Ctirad Hofr

The Solution Structure of the ADAR2 dsRBM-RNA Complex Reveals a Sequence-Specific Readout of the Minor Groove.

Sequence-dependent recognition of dsDNA-binding proteins is well understood, yet sequence-specific recognition of dsRNA by proteins remains largely unknown, despite their importance in RNA maturation pathways. Adenosine deaminases that act on RNA (ADARs) recode genomic information by the site-selective deamination of adenosine. Here, we report the solution structure of the ADAR2 double-stranded RNA-binding motifs (dsRBMs) bound to a stem-loop pre-mRNA encoding the R/G editing site of GluR-2. The structure provides a molecular basis for how dsRBMs recognize the shape, and also more surprisingly, the sequence of the dsRNA. The unexpected direct readout of the RNA primary sequence by dsRBMs is achieved via the minor groove of the dsRNA and this recognition is critical for both editing and binding affinity at the R/G site of GluR-2. More generally, our findings suggest a solution to the sequence-specific paradox faced by many dsRBM-containing proteins that are involved in post-transcriptional regulation of gene expression.

High affinity fucose binding of Pseudomonas aeruginosa lectin PA‐IIL: 1.0 Å resolution crystal structure of the complex combined with thermodynamics and computational chemistry approaches

PA‐IIL is a fucose‐binding lectin from Pseudomonas aeruginosa that is closely related to the virulence factors of the bacterium. Previous structural studies have revealed a new carbohydrate‐binding mode with direct involvement of two calcium ions (Mitchell E, Houles C, Sudakevitz D, Wimmerova M, Gautier C, Peréz S, Wu AM, Gilboa‐Garber N, Imberty A. Structural basis for selective recognition of oligosaccharides from cystic fibrosis patients by the lectin PA‐IIL of Pseudomonas aeruginosa. Nat Struct Biol 2002;9:918–921). A combination of thermodynamic, structural, and computational methods has been used to study the basis of the high affinity for the monosaccharide ligand. A titration microcalorimetry study indicated that the high affinity is enthalpy driven. The crystal structure of the tetrameric PA‐IIL in complex with fucose and calcium was refined to 1.0 Å resolution and, in combination with modeling, allowed a proposal to be made for the hydrogen‐bond network in the binding site. Calculations of partial charges using ab initio computational chemistry methods indicated that extensive delocalization of charges between the calcium ions, the side chains of the protein‐binding site and the carbohydrate ligand is responsible for the high enthalpy of binding and therefore for the unusually high affinity observed for this unique mode of carbohydrate recognition. Proteins 2005.

Serine phosphorylation and proline isomerization in RNAP II CTD control recruitment of Nrd1

Recruitment of appropriate RNA processing factors to the site of transcription is controlled by post-translational modifications of the C-terminal domain (CTD) of RNA polymerase II (RNAP II). Here, we report the solution structure of the Ser5 phosphorylated (pSer5) CTD bound to Nrd1. The structure reveals a direct recognition of pSer5 by Nrd1 that requires the cis conformation of the upstream pSer5-Pro6 peptidyl-prolyl bond of the CTD. Mutations at the complex interface diminish binding affinity and impair processing or degradation of noncoding RNAs. These findings underpin the interplay between covalent and noncovalent changes in the CTD structure that constitute the CTD code.

DNA interactions of antitumor cisplatin analogs containing enantiomeric amine ligands.

Modifications of natural DNA and synthetic oligodeoxyribonucleotide duplexes in a cell-free medium by analogs of antitumor cisplatin containing enantiomeric amine ligands, such as cis-[PtCl(2)(RR-DAB)] and cis-[PtCl(2)(SS-DAB)] (DAB = 2,3-diaminobutane), were studied by various methods of molecular biophysics and biophysical chemistry. These methods include DNA binding studies by pulse polarography and atomic absorption spectrophotometry, mapping of DNA adducts using transcription assay, interstrand cross-linking assay using gel electrophoresis under denaturing conditions, differential scanning calorimetry, chemical probing, and bending and unwinding studies of the duplexes containing single, site-specific cross-link. The major differences resulting from the modification of DNA by the two enantiomers are the thermodynamical destabilization and conformational distortions induced in DNA by the 1,2-d(GpG) intrastrand cross-link. It has been suggested that these differences are associated with a different biological activity of the two enantiomers observed previously. In addition, the results of the present work are also consistent with the view that formation of hydrogen bonds between the carbonyl oxygen of the guanine residues and the quasi equatorial hydrogen of the cis amine in the 1, 2-d(GpG) intrastrand cross-link plays an important role in determining the character of the distortion induced in DNA by this lesion.

