Molecular Switch Mechanism Research Articles

Effect of salt bridge on transcription activation of CRP-dependent lactose operon in Escherichia coli

Expression of catabolite-sensitive operons in Escherichia coli is cAMP-dependent and mediated through the CRP:cAMP complex binding to specific sequences in DNA. Five specific ionic or polar interactions occur in cAMP binding pocket of CRP. E72 interacts with the cAMP 2′ OH, R82 and S83 interact with the negatively charged phosphate moiety, and T127 and S128 interact with the adenine ring. There is evidence to suggest that E72 and R82 may mediate an essential CRP molecular switch mechanism. Therefore, stimulation of CRP transcription activation was examined by perturbing these residues. Further, CRP:cAMP complex was treated with a specific DNA sequence containing the lac CRP binding site along with RNA polymerase to mimic in vivo conditions. Biochemical and biophysical results revealed that regulation of transcription activation depends on alignment of CRP tertiary structure through inter-domain communication and it was concluded that positions 72 and 82 are essential in the activation of CRP by cAMP.

P3-413: Presenilin 1 phosphorylation influences β-catenin stability and signalling

The Alzheimer Disease associated presenilin 1 (PS1) is part of the γ–secretase complex and is implicated in the proteolytic processing of the amyloid precursor protein. In addition, PS1 is also involved in other cellular functions such as apoptosis, neuronal homeostasis and others. Particularly, the β–catenin binding site is located within this loop region and an involvement in the β–catenin signalling was demonstrated. Since phosphorylation sites for GSK3β (Ser353, Ser357) were identified within this binding region, a role of PS1 phosphorylation in β–catenin metabolism and signalling could be hypothesised. In this study, we sought to analyze the role of PS1 phosphorylation on the metabolism of β–catenin and elucidate the molecular mechanisms involved. We used recombinant PS1 loop domains for in vitro phosphorylation and structural investigations. Studies on β–catenin stability and signalling were carried out in Hek293 cells expressing PS1–wt or the phosphorylation site mutants S353/357A or S353/357D. In in vitro phosphorylation experiments we observed the phosphorylation at positions S353 and S357 by GSK3β. PS1 was also phosphorylated in Hek293 cells at these positions. This phosphorylation induced a structural change in the loop domain resulting in changed biochemical properties in SDS–PAGE and size exclusion chromatography. We show that binding of β–catenin is significantly reduced to PS1 phosphorylation site mutants as compared to PS1–wt. Furthermore, β–catenin was stabilized in PS1–S353/357D mutant cells leading to an increased nuclear pool of β–catenin. In reporter assays, transcription of β–catenin target genes was reduced after PS1–wt transfection. Notably, this reduction was suppressed in PS1–S353/357D mutant cells. A PS1–wt induced reduction in cell proliferation and c–myc expression was less pronounced in PS1–S353/357D transfected cells. As shown by in vitro phosphorylation experiments, phosphorylation of β–catenin and PS1 occurs sequentially. The phosphorylation of PS1 at positions Ser353 and Ser357 resulted in a structural change of the loop domain and a concomitant decrease in β–catenin recruitment. This led to a stabilization of β–catenin and increased β–catenin signalling. The sequential phosphorylation of β–catenin and PS1 by GSK3β could indicate a molecular switch mechanism regulating β–catenin signalling. Support Contributed By: The Deutsche Forschungsgemeinschaft

Structure and mechanism of the reversible photoswitch of a fluorescent protein.

Proteins that can be reversibly photoswitched between a fluorescent and a nonfluorescent state bear enormous potential in diverse fields, such as data storage, in vivo protein tracking, and subdiffraction resolution light microscopy. However, these proteins could hitherto not live up to their full potential because the molecular switching mechanism is not resolved. Here, we clarify the molecular photoswitching mechanism of asFP595, a green fluorescent protein (GFP)-like protein that can be transferred from a nonfluorescent "off" to a fluorescent "on" state and back again, by green and blue light, respectively. To this end, we establish reversible photoswitching of fluorescence in whole protein crystals and show that the switching kinetics in the crystal is identical with that in solution. Subsequent x-ray analysis demonstrated that upon the absorption of a green photon, the chromophore isomerizes from a trans (off) to a cis (on) state. Molecular dynamics calculations suggest that isomerization occurs through a bottom hula twist mechanism with concomitant rotation of both bonds of the chromophoric methine ring bridge. This insight into the switching mechanism should facilitate the targeted design of photoswitchable proteins. Reversible photoswitching of the protein chromophore system within intact crystals also constitutes a step toward the use of fluorescent proteins in three-dimensional data recording.

