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Martin Egli

Publications and source records attributed to Martin Egli.

At least 19 recordsLinked to original sources
PubMed2004

Identification of key phosphorylation sites in the circadian clock protein KaiC by crystallographic and mutagenetic analyses.

In cyanobacteria, KaiC is an essential hexameric clock protein that forms the core of a circadian protein complex. KaiC can be phosphorylated, and the ratio of phospho-KaiC to non-phospho-KaiC is correlated with circadian period. Structural analyses of KaiC crystals identify three potential phosphorylation sites within a 10-A radius of the ATP binding regions that are at the T432, S431, and T426 residues in the KaiCII domains. When these residues are mutated by alanine substitution singly or in combination, KaiC phosphorylation is altered, and circadian rhythmicity is abolished. These alanine substitutions do not prevent KaiC from hexamerizing. Intriguingly, the ability of KaiC overexpression to repress its own promoter is also not prevented by alanine substitutions at these sites, implying that the capability of KaiC to repress its promoter is not sufficient to allow the clockwork to oscillate. The KaiC structure and the mutational analysis suggest that S431 and T426 may share a phosphate that can shuttle between these two residues. Because the phosphorylation status of KaiC oscillates over the daily cycle, and KaiC phosphorylation is essential for clock function as shown here, daily modulations of KaiC activity by phosphorylation at T432 and S431/T426 seem to be key components of the circadian clockwork in cyanobacteria.

Amino Acid Sequence↗
PubMed2004

Visualizing a circadian clock protein: crystal structure of KaiC and functional insights.

Circadian (daily) biological clocks express characteristics that are difficult to explain by known biochemical mechanisms, and will ultimately require characterizing the structures, functions, and interactions of their molecular components. KaiC is an essential circadian protein in cyanobacteria that forms the core of the KaiABC clock protein complex. We report the crystal structure of the KaiC homohexameric complex at 2.8 A resolution. The structure resembles a double doughnut with a central pore that is partially sealed at one end. The crystal structure reveals ATP binding, inter-subunit organization, a scaffold for Kai-protein complex formation, the location of critical KaiC mutations, and evolutionary relationships to other proteins. A key auto-phosphorylation site on KaiC (T432) is identified from the crystal structure, and mutation of this residue abolishes circadian rhythmicity. The crystal structure of KaiC will be essential for understanding this circadian clockwork and for establishing its links to global gene expression.

Adenosine Triphosphate↗
PubMed2004

2'-O-[2-(guanidinium)ethyl]-modified oligonucleotides: stabilizing effect on duplex and triplex structures.

[structure: see text] Oligonucleotides with a novel 2'-O-[2-(guanidinium)ethyl] (2'-O-GE) modification have been synthesized using a novel protecting group strategy for the guanidinium group. This modification enhances the binding affinity of oligonucleotides to RNA as well as duplex DNA (DeltaT(m) 3.2 degrees C per modification). The 2'-O-GE modified oligonucleotides exhibited exceptional resistance to nuclease degradation. The crystal structure of a palindromic duplex formed by a DNA oligonucleotide with a single 2'-O-GE modification was solved at 1.16 A resolution.

DNA↗
PubMed2004

"Deoxyribo nanonucleic acid"; antiparallel, parallel, and unparalleled.

The crystal structure of a single-stranded DNA oligonucleotide has revealed formation of a unique three-dimensional array by continuous antiparallel and parallel pairing between monomers. The array is based on tertiary interactions and represents a second-generation nanotechnological system.

Base Sequence↗
PubMed2003

An aminopyridazine-based inhibitor of a pro-apoptotic protein kinase attenuates hypoxia-ischemia induced acute brain injury.

Death associated protein kinase (DAPK) is a calcium and calmodulin regulated enzyme that functions early in eukaryotic programmed cell death, or apoptosis. To validate DAPK as a potential drug discovery target for acute brain injury, the first small molecule DAPK inhibitor was synthesized and tested in vivo. A single injection of the aminopyridazine-based inhibitor administered 6 h after injury attenuated brain tissue or neuronal biomarker loss measured, respectively, 1 week and 3 days later. Because aminopyridazine is a privileged structure in neuropharmacology, we determined the high-resolution crystal structure of a binary complex between the kinase domain and a molecular fragment of the DAPK inhibitor. The co-crystal structure describes a structural basis for interaction and provides a firm foundation for structure-assisted design of lead compounds with appropriate molecular properties for future drug development.

Animals↗
PubMed2003

Face-to-face and edge-to-face pi-pi interactions in a synthetic DNA hairpin with a stilbenediether linker.