Recognition of Transcription Termination Signal by the Nuclear Polyadenylated RNA-binding (NAB) 3 Protein

Non-coding RNA polymerase II transcripts are processed by the poly(A)-independent termination pathway that requires the Nrd1 complex. The Nrd1 complex includes two RNA-binding proteins, the nuclear polyadenylated RNA-binding (Nab) 3 and the nuclear pre-mRNA down-regulation (Nrd) 1 that bind their specific termination elements. Here we report the solution structure of the RNA-recognition motif (RRM) of Nab3 in complex with a UCUU oligonucleotide, representing the Nab3 termination element. The structure shows that the first three nucleotides of UCUU are accommodated on the β-sheet surface of Nab3 RRM, but reveals a sequence-specific recognition only for the central cytidine and uridine. The specific contacts we identified are important for binding affinity in vitro as well as for yeast viability. Furthermore, we show that both RNA-binding motifs of Nab3 and Nrd1 alone bind their termination elements with a weak affinity. Interestingly, when Nab3 and Nrd1 form a heterodimer, the affinity to RNA is significantly increased due to the cooperative binding. These findings are in accordance with the model of their function in the poly(A) independent termination, in which binding to the combined and/or repetitive termination elements elicits efficient termination.

Human Rap1 modulates TRF2 attraction to telomeric DNA

More than two decades of genetic research have identified and assigned main biological functions of shelterin proteins that safeguard telomeres. However, a molecular mechanism of how each protein subunit contributes to the protecting function of the whole shelterin complex remains elusive. Human Repressor activator protein 1 (Rap1) forms a multifunctional complex with Telomeric Repeat binding Factor 2 (TRF2). Rap1–TRF2 complex is a critical part of shelterin as it suppresses homology-directed repair in Ku 70/80 heterodimer absence. To understand how Rap1 affects key functions of TRF2, we investigated full-length Rap1 binding to TRF2 and Rap1–TRF2 complex interactions with double-stranded DNA by quantitative biochemical approaches. We observed that Rap1 reduces the overall DNA duplex binding affinity of TRF2 but increases the selectivity of TRF2 to telomeric DNA. Additionally, we observed that Rap1 induces a partial release of TRF2 from DNA duplex. The improved TRF2 selectivity to telomeric DNA is caused by less pronounced electrostatic attractions between TRF2 and DNA in Rap1 presence. Thus, Rap1 prompts more accurate and selective TRF2 recognition of telomeric DNA and TRF2 localization on single/double-strand DNA junctions. These quantitative functional studies contribute to the understanding of the selective recognition of telomeric DNA by the whole shelterin complex.

Functional characterization of domains in AtTRB1, a putative telomere-binding protein in Arabidopsis thaliana

Telomeres are nucleoprotein structures ensuring the stability of eukaryotic chromosome ends. Two protein families, TRFL (TFL-Like) and SMH (Single-Myb-Histone), containing a specific telobox motif in their Myb domain, have been identified as potential candidates involved in a functional nucleoprotein structure analogous to human shelterin at plant telomeres. We analyze the DNA-protein interaction of the full-length and truncated variants of AtTRB1, a SMH-family member with a typical structure: N-terminal Myb domain, central H1/5 domain and C-terminal coiled-coil. We show that preferential interaction of AtTRB1 with double-stranded telomeric DNA is mediated by the Myb domain, while the H1/5 domain is involved in non-specific DNA-protein interaction and in the multimerization of AtTRB1.

