Structural basis of Nrd1-Nab3 heterodimerization.

Heterodimerization of RNA binding proteins Nrd1 and Nab3 is essential to communicate the RNA recognition in the nascent transcript with the Nrd1 recognition of the Ser5-phosphorylated Rbp1 C-terminal domain in RNA polymerase II. The structure of a Nrd1-Nab3 chimera reveals the basis of heterodimerization, filling a missing gap in knowledge of this system. The free form of the Nrd1 interaction domain of Nab3 (NRID) forms a multi-state three-helix bundle that is clamped in a single conformation upon complex formation with the Nab3 interaction domain of Nrd1 (NAID). The latter domain forms two long helices that wrap around NRID, resulting in an extensive protein-protein interface that would explain the highly favorable free energy of heterodimerization. Mutagenesis of some conserved hydrophobic residues involved in the heterodimerization leads to temperature-sensitive phenotypes, revealing the importance of this interaction in yeast cell fitness. The Nrd1-Nab3 structure resembles the previously reported Rna14/Rna15 heterodimer structure, which is part of the poly(A)-dependent termination pathway, suggesting that both machineries use similar structural solutions despite they share little sequence homology and are potentially evolutionary divergent.

Structural insights into RNA polymerase recognition and essential function of Myxococcus xanthus CdnL.

CdnL and CarD are two functionally distinct members of the CarD_CdnL_TRCF family of bacterial RNA polymerase (RNAP)-interacting proteins, which co-exist in Myxococcus xanthus. While CarD, found exclusively in myxobacteria, has been implicated in the activity of various extracytoplasmic function (ECF) σ-factors, the function and mode of action of the essential CdnL, whose homologs are widespread among bacteria, remain to be elucidated in M. xanthus. Here, we report the NMR solution structure of CdnL and present a structure-based mutational analysis of its function. An N-terminal five-stranded ß-sheet Tudor-like module in the two-domain CdnL mediates binding to RNAP-ß, and mutations that disrupt this interaction impair cell growth. The compact CdnL C-terminal domain consists of five α-helices folded as in some tetratricopeptide repeat-like protein-protein interaction domains, and contains a patch of solvent-exposed nonpolar and basic residues, among which a set of basic residues is shown to be crucial for CdnL function. We show that CdnL, but not its loss-of-function mutants, stabilizes formation of transcriptionally competent, open complexes by the primary σA-RNAP holoenzyme at an rRNA promoter in vitro. Consistent with this, CdnL is present at rRNA promoters in vivo. Implication of CdnL in RNAP-σA activity and of CarD in ECF-σ function in M. xanthus exemplifies how two related members within a widespread bacterial protein family have evolved to enable distinct σ-dependent promoter activity.

Two singular types of CCCH tandem zinc finger in Nab2p contribute to polyadenosine RNA recognition.

The seven C-terminal CCCH-type zinc fingers of Nab2p bind the poly(A) tail of mRNA (∼A25). Using NMR, we demonstrated that the first four (Zf1-Zf4) contain two structurally independent tandems (TZF12 and TZF34) and bind A12 with moderate affinity (KD = 2.3 µM). Nab2p TZF12 contains a long α helix that contacts the zinc fingers Zf1 and Zf2 to arrange them similarly to Zf6-7 in the Nab2p Zf5-7 structure. Nab2p TZF34 exhibits a distinctive two-fold symmetry of the zinc centers with mutual recognition of histidine ligands. Our mutagenesis and NMR data demonstrate that the α helix of TZF12 and Zf3 of TZF34 define the RNA-binding interface, while Zf1, Zf2, and Zf4 seem to be excluded. These results further our understanding of polyadenosine RNA recognition by the CCCH domain of Nab2p. Moreover, we describe a hypothetical mechanism for controlling poly(A) tail length with specific roles for TZF12, TZF34, and Zf5-7 domains.

(1)H, (13)C and (15)N assignments of CdnL, an essential protein in Myxococcus xanthus.

CdnL, an essential protein in Myxococcus xanthus and several other bacteria, is a member of the large CarD_TRCF family of bacterial proteins that interact with RNA polymerase. Structural analyses of the 164-residue M. xanthus CdnL by NMR is complicated because of broadening, and hence overlap, of the signals due to the self-association and the monomer-dimer equilibrium that occurs in solution. Here, we report (1)H, (13)C and (15)N assignments for CdnL achieved by analyzing its NMR spectra on the basis of the complete assignment obtained in this study for the 68-residue N-terminal fragment of CdnL (CdnLNt) together with those we described previously for the stable, protease-resistant, 110-residue C-terminal domain (CdnLCt). This approach relied on our observation that many of the CdnLNt and CdnLCt chemical shifts matched closely with those of the equivalent residues in the full-length protein. Our assignments provide the crucial first step in the structural analysis of CdnL and this functionally important family of bacterial proteins.