Towards understanding a molecular switch mechanism: thermodynamic and crystallographic studies of the signal transduction protein CheY

The signal transduction protein CheY displays an α/β-parallel polypeptide folding, including a highly unstable helix α4 and a strongly charged active site. Helix α4 has been shown to adopt various positions and conformations in different crystal structures, suggesting that it is a mobile segment. Furthermore, the instability of this helix is believed to have functional significance because it is involved in protein-protein contacts with the transmitter protein kinase CheA, the target protein FliM and the phosphatase CheZ. The active site of CheY comprises a cluster of three aspartic acid residues and a lysine residue, all of which participate in the binding of the Mg 2+ needed for the protein activation. Two steps were followed to study the activation mechanism of CheY upon phosphorylation: first, we independently substituted the three aspartic acid residues in the active site with alanine; second, several mutations were designed in helix α4, both to increase its level of stability and to improve its packing against the protein core. The structural and thermodynamic analysis of these mutant proteins provides further evidence of the connection between the active-site area and helix α4, and helps to understand how small movements at the active site are transmitted and amplified to the protein surface.

Ab Initio Study of the Role of Lysine 16 for the Molecular Switching Mechanism of Ras Protein p21

Quantum chemical computations using the ab initio molecular orbital (MO) method have been performed to investigate the molecular switching mechanism of Ras protein p21, which has an important role in intracellular signal cascades. Lys 16 was demonstrated to be crucial to the function of Ras p21, and the hydrolysis of GTP to GDP was found to be an one-step reaction. The potential energy barrier of this hydrolysis reaction from GTP to (GDP + P) was calculated to be ∼42 kcal/mol. The role of GAP (GTPase-activating protein) was also discussed in terms of the delivery of the water molecules required for the hydrolysis.

An ATP/ADP-Dependent Molecular Switch Regulates the Stability of p53-DNA Complexes

Interaction with DNA is essential for the tumor suppressor functions of p53. We now show, for the first time, that the interaction of p53 with DNA can be stabilized by small molecules, such as ADP and dADP. Our results also indicate an ATP/ADP molecular switch mechanism which determines the off-on states for p53-DNA binding. This ATP/ADP molecular switch requires dimer-dimer interaction of the p53 tetramer. Dissociation of p53-DNA complexes by ATP is independent of ATP hydrolysis. Low-level ATPase activity is nonetheless associated with ATP-p53 interaction and may serve to regenerate ADP-p53, thus recycling the high-affinity DNA binding form of p53. The ATP/ADP regulatory mechanism applies to two distinct types of p53 interaction with DNA, namely, sequence-specific DNA binding (via the core domain of the p53 protein) and binding to sites of DNA damage (via the C-terminal domain). Further studies indicate that ADP not only stabilizes p53-DNA complexes but also renders the complexes susceptible to dissociation by specific p53 binding proteins. We propose a model in which the DNA binding functions of p53 are regulated by an ATP/ADP molecular switch, and we suggest that this mechanism may function during the cellular response to DNA damage.

Switching fungi

The human pathogenic fungus Candida albicans can undergo transitions between budding and filamentous forms, as well as another morphogenetic process termed phenotypic switching. Phenotypic switching occurs spontaneously in the absence of environmental signals and can give rise to several different colony morphologies. The molecular mechanism of phenotypic switching in C. albicans is not well understood. The role of genomic rearrangements has been the primary focus of molecular switching models. Recently, however, epigenetic mechanisms have been considered, based on observations of heritable genetic changes in Saccharomyces cerevisiae. Many of these epigenetic changes are controlled by the SIR2 (silent information regulator) gene. It now appears that epigenetic mechanisms might play a role in regulating morphogenesis in C. albicans1 Perez-Martin J. et al. Phenotypic switching in Candida albicans is controlled by a SIR2 gene. EMBO J. 1999; 18: 2580-2592 Crossref PubMed Scopus (112) Google Scholar .

Differentiation-associated surface antigen variation in the ancient eukaryote Giardia lamblia.

