Thorsten Dieckmann

Solution structure of protegrin-1, a broad-spectrum antimicrobial peptide from porcine leukocytes.

BACKGROUND The protegrins are a family of arginine- and cysteine-rich cationic peptides found in porcine leukocytes that exhibit a broad range of antimicrobial and antiviral activities. They are composed of 16-18 amino-acid residues including four cysteines, which form two disulfide linkages. To begin to understand the mechanism of action of these peptides, we set out to determine the structure of protegrin-1 (PG-1). RESULTS We used two-dimensional homonuclear nuclear magnetic resonance spectroscopy to study the conformation of both natural and synthetic PG-1 under several conditions. A refined three-dimensional structure of synthetic PG-1 is presented. CONCLUSIONS Both synthetic and natural protegrin-1 form a well-defined structure in solution composed primarily of a two-stranded antiparallel beta sheet, with strands connected by a beta turn. The structure of PG-1 suggests ways in which the peptide may interact with itself or other molecules to form the membrane pores and the large membrane-associated assemblages observed in protegrin-treated, gram-negative bacteria.

Molecular basis of sequence-specific recognition of pre-ribosomal RNA by nucleolin

The structure of the 28 kDa complex of the first two RNA binding domains (RBDs) of nucleolin (RBD12) with an RNA stem–loop that includes the nucleolin recognition element UCCCGA in the loop was determined by NMR spectroscopy. The structure of nucleolin RBD12 with the nucleolin recognition element (NRE) reveals that the two RBDs bind on opposite sides of the RNA loop, forming a molecular clamp that brings the 5′ and 3′ ends of the recognition sequence close together and stabilizing the stem–loop. The specific interactions observed in the structure explain the sequence specificity for the NRE sequence. Binding studies of mutant proteins and analysis of conserved residues support the proposed interactions. The mode of interaction of the protein with the RNA and the location of the putative NRE sites suggest that nucleolin may function as an RNA chaperone to prevent improper folding of the nascent pre‐rRNA.

Solution structure of the HMG protein NHP6A and its interaction with DNA reveals the structural determinants for non-sequence-specific binding.

NHP6A is a chromatin‐associated protein from Saccharomyces cerevisiae belonging to the HMG1/2 family of non‐specific DNA binding proteins. NHP6A has only one HMG DNA binding domain and forms relatively stable complexes with DNA. We have determined the solution structure of NHP6A and constructed an NMR‐based model structure of the DNA complex. The free NHP6A folds into an L‐shaped three α‐helix structure, and contains an unstructured 17 amino acid basic tail N‐terminal to the HMG box. Intermolecular NOEs assigned between NHP6A and a 15 bp 13C, 15N‐labeled DNA duplex containing the SRY recognition sequence have positioned the NHP6A HMG domain onto the minor groove of the DNA at a site that is shifted by 1 bp and in reverse orientation from that found in the SRY–DNA complex. In the model structure of the NHP6A–DNA complex, the N‐terminal basic tail is wrapped around the major groove in a manner mimicking the C‐terminal tail of LEF1. The DNA in the complex is severely distorted and contains two adjacent kinks where side chains of methionine and phenylalanine that are important for bending are inserted. The NHP6A–DNA model structure provides insight into how this class of architectural DNA binding proteins may select preferential binding sites.

Structure of a human DNA repair protein UBA domain that interacts with HIV-1 Vpr.

The HIV-1 protein Vpr is critical for a number of viral functions including a unique ability to arrest T-cells at a G2/M checkpoint and induce subsequent apoptosis. It has been shown to interact specifically with the second UBA (ubiquitin associated) domain found in the DNA repair protein HHR23A, a highly evolutionarily conserved protein. This domain is a commonly occurring sequence motif in some members of the ubiquitination pathway, UV excision repair proteins, and certain protein kinases. The three dimensional structure of the UBA domain, determined by NMR spectroscopy, is presented. The protein domain forms a compact three-helix bundle. One side of the protein has a hydrophobic surface that is the most likely Vpr target site.

Aptamer structures from A to ζ

Solution structures of RNA aptamers for FMN, ATP, arginine, and citrulline reveal how oligonucleotides can fold to form selective binding pockets for biological cofactors and amino acids. These structures confirm old ideas and provide new insights about three-dimensional structures of nucleic acids and their possible role in chemical reactions.

Solution structure of a GAAA tetraloop receptor RNA

The GAAA tetraloop receptor is an 11‐nucleotide RNA sequence that participates in the tertiary folding of a variety of large catalytic RNAs by providing a specific binding site for GAAA tetraloops. Here we report the solution structure of the isolated tetraloop receptor as solved by multidimensional, heteronuclear magnetic resonance spectroscopy. The internal loop of the tetraloop receptor has three adenosines stacked in a cross‐strand or zipper‐like fashion. This arrangement produces a high degree of base stacking within the asymmetric internal loop without extrahelical bases or kinking the helix. Additional interactions within the internal loop include a U·U mismatch pair and a G·U wobble pair. A comparison with the crystal structure of the receptor RNA bound to its tetraloop shows that a conformational change has to occur upon tetraloop binding, which is in good agreement with previous biochemical data. A model for an alternative binding site within the receptor is proposed based on the NMR structure, phylogenetic data and previous crystallographic structures of tetraloop interactions.

