Interaction of the intrinsically unstructured phage lambda N Protein with Escherichia coli NusA.

N protein of the Escherichia coli phage lambda (lambdaN) is involved in antitermination, a transcription regulatory process that is essential for the expression of delayed early genes during phage lytic development. lambdaN is an intrinsically unstructured protein that possesses three distinct binding sites interacting with the carboxy terminus of the E. coli host factor protein NusA, the viral nutBoxB-RNA, and RNA polymerase, respectively. Heteronuclear NMR experiments with lambdaN(1-53) in complex with NusA(339-495) revealed that upon complex formation the lambdaN-binding interface, lambdaN(34-47), adopts a rigid structure. NMR data also indicate the induction of a weak helical structure in the nutboxB RNA-binding region lambdaN(1-22) upon binding to NusA(339-495) even in the absence of RNA. Titration experiments of the lambdaN(1-53)-nutBoxB RNA complex with NusA(339-495) revealed that the ternary complex can be described in terms of two structurally independent binary interactions. Furthermore, chemical-shift perturbation experiments with different NusA constructs and different lambdaN peptides showed that only NusA(353-416) is involved in lambdaN binding. We found that only one molecule of NusA(339-426) binds to one molecule of lambdaN(1-53). We also clarified the role of the lambdaN-binding region and could show that N41-R47 also binds to NusA(339-495). Furthermore, we observe that lambdaN(1-22) adopts a helical fold upon binding to NusA(339-495), in agreement with one of the theoretical models of lambdaN action.

The E. coli NusA carboxy-terminal domains are structurally similar and show specific RNAP- and lambdaN interaction.

The carboxy-terminal domain of the transcription factor Escherichia coli NusA, NusACTD, interacts with the protein N of bacteriophage lambda, lambdaN, and the carboxyl terminus of the E. coli RNA polymerase alpha subunit, alphaCTD. We solved the solution structure of the unbound NusACTD with high-resolution nuclear magnetic resonance (NMR). Additionally, we investigated the binding sites of lambdaN and alphaCTD on NusACTD using NMR titrations. The solution structure of NusACTD shows two structurally similar subdomains, NusA(353-416) and NusA(431-490), matching approximately two homologous acidic sequence repeats. Further characterization of NusACTD with 15N NMR relaxation data suggests that the interdomain region is only weakly structured and that the subdomains are not interacting. Both subdomains adopt an (HhH)2 fold. These folds are normally involved in DNA-protein and protein-protein interactions. NMR titration experiments show clear differences of the interactions of these two domains with alphaCTD and lambdaN, in spite of their structural similarity.

Solution structure, dynamics, and hydrodynamics of the calcium-bound cross-reactive birch pollen allergen Bet v 4 reveal a canonical monomeric two EF-hand assembly with a regulatory function.

Birch pollinosis is one of the prevailing allergic diseases. In all, 5-20% of birch pollinotics mount IgE antibodies against the minor birch pollen allergen Bet v 4, a Ca2+-binding polcalcin. Due to IgE cross-reactivity among the polcalcins these patients are polysensitized to various plant pollens. Determination of the high-resolution structure of holo Bet v 4 by heteronuclear NMR spectroscopy reveals a canonical two EF-hand assembly in the open conformation with interhelical angles closely resembling holo calmodulin. The polcalcin-specific amphipathic COOH-terminal alpha-helix covers only a part of the hydrophobic groove on the molecular surface. Unlike the polcalcin Phl p 7 from timothy grass, which was recently shown to form a domain-swapped dimer, the hydrodynamic parameters from NMR relaxation, NMR translational diffusion, and analytical ultracentrifugation indicate that both apo and holo Bet v 4 are predominantly monomeric, raising the question of the physiological and immunological significance of the dimeric form of these polcalcins, whose physiological function is still unknown. The reduced helicity and heat stability in the CD spectra, the poor chemical shift dispersion of the NMR spectra, and the slightly increased hydrodynamic radius of apo Bet v 4 indicate a reversible structural transition upon Ca2+ binding, which explains the reduced IgE binding capacity of apo Bet v 4. The remarkable structural similarity of holo Bet v 4 and holo Phl p 7 in spite of different oligomerization states explains the IgE cross-reactivity and indicates that canonical monomers and domain-swapped dimers may be of similar allergenicity. Together with the close structural homology to calmodulin and the hydrophobic ligand binding groove this transition suggests a regulatory function for Bet v 4.