Encystation of Giardia lamblia is required for survival outside the host, whereas excystation initiates infection. The dormant cyst was considered an adaptation to external survival and passage through the stomach. However, we found previously that trophozoites which had recovered after completion of the life cycle had switched their major variant surface protein (VSP), called TSA 417, but neither the timing nor the molecular mechanism of switching had been elucidated. Here we demonstrate that TSA 417 predominates in cysts, but is downregulated during the stage of excystation that models cyst arrival in the small intestine. Transcripts of new VSPs appear late in encystation, and during and after excystation. Trophozoites appear to prepare for switching during encystation, when the major VSP on the cell surface diminishes and is internalized in lysosome-like vacuoles. As short-range DNA rearrangements were not detected, giardial VSP switching during differentiation appears to resemble the in situ switching of surface glycoproteins in African trypanosomes. We also report a unique extended 15 nucleotide polyadenylation signal in all VSP transcripts, but not in other known giardial genes. Antigenic variation during encystation-excystation may be a novel form of immune evasion that could help explain the common occurrence of reinfection by Giardia and other parasites with similar life cycles.

Involvement of calcium/calmodulin-dependent protein kinases in the setting of a molecular switch involved in hippocampal LTP

Long-term potentiation (LTP) is the form of synaptic plasticity most commonly associated with learning and memory. Studies using protein kinase inhibitors have suggested functional roles for several kinases in the induction of LTP in the CA1 region of the hippocampus, though the precise role of any given kinase has yet to be fully established. Here we report that the selective calcium/calmodulin-dependent protein kinase (CaMK) inhibitor KN-62 has two distinct actions on LTP. As reported previously, KN-62 (3 μM) prevented the induction of LTP. Here we show that KN-62 also prevents the setting of a molecular switch, initiated by the synaptic activation of ( S)- α-methyl-4-carboxyphenylglycine (MCPG)-sensitive metabotropic glutamate (mGlu) receptors. There are two aspects of this work which might be considered surprising. First, the setting of the molecular switch was prevented by a concentration of KN-62 (1 μM) subthreshold for the inhibition of the induction of LTP per se. Second, the setting of the molecular switch, by the delivery of a tetanus (100 Hz, 1 s) in the presence of a specific NMDA receptor antagonist ( R)-2-amino-5-phosphonopentanoate (AP5), reduced the sensitivity of LTP to KN-62, such that at a concentration of 3 μM it no longer blocked induction (though at 10 μM it did). This conditioning effect of a tetanus, delivered in the presence of AP5, was prevented by MCPG (200 μM). These data reveal unexpected complexities in the involvement of KN-62-sensitive processes (presumably CaMKII) in the induction of LTP. They suggest that activation of KN-62-sensitive processes leads to (at least) two phosphorylation steps with fundamentally different roles in synaptic plasticity within a single synapse. They also raise the possibility that CaMKII is an integral part of the MCPG-sensitive molecular switch mechanism.

Isolation of differentially expressed genes upon immunoglobulin class switching by a subtractive hybridization method using uracil DNA glycosylase.

Immunoglobulin class switch recombination enables B lymphocytes to sequentially express antibodies that have identical specificities but that differ in class and effector function. Although several cis elements required for class switch recombination have been identified, few trans -acting factors which are directly related to class switching have been found. Previously we have developed an efficient in vitro class switching system using a cell line, CH12F3-2. To clarify the molecular mechanism of class switching, we intended to isolate genes induced with interleukin (IL)-4, transforming growth factor (TGF)-beta and CD40L using the in vitro class switching system. For that purpose, an improved method for making subtracted cDNA libraries, using uracil DNA glycosylase, has been developed. This method can overcome a general problem of subtraction, that rare cDNAs are easily lost. This new subtraction method was applied to the CH12F3-2 switching system to isolate genes induced by stimulations with IL-4, TGF-beta and CD40L, and cDNAs encoding deiodinase 1 and SS32, an alternatively spliced form of the muscle LIM protein, were obtained. Their expression patterns in response to various combinations of stimuli and the time courses of the induction supported the usefulness of this method.

Isolation and Characterization of cDNA Clones Encoding Embryonic and Adult Isoforms of Rat Cardiac Troponin T

