Oliver Ohlenschläger

The NMR structure of the 5S rRNA E-domain-protein L25 complex shows preformed and induced recognition

The structure of the complex between ribosomal protein L25 and a 37 nucleotide RNA molecule, which contains the E‐loop and helix IV regions of the E‐domain of Escherichia coli 5S rRNA, has been determined to an overall r.m.s. displacement of 1.08 Å (backbone heavy atoms) by heteronuclear NMR spectroscopy (Protein Databank code 1d6k). The interacting molecular surfaces are bipartite for both the RNA and the protein. One side of the six‐stranded β‐barrel of L25 recognizes the minor groove of the E‐loop with very little change in the conformations of either the protein or the RNA and with the RNA–protein interactions occurring mainly along one strand of the E‐loop duplex. This minor groove recognition module includes two parallel β‐strands of L25, a hitherto unknown RNA binding topology. Binding of the RNA also induces conversion of a flexible loop to an α‐helix in L25, the N‐terminal tip of which interacts with the widened major groove at the E‐loop/helix IV junction of the RNA. The structure of the complex reveals that the E‐domain RNA serves as a preformed docking partner, while the L25 protein has one preformed and one induced recognition module.

Highly modular structure and ligand binding by conformational capture in a minimalistic riboswitch.

Riboswitches are highly structured RNA motifs with gene regulatory activity located in the untranslated regions of mRNAs. They either modulate transcription termination or translation initiation through conformational changes triggered by direct interactions with small metabolite ligands. Many naturally occurring riboswitches are large and structurally very complex. In contrast, synthetic riboswitches—tailored gene regulatory elements for synthetic biology applications—are based on small in vitro selected RNA aptamers. Yet, despite a ligand affinity and specificity comparable to their natural counterparts only a few in vitro selected aptamers are regulatory active in vivo. Recently, Suess et al. engineered a riboswitch for the aminoglycoside antibiotic neomycin B by subjecting an in vitro SELEX-pool to an in vivo screening for gene regulatory activity in a yeastbased reporter gene assay. The resulting neomycin B and ribostamycin (Figure 1a) responsive RNA-element (N1) contains only 27 nucleotides in a bulged hairpin secondary structure (Figure 1b)—the smallest riboswitch functional in vivo identified to date. In sequence and secondary structure, N1 differs completely from an in vitro selected but regulatory inactive RNA-aptamer for the same ligand (R23). Instead it partially resembles the ribosomal A-site, the natural target for aminoglycoside antibiotics (Figure 1b). The NMR spectroscopic analysis of the N1 riboswitch complexed with ribostamycin identifies structural determinants for its regulatory activity and suggests a ligand binding mechanism based on conformational capture. Our results provide insights into the modularity of ligand binding sites in RNA and highlight structural and dynamic features N1 shares with the larger naturally occurring riboswitches as well as with other regulatory active aptamers. This knowledge may guide the future design of novel synthetic riboswitches for targeted in vivo applications. Structure of the N1–ligand complex—the “OFF”-state of the riboswitch: N1 represses gene expression upon binding to either neomycin B or the closely related but smaller ribostamycin. NMR spectra of N1 bound to either ligand (Supporting Information Figure S1) indicate that both complexes are formed with similarly high affinity and display a high degree of structural similarity suggesting that the contribution of ring IV of neomycin to the interaction is negligible. Thus, we determined the structure of the N1–ribostamycin complex, because of its superior spectral resolution for the ligand resonances, by NMR spectroscopy (see Table 1). Chemical shift assignments and coordinates have been deposited (BMRB code: 16609, pdb-code: 2kxm). The structure of ribostamycin-bound N1 consists of a continuous helical stem with canonical stacking interactions between the G5:C23 and the G9:C22 base pair despite the presence of a flexible three-nucleotide bulge (C6–U8) and a compactly folded apical hexaloop organized around a U-turn motif (U14–A16) closed by the U13:U18 base pair (Figure 1c–e). Ribostamycin rings I and II are sandwiched between the N1 major groove, in the region from G5:C23 to U13:U18 and A17 protruding from the apical loop (Figure 2). Ring III is located close to the backbone of the 3’-strand (U18 to G20). Simultaneous contacts of the ligand with the G5:C23 base pair below and G9:C22 above the bulge (Figure 2b) clamp together the lower and upper helical stem and thus enforce the uninterrupted coaxial helical stacking across the flexible C6–U8 internal bulge. The bulge itself is not interacting with the ligand. A detailed structural description of the N1– ribostamycin complex is given in the Supporting Information. A comparison of the N1–ribostamycin complex with other aminoglycoside binding RNAs reveals partial similarities to known aminoglycoside binding sub-motifs: The helical stem centered at the U10:U21 base pair is similar to the ribosomal [*] Dr. E. Duchardt-Ferner, S. R. Schmidtke, Prof. Dr. J. W hnert Institute for Molecular Biosciences, Center for Biomolecular Magnetic Resonance (BMRZ), Johann-Wolfgang-Goethe-University Frankfurt Max-von-Laue-Strasse 9, 60438 Frankfurt (Germany) Fax: (+49)69-798-29527 E-mail: woehnert@bio.uni-frankfurt.de