Recognition of Planar and Nonplanar Ligands in the Malachite Green–RNA Aptamer Complex

Ribonucleic acids are an attractive drug target owing to their central role in many pathological processes. Notwithstanding this potential, RNA has only rarely been successfully targeted with novel drugs. The difficulty of targeting RNA is at least in part due to the unusual mode of binding found in most small‐molecule–RNA complexes: the ligand binding pocket of the RNA is largely unstructured in the absence of ligand and forms a defined structure only with the ligand acting as scaffold for folding. Moreover, electrostatic interactions between RNA and ligand can also induce significant changes in the ligand structure due to the polyanionic nature of the RNA. Aptamers are ideal model systems to study these kinds of interactions owing to their small size and the ease with which they can be evolved to recognize a large variety of different ligands. Here we present the solution structure of an RNA aptamer that binds triphenyl dyes in complex with malachite green and compare it with a previously determined crystal structure of a complex formed with tetramethylrosamine. The structures illustrate how the same RNA binding pocket can adapt to accommodate both planar and nonplanar ligands. Binding studies with single‐ and double‐substitution mutant aptamers are used to correlate three‐dimensional structure with complex stability. The two RNA–ligand complex structures allow a discussion of structural changes that have been observed in the ligand in the context of the overall complex structure. Base pairing and stacking interactions within the RNA fold the phosphate backbone into a structure that results in an asymmetric charge distribution within the binding pocket that forces the ligand to adapt through a redistribution of the positive partial charge.

Assignment methodology for larger RNA oligonucleotides: Application to an ATP-binding RNA aptamer

The use of uniform 13C,15N labeling in the NMR spectroscopic study of RNA structures hasgreatly facilitated the assignment process in small RNA oligonucleotides. For ribose spinsystem assignments, exploitation of these labels has followed previously developed methodsfor the study of proteins. However, for sequential assignment of the exchangeable andnonexchangeable protons of the nucleotides, it has been necessary to develop a variety of newNMR experiments. Even these are of limited utility in the unambiguous assignment of largerRNAs due to the short carbon relaxation times and extensive spectral overlap for all nuclei.These problems can largely be overcome by the additional use of base-type selectively13C,15N-labeled RNA in combination with a judicious use of related RNAs with basesubstitutions. We report the application of this approach to a 36-nucleotide ATP-binding RNAaptamer in complex with AMP. Complete sequential 1H assignments, as well as the majorityof 13C and 15N assignments, were obtained.

Secondary structure and dynamics of the r(CGG) repeat in the mRNA of the fragile X mental retardation 1 (FMR1) gene.

The CGG triplet repeat found within the 5’UTR of the FMR1 gene is involved in the pathogenesis of both fragile X syndrome and fragile X-associated tremor/ataxia syndrome (FXTAS). The repeat has been shown to form both hairpins and tetraplexes in DNA; however, the secondary structure of CGG-repeat RNA has not been well defined. To this end, we have performed NMR spectroscopy on in vitro transcribed CGG-repeat RNAs and see clear evidence of intramolecular hairpins, with no evidence of tetraplex structures. Both C•G and G◦G base pairs form in the hairpin stem, though in a dynamic equilibrium of conformations. In addition, we investigated the effect of an AGG repeat interruption on hairpin stability; such interruptions are often interspersed within the CGG repeat element and are thought to modulate secondary structure of the RNA. While the AGG repeat lowers the Tm of the hairpin at low Mg2+ concentrations, this difference disappears at physiological Mg2+ levels.

Extraction of spectral information from a short-time signal using filter-diagonalization: Recent developments and applications to semiclassical reaction dynamics and nuclear magnetic resonance signals

Filter-diagonalization [M. R. Wall and D. Neuhauser, J. Chem. Phys. 102, 8011 (1995)] is a new method for extracting frequencies and damping constants from a short-time segment of any time-dependent signal, whether of quantum origin or not. The method is efficient and able to handle signals with, e.g., millions of (possibly overlapping) frequencies, since it concentrates on specific spectral ranges. The method was shown to be a powerful tool for extracting eigenstates and normal-modes, and for reducing propagation times, in several recent works by us, by Mandelshtam and Taylor (who recently introduced the box filter) and by other groups. Here we extend the method in several directions: first, we show how it can be used with a filter of any form. Next, we show how the methodology may be extended to treat multi-dimensional signals, of the type that appears, e.g., in 2-D nuclear magnetic resonance (NMR). Finally, we exemplify the performance of the various filters for two types of signals where the time-redu...