We have previously reported that troponin T (TnT), a regulatory protein in muscle contraction, undergoes a switch from the larger, acidic embryonic form to the smaller, basic adult form during rat heart development (Jin, J.-P., and Lin, J.J.-C. (1988) J. Biol. Chem. 263, 7309-7315). To investigate the significance and the molecular mechanism of this isoform switching, cDNA clones encoding rat cardiac TnT were obtained by screening a cDNA library constructed from young rat cardiac poly(A)+ RNAs using the expression vector lambda gt11. Clone RCT10 proved to be a full length clone containing 50 base pairs (bp) of 5'-untranslated sequence, 870 bp of coding sequence, and 196 bp plus poly(A) tail at the 3'-untranslated region. In the other cDNA clone (RCT11), the entire 3'-untranslated sequence and most of the coding sequence were identical with that of RCT10, but an additional 30-bp insert was present in the coding region from residues 18 to 27. This insertion sequence appeared to code for a fragment (EDWSEEEEDE) highly enriched in acidic amino acid residues. Thus, RCT11 might represent a clone encoding the embryonic isoform of rat cardiac TnT. S1 nuclease RNA mapping analysis using end-labeled RCT11 cDNA probes confirmed that this region of the sequence is different in embryonic and adult isoform mRNAs. These results suggest that both embryonic and adult isoforms of rat cardiac TnT are generated from the same primary transcript by developmentally regulated alternative splicing. The amino acid sequence deduced from RCT10 cDNA exhibits 87%, 85%, and 72% homology with bovine, rabbit, and chicken cardiac TnTs, respectively, but less homology (57-59%) with the known skeletal TnTs from human, rat, rabbit, and chicken. Moreover, both the 5'- and the 3'-untranslated sequences of rat cardiac TnT mRNA are completely different from those reported for rat skeletal TnT mRNA, suggesting that rat cardiac TnT is coded from a gene distinct from the rat skeletal TnT gene.

Macromolecular switches for bilayer membranes

Controlled polyelectrolyte adsorption provides a powerful tool for use in the design of thin molecular films. The adsorption of polyelectrolyte chains would be expected to cause large changes in the delicate balance of forces that determines the structural and functional properties of organized assemblies, including monolayer, bilayer and multilayer films. But in addition, polyelectrolyte adsorption is exceedingly sensitive to environment, e.g., to pH, temperature and ionic strength. Controlled adsorption can therefore serve as a molecular switching mechanism, rendering film properties sensitive to selected chemical and physical stimuli. We have used this approach to effect molecular switching in mixtures of poly(2-ethylacrylic acid) with bilayer vesicles of natural or synthetic phosphatidylcholines. In particular, we have prepared phosphatidylcholine vesicles that release their contents rapidly and quantitatively in response to changes in pH, temperature or glucose concentration. The design and preparation of responsive bilayer membranes, and the kinetics and mechanisms of the associated molecular switching processes, are discussed herein.

A Molecular Switching Mechanism in Differentiation: Induction of Polarity in Cambial Cells

The selection of polarity in cells of the cambium in higher plants is a regulated process of development which results in horizontally or vertically oriented cells. A set of mathematical equations suggestive of this developmental dichotomy is given a new biologic interpretation. As a result, a molecular scheme capable of acting as a biochemical switch is suggested. The model features two structural protein monomers whose synthesis is controlled autogenously by feedback repression. The implications of this and similar mechanisms to other differentiating systems is discussed.

The study of molecular switching mechanisms

High resolution NMR studies of proteins reveal differences in mobility in different segments and side chains. These differences in mobility may be related to those features of the protein structure which are important in the function of these proteins as regulatory units--switches or transducers.

Effect of azide on free haematins

MHC class I molecules present antigenic peptides to cytotoxic T-cells at the cell surface. Peptide loading of class I molecules in the endoplasmatic reticulum can involve interaction with the tapasin chaperone protein. The human class I allotype HLA-B*44:02 with an Asp at position 116 at the floor of the F pocket (which binds the peptide C-terminal residues) depends on tapasin for efficient peptide loading. However, HLA-B*44:05 (identical to B*44:02 except for tyrosine 116) can efficiently load peptides in the absence of tapasin. Both allotypes adopt very similar structures in the presence of the same peptide. Molecular dynamics simulations indicate a significantly higher conformational flexibility of the F pocket in the absence of a peptide for B*44:02 compared to B*44:05. Free energy simulations to open the F pocket indicate a molecular side chain switch mechanism that underlies the global opening motion. This side chain switch involves the rearrangement of salt bridges and hydrogen bonding of the basic arginine 97 with three acidic aspartate residues 114, 116 and 156 near the F pocket. A replica exchange simulation to specifically accelerate side chain motions demonstrates that the same side chain rearrangements induce global opening motions of the F pocket. In case of B*44:05 the free energy barrier for F pocket opening was significantly higher compared to B*44:02 and no associated side chain rearrangement was observed. Such coupling of local side chain rearrangements with global conformational changes might be the basis for allosteric changes in other class I allotypes as well as for allosteric changes in other proteins.