Structural Basis of β‐Amyloid‐Dependent Synaptic Dysfunctions

Aggregation of b-amyloid (Ab) peptide into oligomers and protofibrils is a hallmark of Alzheimer s disease (AD). Increasing evidence shows that the primary insult in AD is caused by oligomeric species that impair the ordered function of synaptic networks. Consistent with this view, oligomers were shown to affect synaptic plasticity, and they impair the long-term potentiation (LTP) in living brain tissues, a widely used model system of brain memory functions. Using solidstate NMR spectroscopy, we here determined the residuespecific molecular conformation of a highly synaptotoxic bamyloid oligomer structure. Our measurements reveal a stable N-terminal b strand that controls the partitioning between oligomer and protofibril formation, whereas targeting the peptide N-terminus ameliorates Ab-dependent neuronal dysfunctions. The presently investigated, chemically well-defined Ab oligomers faithfully reproduce the hallmark characteristics of AD-related oligomers. Living hippocampal brain slices were exposed to different Ab conformers (Figure 1A), and a series of tetanic electrical stimuli were applied to evoke a longlasting increase of the synaptic transmission, termed LTP. Oligomers, but not freshly dissolved, that is, primarily monomeric, Ab peptide or fibrils, reduce the LTP response and therefore disturb the brain memory functions within these tissue samples (Figure 1B). A similar oligomer-specificity is seen with cultured primary neurons, which present a significant oligomer-dependent decrease ( 40%) of their

Conformational analysis of protein and nucleic acid fragments with the new grid search algorithm FOUND.

The new computer algorithm FOUND, which is implemented as an integrated module of the DYANA structure calculation program, is capable of performing systematic local conformation analyses by exhaustive grid searches for arbitrary contiguous fragments of proteins and nucleic acids. It uses torsion angles as the only degrees of freedom to identify all conformations that fulfill the steric and NMR-derived conformational restraints within a contiguous molecular fragment, as defined either by limits on the maximal restraint violations or by the fragment-based DYANA target function value. Sets of mutually dependent torsion angles, for example in ribose rings, are treated as a single degree of freedom. The results of the local conformation analysis include allowed torsion angle ranges and stereospecific assignments for diastereotopic substituents, which are then included in the input of a subsequent structure calculation. FOUND can be used for grid searches comprising up to 13 torsion angles, such as the backbone of a complete α-helical turn or dinucleotide fragments in nucleic acids, and yields a significantly higher number of stereospecific assignments than the precursor grid search algorithm HABAS.

Interaction of Kazal-type inhibitor domains with serine proteinases: biochemical and structural studies.

The interaction of domains of the Kazal-type inhibitor protein dipetalin with the serine proteinases thrombin and trypsin is studied. The functional studies of the recombinantly expressed domains (Dip-I+II, Dip-I and Dip-II) allow the dissection of the thrombin inhibitory properties and the identification of Dip-I as a key contributor to thrombin/dipetalin complex stability and its inhibitory potency. Furthermore, Dip-I, but not Dip-II, forms a complex with trypsin resulting in an inhibition of the trypsin activity directed towards protein substrates. The high resolution NMR structure of the Dip-I domain is determined using multi-dimensional heteronuclear NMR spectroscopy. Dip-I exhibits the canonical Kazal-type fold with a central alpha-helix and a short two-stranded antiparallel beta-sheet. Molecular regions essential for inhibitor complex formation with thrombin and trypsin are identified. A comparison with molecular complexes of other Kazal-type thrombin and trypsin inhibitors by molecular modeling shows that the N-terminal segment of Dip-I fulfills the structural prerequisites for inhibitory interactions with either proteinase and explains the capacity of this single Kazal-type domain to interact with different proteinases.

