essential interaction domains of signaling proteins
DNA Zipper-Based Tweezers
No full textHere we report the design and development of DNA zippers and tweezers. Essentially a zipper system consists of a normal strand (N), a weak strand (W), and an opening strand (O). N strand is made up of normal DNA bases, while W is engineered to have inosine substituting for guanine. By altering the number and order of inosine, W is engineered to provide less than natural bonding affinities to N in forming the [N:W] helix. When O is introduced (a natural complement of N), it competitively displaces W from [N:W] and forms [N:O]. This principle is incorporated in the development of a molecular device that can perform the functions of tweezers (sense, hold, and release). Tweezers were constructed by holding N and W together using a hinge at one end. Thus, when the tweezers open, N and W remain in the same vicinity. This allows the tweezers to cycle among open and close positions by their opening and closing strands. Control over their opening and closing kinetics is demonstrated. In contrast to the previously reported DNA tweezers, the zipper mechanism makes it possible to operate them with opening strands that do not contain single-stranded DNA overhangs. Our approach yields a robust, compact, and regenerative tweezer system that could potentially be integrated into complex nanomachines
A technique of releasing a prepuce caught in a zipper
No full textA young boy may sometimes be the victim of an accident in which his prepuce is caught in a zipper. The author describes herein a technique for the release which has been found to be simple, painless and effective
Structural and dynamical properties of central nervous system proteins with pharmaceutical and biotechnological potential.
No full textNeurodegenerative diseases are widespread pathologies of large social impact that include: prion, Alzheimer and Parkinson disease, Huntington chorea and amyotrophic lateral sclerosis. The onset of such diseases is commonly associated with the accumulation of insoluble amyloid plaques in specific neuronal population. In this scenario, research activities for the prevention and the treatment of these diseases are focused on two distinct directions: (a) the enhancement of factors that promote the survival and maintenance of nerve cells and (b) the definition of the molecular processes that lead to onset of neurodegenerative diseases. In this framework, the main scopes of the present PhD project have been the analysis of structural/dynamic determinants of the function of neuroprotective proteins (neurotrophins) and the study of structural properties of amyloid aggregates and their toxic precursors.
Neurotrophins (NTs) are homodimeric proteins that play a key role in the differentiation, survival and maintenance of nerve cells. This class of proteins include: nerve growth factor (NGF), Brain-Derived Factor (BDNF), neurotrophin 3 (NT3), neurotrophin 4 (NT4), and neurotrophin 6 (NT6). NTs act by binding to two distinct classes of transmembrane receptors. One is the p75NTR neurotrophin receptor and the other is the Trk family of tyrosine kinase receptors, which includes TrkA, TrkB, and TrkC. All mature NTs bind to p75NTR, but Trks are more selective. NGF interacts selectively with TrkA receptors, NT4 and BDNF selectively with TrkB receptors, and NT3 interacts with TrkC receptors.
During the PhD, a detailed investigation of the dynamical properties of different regions of NTs was carried out by molecular dynamics techniques. Initially, these studies were focussed on the intrinsic conformational preferences of N-terminal region of the NTs. These N-terminal regions are important for the recognition and the specificity of NT-Trk binding. Indeed, N-terminal region of NGF in complex with TrkA has an α-helical conformation, whereas the NT4 in complex with TrkB receptor is in 3/10 helix conformation. However, both N-terminal regions of the two NTs are absent in the crystallographic models of isolated dimers and in complex with the p75NTR receptor, revealing their flexibility in the absence of receptor and a conformational transitions in the interaction with the Trk receptor. Our calculations unveil that for NT4-Nter, and to a lesser extent for NGF-Nter, the conformation of the peptide that is prone to the Trk binding is already present among the states that are energetically accessible to the isolated peptide. This consideration has suggested feasible strategies for the design of effective NT agonist/antagonists. Indeed, variants of these peptides with an increased helical propensity will better mimic the NT functions. Successive simulations carried out on the main body of NGF have provided a detailed picture of mechanisms of interaction with the p75NTR receptor, whose stoichiometry of binding is controversial. These results provided important information on the correlated motions of distant region of the protein. Moreover, essential dynamics analyses clearly indicate that most of the motions of the protein are highly symmetrical. On the basis of results, it has been concluded that the binding of p75NTR to NGF induces a significant "induced-fit” from symmetric structures to asymmetric structures.
In the last years, enormous efforts have been made to obtain insights into the structure of the amyloid-like forms of proteins and peptides involved in the insurgence of neurodegenerative diseases. A characterization of the structure and dynamic properties of these aggregates is required to define the molecular mechanisms underlying these diseases for the development of effective therapeutic strategies. In addition, the considerable resistance of amyloid-like fibrils, combined with their flexibility, versatility and ability to self-assemble has stimulated a growing interest in the potential of these fibers in biotechnological fields as nanobiomaterials. Previous MD simulations have shown that some steric zipper models are endowed with a remarkable stability also in a crystal-free context. However, MD simulations were limited to peptides with polar and/or aromatic dry interfaces. In this scenario, a section of my PhD project was focused on MD simulations of various amyloidogenic structures recently determined. Primarily, were carried out MD studies of steric zipper assemblies whose dry interface involves exclusively aliphatic residues. These simulations have highlighted the key role of residues involved in the steric zipper interface. Indeed, aliphatic residues are not able to form the intra-sheet and inter-sheet interactions formed by polar and aromatic residues that likely provide a strong contribution to the steric zipper motif stability. Along this line, amyoid-like assemblies endowed with hydrophobic residues presumably require larger interfaces, as it is shown by the stability of MD simulation of HET-s protein with a larger steric zippers interface.