Contributions of the TEL-patch amino acid cluster on TPP1 to telomeric DNA synthesis by human telomerase.

Telomere maintenance is a highly coordinated process, and its misregulation is linked to cancer and telomere-shortening syndromes. Recent studies have shown that the TEL-patch--a cluster of amino acids on the surface of the shelterin component TPP1--is necessary for the recruitment of telomerase to the telomere in human cells. However, there has been only basic biochemical analysis of the role of TPP1 in the telomerase recruitment process. Here we develop an in vitro assay to quantitatively measure the contribution of the TEL-patch to telomerase recruitment--binding and extension of the first telomeric repeat. We also demonstrate that the TEL-patch contributes to the translocation step of the telomerase reaction. Finally, our quantitative observations indicate that the TEL-patch stabilizes the association between telomerase and telomeric DNA substrates, providing a molecular explanation for its contributions to telomerase recruitment and action.

Single-Myb-histone proteins from Arabidopsis thaliana: a quantitative study of telomere-binding specificity and kinetics.

Proteins that bind telomeric DNA modulate the structure of chromosome ends and control telomere function and maintenance. It has been shown that AtTRB (Arabidopsis thaliana telomere-repeat-binding factor) proteins from the SMH (single-Myb-histone) family selectively bind double-stranded telomeric DNA and interact with the telomeric protein AtPOT1b (A. thaliana protection of telomeres 1b), which is involved in telomere capping. In the present study, we performed the first quantitative DNA-binding study of this plant-specific family of proteins. Interactions of full-length proteins AtTRB1 and AtTRB3 with telomeric DNA were analysed by electrophoretic mobility-shift assay, fluorescence anisotropy and surface plasmon resonance to reveal their binding stoichiometry and kinetics. Kinetic analyses at different salt conditions enabled us to estimate the electrostatic component of binding and explain different affinities of the two proteins to telomeric DNA. On the basis of available data, a putative model explaining the binding stoichiometry and the protein arrangement on telomeric DNA is presented.

Modification of natural, double-helical DNA by antitumor cis- and trans-[Cl2(Me2SO4)4Ru] in cell-free media

Modifications of natural DNA in cell-free media by the antitumor ruthenium compounds cis- and trans-[Cl(2)(Me(2)SO(4))(4)Ru] were studied by various biochemical and biophysical methods. These methods included: binding studies by means of flameless atomic absorption spectrophotometry, mapping of DNA adducts by means of transcription assay, use of ethidium bromide as a fluorescent probe of DNA adducts of metal complexes, an interstrand cross-linking assay employing gel electrophoresis under denaturing conditions, measurements of DNA unwinding by gel electrophoresis, differential pulse polarographic analysis of DNA conformation, and analysis of liquid crystalline dispersions of DNA by circular dichroism. The results indicated that both ruthenium compounds irreversibly coordinated to DNA; the rate of binding of the cis isomer was considerably lower than that of the trans isomer. The DNA-binding mode of trans-[Cl(2)(Me(2)SO(4))(4)Ru] included formation of bifunctional adducts such as intrastrand cross-links between neighboring purine residues and a small amount ( approximately 1%) of interstrand cross-links. cis-[Cl(2)(Me(2)SO(4))(4)Ru] formed mainly monofunctional lesions on natural DNA. Both ruthenium isomers induced conformational alterations of non-denaturational character in DNA, the trans compound being more effective. In addition, DNA adducts of trans-[Cl(2)(Me(2)SO(4))(4)Ru] were capable of inhibiting RNA synthesis by DNA-dependent RNA polymerases, while the adducts of the cis isomer were not. Thus, several features of the DNA-binding mode of trans-[Cl(2)(Me(2)SO(4))(4)Ru] were similar to those of antitumor cis-diamminedichloroplatinum (II), which may be relevant to the biological effects of this antitumor ruthenium drug. On the other hand, the different DNA-binding mode of cis-[Cl(2)(Me(2)SO(4))(4)Ru] was consistent with its less pronounced biological effects.