Synthetic conjugates possessing bis(2-hydroxyethyl)stilbene-4,4'-diether linkers (Sd2) form the most stable DNA hairpins reported to date. Factors that affect stability are length and flexibility of the linkers and pi-stacking of the stilbene moiety on the adjacent base pair. The crystal structure of the hairpin d(GT(4)G)-Sd2-d(CA(4)C) was determined at 1.5 A resolution. The conformations of the two molecules in the asymmetric unit differ both in the linker and the stem portions. One of them shows a planar stilbene that is stacked on the adjacent G:C base pair. The other displays considerable rotation between the phenyl rings and an unprecedented edge-to-face orientation of stilbene and base pair. The observation of considerable variations in the conformation of the Sd moiety in the crystal structure allows us to exclude restriction of motion as the reason for the absence of Sd photoisomerization in the hairpins. Conformational differences in the stem portion of the two hairpin molecules go along with different Mg(2+) binding modes. Most remarkable among them is the sequence-specific coordination of a metal ion in the narrow A-tract minor groove. The crystal structure provides unequivocal evidence that a fully hydrated Mg(2+) ion can penetrate the narrow A-tract minor groove, causing the groove to further contract. Overall, the structural data provide a better understanding of the origins of hairpin stability and their photochemical behavior in solution.

Cations↗
PubMed2003

Water-nucleobase "stacking": H-pi and lone pair-pi interactions in the atomic resolution crystal structure of an RNA pseudoknot.

The hydrogen bond acceptor capability of aromatic rings has been demonstrated both by experimental and theoretical studies, and D-H...pi interactions (H-pi interaction), where D is mainly N, O, and C, are ubiquitous in structures of macromolecules. By comparison, the interaction of a lone pair of water directly with the face of a pi-system (l.p.-pi interaction) seems counterintuitive and to date has only been studied theoretically. In the crystal structure of an RNA pseudoknot at atomic resolution, all nucleobases not involved in either intra- or intermolecular base-base stacking interactions exhibit stacking with water molecules either of the H-pi or the l.p.-pi type. The geometry observed for the l.p.-pi type water-nucleobase stacking is consistent with that predicted in recent ab initio studies.

Crystallography, X-Ray↗
PubMed2003

2'-O-[2-[2-(N,N-dimethylamino)ethoxy]ethyl] modified oligonucleotides: symbiosis of charge interaction factors and stereoelectronic effects.

[structure: see text] Oligonucleotides with a novel, 2'-O-[2-[2-(N,N-dimethylamino)ethoxy]ethyl] (2'-O-DMAEOE) modification have been synthesized. This modification, a cationic analogue of the 2'-O-(2-methoxyethyl) (2'-O-MOE) modification, exhibits high binding affinity to target RNA (but not to DNA) and exceptional resistance to nuclease degradation. Analysis of the crystal structure of a self-complementary oligonucleotide containing a single 2'-O-DMAEOE modification explains the importance of charge factors and gauche effects on the observed antisense properties. 2'-O-DMAEOE modified oligonucleotides are ideal candidates for antisense drugs.

Animals↗
PubMed2002

Selenium-assisted nucleic acid crystallography: use of phosphoroselenoates for MAD phasing of a DNA structure.

The combination of synchrotron radiation and a variety of atoms or ions (either covalently attached to the biomolecule prior to crystallization or soaked into crystals) that serve as anomalous scatterers constitutes a powerful tool in the X-ray crystallographer's repertoire of structure determination techniques. Phosphoroselenoates in which one of the nonbridging phosphate oxygens in the backbone is replaced by selenium offer a simplified means for introducing an anomalous scatterer into oligonucleotides by conventional solid-phase synthesis. Unlike other methods that are used to derivatize DNA or RNA by covalent attachment of a heavy atom (i.e., bromine at the C5 position of pyrimidines), tedious synthesis of specialized nucleosides is not required. Introduction of selenium is readily accomplished in solid-phase oligonucleotide synthesis by replacing the standard oxidation agent with a solution of potassium selenocyanide. This results in a diastereomeric mixture of phosphoroselenoates that can be separated by strong anion-exchange HPLC. As a test case, all 10 DNA hexamers of the sequence CGCGCG containing a single phosphoroselenoate linkage (PSe) were prepared. Crystals were grown for a subset of them, and the structure of [d(C(PSe)GCGCG)](2) was determined by the multiwavelength anomalous dispersion technique and refined to 1.1 A resolution.

Crystallization↗
PubMed2002

Crystal structure of a B-form DNA duplex containing (L)-alpha-threofuranosyl (3'-->2') nucleosides: a four-carbon sugar is easily accommodated into the backbone of DNA.