Disulfide and amide-bridged cyclic peptide analogues of the VEGF81₋91 fragment: synthesis, conformational analysis and biological evaluation.

The design, synthesis, conformational studies and binding affinity for VEGFR-1 receptors of a collection of linear and cyclic peptide analogues of the ß-hairpin fragment VEGF(81-91) are described. Cyclic 11-mer peptide derivatives were prepared from linear precursors with conveniently located Cys, Asp or Dap residues, by the formation of disulfide and amide bridges, using solid-phase synthesis. Molecular modelling studies indicated a tendency to be structured around the central ß-turn of the VEGF(81-91) ß-hairpin in most synthesized cyclic compounds. This structural behavior was confirmed by NMR conformational analysis. The NHCO cyclic derivative 7 showed significant affinity for VEGFR-1, slightly higher than the native linear fragment, thus supporting the design of mimics of this fragment as a valid approach to disrupt the VEGF/VEGFR-1 interaction.

Pub1p C-terminal RRM domain interacts with Tif4631p through a conserved region neighbouring the Pab1p binding site.

Pub1p, a highly abundant poly(A)+ mRNA binding protein in Saccharomyces cerevisiae, influences the stability and translational control of many cellular transcripts, particularly under some types of environmental stresses. We have studied the structure, RNA and protein recognition modes of different Pub1p constructs by NMR spectroscopy. The structure of the C-terminal RRM domain (RRM3) shows a non-canonical N-terminal helix that packs against the canonical RRM fold in an original fashion. This structural trait is conserved in Pub1p metazoan homologues, the TIA-1 family, defining a new class of RRM-type domains that we propose to name TRRM (TIA-1 C-terminal domain-like RRM). Pub1p TRRM and the N-terminal RRM1-RRM2 tandem bind RNA with high selectivity for U-rich sequences, with TRRM showing additional preference for UA-rich ones. RNA-mediated chemical shift changes map to ß-sheet and protein loops in the three RRMs. Additionally, NMR titration and biochemical in vitro cross-linking experiments determined that Pub1p TRRM interacts specifically with the N-terminal region (1-402) of yeast eIF4G1 (Tif4631p), very likely through the conserved Box1, a short sequence motif neighbouring the Pab1p binding site in Tif4631p. The interaction involves conserved residues of Pub1p TRRM, which define a protein interface that mirrors the Pab1p-Tif4631p binding mode. Neither protein nor RNA recognition involves the novel N-terminal helix, whose functional role remains unclear. By integrating these new results with the current knowledge about Pub1p, we proposed different mechanisms of Pub1p recruitment to the mRNPs and Pub1p-mediated mRNA stabilization in which the Pub1p/Tif4631p interaction would play an important role.

1H, 13C and 15N backbone and side chain resonance assignments of the C-terminal domain of CdnL from Myxococcus xanthus.

CdnL, a 164-residue protein essential for Myxococcus xanthus viability, is a member of a large family of bacterial proteins of unknown structure and function. Here, we report the (1)H, (13)C and (15)N backbone and side chain assignments for the stable C-terminal domain of CdnL identified by limited proteolysis.

Disulfide bonds versus TrpTrp pairs in irregular beta-hairpins: NMR structure of vammin loop 3-derived peptides as a case study.

Structural studies on model peptides have led to a good understanding of the rules behind the formation and stability of regular beta-hairpins. To test their applicability to the successful design of irregular beta-hairpins with long loops and/or beta-bulges at the strands, we mimicked loop 3 of vammin, a 4:6 beta-hairpin with a non-Gly beta-bulge. The most stabilising cross-strand pairs, disulfide bonds or/and TrpTrp pairs, were incorporated at non-hydrogen-bonded sites in peptides spanning the 69-80 region of vammin. According to NMR data, these modified peptides adopt beta-hairpin conformations as intended by design. The Trp-containing peptides reproduce even the unusual positive phi angle for the Gln residue, with the indole rings in the preferred edge-to-face orientation. For the first time the beta-hairpin-stabilising capacities of a disulfide bond and a TrpTrp pair are compared in the same model system. We found that the contribution to stability of the noncovalent indole-indole interaction is larger than that of the covalent disulfide bond, and that their combination gives rise to an even more stable beta-hairpin.