Very recent crystallographic studies have shown that the same amyloidogenic peptide can adopt distinct steric zipper assemblies (polymorphs). Intriguingly, it has been postulated that the different polymorphs of the same polypeptide sequence may be representative of distinct strains. In this framework, a detailed analysis of dynamical properties of two polymorphic structures formed by a segment of the islet amyloid polypeptide (IAPP) was carried out during the PhD. The analyses of the MD simulations show that the two IAPP distinct polymorphs are stable in a crystalline-free environment. This finding supports the hypothesis that the occurrence of strains in neurodegenerative diseases may be related to the possibility that a single peptide/protein chain may self-associate in alternative steric zipper-based assemblies.
The last section of present thesis was dedicated to the studies of human prion protein (HPrP) properties. These studies were conducted in collaboration with the University of Cambridge. Independent crystallographic studies have shown the involvement of the β-sheet of the HPrP in intermolecular interactions that lead to the association of two different molecules HPrP in the crystalline state. These observations suggest that this association may be representative of the early stages of aggregation of HPrP. In this framework, during the PhD project detailed replica exchange molecular dynamics (REMD) studies on the intrinsic stability of HPrP β-structure were conducted. In particular, simulations were conducted on different β-strand combinations taken either from HPrP monomer or dimeric crystalline assemblies. The REMD simulations conducted on the isolated two stranded β-sheet of the protein monomer indicate that this structure is remarkably stable. The stability of larger aggregates formed by the juxtaposition of two of these sheets, as detected in the crystalline state, is very limited stability. Interestingly, additional simulations indicate that these aggregates are stabilized by mutations linked to the insurgence of pathological states. The observation that the two stranded β-sheet of the prion monomer are intrinsically stable hold important implications for prion polymerization process and for the design of synthetic peptides that potentially can inhibit the aggregation process of human prion protein
Photosystem I reaction-centre proteins contain leucine zipper motifs A proposed role in dimer formation
No full textAbstractThe photosystem I (PS I) reaction-centre polypeptides, encoded by the psaA and psaB genes, are shown to contain several highly conserved leucine repeats, consisting of a leucine residue every seventh amino acid, similar to the leucine zipper motifs known to mediate DNA-binding polypeptide dimerisation. In each of the PSI reaction-centre subunits the leucine zipper motif precedes highly conserved cysteine residues which have been proposed to ligate the interpolypeptide [4Fe-4S] centre, Fx. We propose that PS I reaction-centre dimerisation and [4Fe-4S] centre formation are mediated through the leucine zipper
Solution structure of O-glycosylated C-terminal leucine zipper domain of human salivary mucin (MUC7)
No full textSolution structures of a 23 residue glycopeptide II (KIS* RFLLYMKNLLNRIIDDMVEQ, where * denotes the glycan Gal-beta-(1-3)-alpha-GalNAc) and its deglycosylated counterpart I derived from the C-terminal leucine zipper domain of low molecular weight human salivary mucin (MUC7) were studied using CD, NMR spectroscopy and molecular modeling. The peptide I was synthesized using the Fmoc chemistry following the conventional procedure and the glycopeptide II was synthesized incorporating the O-glycosylated building block (N alpha-Fmoc-Ser-[Ac-4,-beta-D-Gal-(1,3)-Ac(2)alpha-D-GalN(3)]-OPfp) at the appropriate position in stepwise assembly of peptide chain. Solution structures of these glycosylated and nonglycosylated peptides were studied in water and in the presence of 50% of an organic cosolvent, trifluoroethanol (TFE) using circular dichroism (CD), and in 50% TFE using two-dimensional proton nuclear magnetic resonance (2D H-1 NMR) spectroscopy. CD spectra in aqueous medium indicate that the apopeptide I adapts, mostly, a beta-sheet conformation whereas the glycopeptide II assumes helical structure. This transition in the secondary structure, upon glycosylation, demonstrates that the carbohydrate moiety exerts significant effect on the peptide backbone conformation. However, in 50% TFE both the peptides show pronounced helical structure. Sequential and medium range NOEs, C alpha H chemical shift perturbations, (3)J(NH:C alpha H) couplings and deuterium exchange rates of the amide proton resonances in water containing 50% TFE indicate that the peptide I adapts alpha-helical structure from Ile2-Val21 and the glycopeptide II adapts alpha-helical structure from Ser3-Glu22. The observation of continuous stretch of helix in both the peptides as observed by both NMR and CD spectroscopy strongly suggests that the C-terminal domain of MUC7 with heptad repeats of leucines or methionine residues may be stabilized by dimeric leucine zipper motif. The results reported herein may be invaluable in understanding the aggregation (or dimerization) of MUC7 glycoprotein which would eventually have implications in determining its structure-function relationship
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