Juli Feigon

Interactions of antitumor drugs with natural DNA: 1H NMR study of binding mode and kinetics.

1H NMR has been used to study the interactions of over 70 clinical and experimental antitumor drugs with DNA. Spectra of the low-field (H-bonded imino proton) resonances of DNA were studied as a function of drug per base pair ratio. From the spectral changes observed, it was possible to distinguish three modes of drug binding (intercalation, groove binding, and nonspecific outside binding), to determine the kinetics of drug binding (approximate lifetime of the bound drug), and, in favorable cases, to determine the specificity of the drugs for A X T or G X C base pairs. This method is a useful assay for general drug-binding characteristics. For the intercalating compounds there appears to be a correlation between drug-binding kinetics and useful antitumor activity.

Multistranded DNA structures.

DNA oligonucleotides can form multistranded helices through either the folding of a single strand or the association of two, three or four strands of DNA. Structures of several new DNA triplexes, G-quartet DNA quadruplexes and I-motif DNA quadruplexes have been reported recently. These structures provide new insights into helix stability and folding, loop conformations and cation interactions.

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 structures of UBA domains reveal a conserved hydrophobic surface for protein-protein interactions.

UBA domains are a commonly occurring sequence motif of approximately 45 amino acid residues that are found in diverse proteins involved in the ubiquitin/proteasome pathway, DNA excision-repair, and cell signaling via protein kinases. The human homologue of yeast Rad23A (HHR23A) is one example of a nucleotide excision-repair protein that contains both an internal and a C-terminal UBA domain. The solution structure of HHR23A UBA(2) showed that the domain forms a compact three-helix bundle. We report the structure of the internal UBA(1) domain of HHR23A. Comparison of the structures of UBA(1) and UBA(2) reveals that both form very similar folds and have a conserved large hydrophobic surface patch. The structural similarity between UBA(1) and UBA(2), in spite of their low level of sequence conservation, leads us to conclude that the structural variability of UBA domains in general is likely to be rather small. On the basis of the structural similarities as well as analysis of sequence conservation, we predict that this hydrophobic surface patch is a common protein-interacting surface present in diverse UBA domains. Furthermore, accumulating evidence that ubiquitin binds to UBA domains leads us to the prediction that the hydrophobic surface patch of UBA domains interacts with the hydrophobic surface on the five-stranded beta-sheet of ubiquitin. Detailed comparison of the structures of the two UBA domains, combined with previous mutagenesis studies, indicates that the binding site of HIV-1 Vpr on UBA(2) does not completely overlap the ubiquitin binding site.

Mutations linked to dyskeratosis congenita cause changes in the structural equilibrium in telomerase RNA

Autosomal dominant dyskeratosis congenita (DKC), as well as aplastic anemia, has been linked to mutations in the RNA component of telomerase, the ribonucleoprotein responsible for telomere maintenance. Here we examine the effect of the DKC mutations on the structure and stability of human telomerase RNA pseudoknot and CR7 domains by using NMR and thermal melting. The CR7 domain point mutation decreases stability and alters a conserved secondary structure thought to be involved in human telomerase RNA accumulation in vivo. We find that pseudoknot constructs containing the conserved elements of the pseudoknot domain are in equilibrium with a hairpin conformation. The solution structure of the wild-type hairpin reveals that it forms a continuous helix containing a novel run of three consecutive U⋅U and a U⋅C base pairs closed by a pentaloop. The six base pairs unique to the hairpin conformation are phylogenetically conserved in mammals, suggesting that this conformation is also functionally important. The DKC mutation in the pseudoknot domain results in a shift in the equilibrium toward the hairpin form, primarily due to destabilization of the pseudoknot. Our results provide insight into the effect of these mutations on telomerase structure and suggest that the catalytic cycle of telomerase involves a delicate interplay between RNA conformational states, alteration of which leads to the disease state.

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.

Formation of a stable triplex from a single DNA strand

HOMOPURINE·homopyrimidine DNA sequences have been shown to form triple-stranded structures readily under appropriate conditions1–5. Interest in DNA triplexes arises from potential applications of intermolecular triplexes as antisense inhibitors of gene expression6–8 and from the possibility that intramolecular triplexes may have a role in gene expression and recombination1. We recently presented NMR evidence for triplex formation from the DNA oligonucleotides d(GA)4 and d(TC)4, which showed unambiguously that the second pyrimidine strand is Hoogsteen base paired and the cytosines are protonated at N3 as required9,10. To obtain a more well defined triplex, and to provide a model for in vivo triplex structures, we have designed and synthesized a 28-base DNA oligomer with a sequence that could potentially fold to form a triplex containing both T·A·T and C +·G·C triplets. Our NMR results indicate that the conformation at pH5.5 is an intramolecular triplex and that a significant amount of triplex remains even at pH 8.0.

Solution structure of the loop B domain from the hairpin ribozyme.

The hairpin ribozyme is a small catalytic RNA with a unique two-domain structure. Here we present the solution structure of the loop B domain of the hairpin ribozyme, which contains most of the catalytically essential nucleotides. The 38-nucleotide domain contains a 16-nucleotide internal loop that forms one of the largest non-Watson–Crick segments of base pairing thus far determined by either NMR or crystallography. Since the solution structure of the smaller loop A domain has been previously solved, an NMR structure-based model of the 22,000 Mr hairpin ribozyme–substrate open complex emerges by joining the two domain structures. Strikingly, catalytically essential functional groups for the loop B domain are concentrated within an expanded minor groove, presenting a clear docking surface for the loop A domain.