(L)-alpha-Threofuranosyl-(3'-->2')-oligonucleotides (TNA) containing vicinally connected phosphodiester linkages undergo informational base pairing in an antiparallel strand orientation and are capable of cross-pairing with RNA and DNA. TNA is derived from a sugar containing only four carbon atoms and is one of the simplest potentially natural nucleic acid alternatives investigated thus far in the context of a chemical etiology of nucleic acid structure. Compared to DNA and RNA that contain six covalent bonds per repeating nucleotide unit, TNA contains only five. We have determined the atomic-resolution crystal structure of the B-form DNA duplex [d(CGCGAA)Td(TCGCG)](2) containing a single (L)-alpha-threofuranosyl thymine (T) per strand. In the modified duplex base stacking interactions are practically unchanged relative to the reference DNA structure. The orientations of the backbone at the TNA incorporation sites are slightly altered in order to accommodate fewer atoms and covalent bonds. The conformation of the threose is C4'-exo with the 2'- and 3'-substituents assuming quasi-diaxial orientation.

Carbohydrate Conformation↗
PubMed2002

2'-O-[2-(methylthio)ethyl]-modified oligonucleotide: an analogue of 2'-O-[2-(methoxy)-ethyl]-modified oligonucleotide with improved protein binding properties and high binding affinity to target RNA.

A novel 2'-modification, 2'-O-[2-(methylthio)ethyl] or 2'-O-MTE, has been incorporated into oligonucleotides and evaluated for properties relevant to antisense activity. The results were compared with the previously characterized 2'-O-[2-(methoxy)ethyl] 2'-O-MOE modification. As expected, the 2'-O-MTE modified oligonucleotides exhibited improved binding to human serum albumin compared to the 2'-O-MOE modified oligonucleotides. The 2'-O-MTE oligonucleotides maintained high binding affinity to target RNA. Nuclease digestion of 2'-O-MTE oligonucleotides showed that they have limited resistance to exonuclease degradation. We analyzed the crystal structure of a decamer DNA duplex containing the 2'-O-MTE modifcation. Analysis of the crystal structure provides insight into the improved RNA binding affinity, protein binding affinity and limited resistance of 2'-O-MTE modified oligonucleotides to exonuclease degradation.

Binding Sites↗
PubMed2002

Metal ions and flexibility in a viral RNA pseudoknot at atomic resolution.

Many pathogenic viruses use programmed -1 ribosomal frameshifting to regulate translation of their structural and enzymatic proteins from polycistronic mRNAs. Frameshifting is commonly stimulated by a pseudoknot located downstream from a slippery sequence, the latter positioned at the ribosomal A and P sites. We report here the structures of two crystal forms of the frameshifting RNA pseudoknot from beet western yellow virus at resolutions of 1.25 and 2.85 A. Because of the very high resolution of 1.25 A, ten mono- and divalent metal ions per asymmetric unit could be identified, giving insight into potential roles of metal ions in stabilizing the pseudoknot. A magnesium ion located at the junction of the two pseudoknot stems appears to play a crucial role in stabilizing the structure. Because the two crystal forms exhibit mostly unrelated packing interactions and local crystallographic disorder in the high-resolution form was resolvable, the two structures offer the most detailed view yet of the conformational preference and flexibility of an RNA pseudoknot.

Crystallization↗
PubMed2002

Covalent incorporation of selenium into oligonucleotides for X-ray crystal structure determination via MAD: proof of principle. Multiwavelength anomalous dispersion.

Selenium was incorporated into an oligodeoxynucleotide in the form of 2'-methylseleno-uridine (U(Se)). The X-ray crystal structure of the duplex left open bracket d(GCGTA)U(Se)d(ACGC) right open bracket (2) was determined by the multiwavelength anomalous dispersion (MAD) technique and refined to a resolution of 1.3 A, demonstrating that selenium can selectively substitute oxygen in DNA and that the resulting compounds are chemically stable. Since derivatization at the 2'-alpha-position with selenium does not affect the preference of the sugar for the C3'-endo conformation, this strategy is suitable for incorporating selenium into RNA. The availability of selenium-containing nucleic acids for crystallographic phasing offers an attractive alternative to the commonly used halogenated pyrimidines.

Base Pairing↗
PubMed2002

DNA-cation interactions: quo vadis?

The interactions between double helical DNA and cations, specifically mono- and divalent metal ions, have recently received increased attention. Molecular dynamics simulations, solution NMR, and X-ray crystallography have all shed light on the coordination of ions in the major and minor grooves of DNA. Metal ion interactions may play key roles in the control of DNA conformation and topology, but despite progress in locating the ions and determining their precise binding modes, it remains difficult to figure out just how important ions really are. What have we learned and what remains to be done?

Cations↗