Solution Structure of a Functional Biomimetic and Mechanistic Implications for Nickel Superoxide Dismutases

The nickel complex of a synthetic nonapeptide (HCDLPCFVY‐NH2) is capable of catalytically disproportionating O2.− and is thus a functional biomimetic for nickel superoxide dismutases. This represents a simplification as compared to a NiSOD “maquette” that is based on a dodecapeptide that was recently reported [Inorg. Chem. 2006, 45, 2358]. The 3D solution structure reveals that the first six residues form a stable macrocyclic structure with a preformed binding site for NiII. Proline 5 exhibits a trans peptide linkage in the biomimetic and a cis conformation in NiSOD enzymes. DFT calculations reveal the source of this preference. Mechanistic consequences for the mode of action (identity of the fifth ligand) are discussed. The SOD activity is compared to enzymatic systems, and selected modifications allowed the biomimetic to be reduced to a functional minimal motif of only six amino acids (ACAAPC‐NH2).

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]

Analysis of Fe(III) Heme Binding to Cysteine-Containing Heme-Regulatory Motifs in Proteins

Regulatory heme binds to specific motifs in proteins and controls a variety of biochemical processes. Several of these proteins were recently shown to form complexes with ferric and/or ferrous heme via a cysteine residue as axial ligand. The objective of this study was to examine the heme-binding properties of a series of cysteine-containing peptides with focus on CP motif sequences. The peptides displayed different binding behavior upon Fe(III) heme application with characteristic wavelength shifts of the Soret band to 370 nm or 420-430 nm and in some cases to both wavelengths. Whereas for most of the peptides containing a cysteine only a shift to 420-430 nm was observed, CP-containing peptides exhibited a preference for a shift to 370 nm. Detailed structural investigation using Raman and NMR spectroscopy on selected representatives revealed different binding modes with respect to iron ion coordination, which reflected the results of the UV-vis studies. A predicted short sequence stretch derived from dipeptidyl peptidase 8 was additionally examined with respect to CP motif binding to heme on the peptide as well as on the protein level. The heme association was confirmed with the first solution structure of a CP-peptide-heme complex and, moreover, an inhibitory effect of Fe(III) heme on the enzymes activity. The relevance of both the use of model compounds to elucidate the molecular mechanism underlying regulatory heme binding and its potential for the investigation of regulatory heme control is discussed.

The intrinsically disordered amino-terminal region of human RecQL4: multiple DNA-binding domains confer annealing, strand exchange and G4 DNA binding

Human RecQL4 belongs to the ubiquitous RecQ helicase family. Its N-terminal region represents the only homologue of the essential DNA replication initiation factor Sld2 of Saccharomyces cerevisiae, and also participates in the vertebrate initiation of DNA replication. Here, we utilized a random screen to identify N-terminal fragments of human RecQL4 that could be stably expressed in and purified from Escherichia coli. Biophysical characterization of these fragments revealed that the Sld2 homologous RecQL4 N-terminal domain carries large intrinsically disordered regions. The N-terminal fragments were sufficient for the strong annealing activity of RecQL4. Moreover, this activity appeared to be the basis for an ATP-independent strand exchange activity. Both activities relied on multiple DNA-binding sites with affinities to single-stranded, double-stranded and Y-structured DNA. Finally, we found a remarkable affinity of the N-terminus for guanine quadruplex (G4) DNA, exceeding the affinities for other DNA structures by at least 60-fold. Together, these findings suggest that the DNA interactions mediated by the N-terminal region of human RecQL4 represent a central function at the replication fork. The presented data may also provide a mechanistic explanation for the role of elements with a G4-forming propensity identified in the vicinity of vertebrate origins of DNA replication.