Comparison between the PK1 and the PLRV pseudoknots: Schematics using same color coding as in with the exception of ‘rejected’ residues which are displayed in grey

<p><b>Copyright information:</b></p><p>Taken from "NMR structure of the tmRNA pseudoknot PK1: new insights into the recoding event of the ribosomal trans-translation"</p><p>Nucleic Acids Research 2006;34(6):1847-1853.</p><p>Published online 4 Apr 2006</p><p>PMCID:PMC1428798.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> () NMR structure PK1. () X-ray structure of the PLRV (). An arrow points the important looped-out residues

Reverse NOE-pumping experiments.

<p>Expanded 1D NMR spectra recorded in D₂O at 600 MHz and 308 K, pH 6.5 of c-di-GMP and 5′-pGpG alone (respectively left panels A-B and E-F) and with FimX-EAL (right panels C-D and G-H). Panels A, C, E and G: reference 1D-esgp experiments. Panels B, D, F and H: RNP experiments. (S = signals arising from buffer).</p

Crystal Structure of an EAL Domain in Complex with Reaction Product 5′-pGpG

<div><p>FimX is a large multidomain protein containing an EAL domain and involved in twitching motility in <em>Pseudomonas aeruginosa</em>. We present here two crystallographic structures of the EAL domain of FimX (residues 438–686): one of the apo form and the other of a complex with 5′-pGpG, the reaction product of the hydrolysis of c-di-GMP. In both crystal forms, the EAL domains form a dimer delimiting a large cavity encompassing the catalytic pockets. The ligand is trapped in this cavity by its sugar phosphate moiety. We confirmed by NMR that the guanine bases are not involved in the interaction in solution. We solved here the first structure of an EAL domain bound to the reaction product 5′-pGpG. Though isolated FimX EAL domain has a very low catalytic activity, which would not be significant compared to other catalytic EAL domains, the structure with the product of the reaction can provides some hints in the mechanism of hydrolysis of the c-di-GMP by EAL domains.</p> </div

Stereo view of the active site of FimX-EAL domain.

<p>(A) The residual density Fo-Fc calculated after molecular replacement with no ligand in the model is contoured in orange at 3 σ level (A). The FimX backbone is represented as green cartoon. (B) Interpretation of the density as the bound 5′-pGpG, shown as black sticks. The magnesium ion is shown as a magenta sphere and the catalytic water involved in its coordination as a red sphere. Side-chains of residues involved in ligand binding are shown. The final electron density map 2Fo-Fc contoured at 1.2 σ level is traced around the ligand.</p

Enzymatic activity of hydrolysis of BpNPP by FimX.

<p>(A) Kinetics parameters of full-length FimX. Initial velocities are expressed in arbitrary units. (B) Relative activity of B<i>p</i>NPP hydrolysis after 9 hours of full-length FimX (FimX-FL), GGDEF-EAL domains (Dual) and EAL domain (EAL). Catalytic activities are normalised against that of FimX-FL. Dual has 18.1% activity and the EAL domain alone retains 6.6% activity.</p

Representation of the cavity in the dimer of FimX-EAL bound to pGpG.

<p>(A) The cavity is presented as an orange surface inside the FimX-EAL dimer. It is connected to the outside by two channels of different size. Left and right views are rotated by 180°. The corresponding electrostatic surface is represented in (B). The edges of both channels are negatively charged and forbid the ligand to escape from the cavity.</p

Schematics view of the interactions between FimX-EAL and 5′-pG-pG.

<p>5′-pGpG is shown as black sticks. Residues of FimX-EAL involved in polar contacts are shown in green sticks and residues involved in hydrophobic interactions are shown as purple spheres. Polar contacts are shown as red dotted lines.</p

Sequence alignments of EAL domains from various proteins.

<p>Alignement of EAL domains was done with ClustalW. Conserved residues important for the PDE-A activity are numbered #1 to #10. Residues conserved in FimX-EAL are boxed in red. The secondary structure is indicated for FimX-EAL structure.</p

Structure of the homodimer of EAL domain of FimX with bound 5′-pGpG.

<p>(A) Structure of the homodimer of EAL domain of FimX with bound 5′-pGpG. The secondary structure elements are labelled on one monomer. (B) Stereo view of the superposition of homodimer of EAL domain of FimX in apo form crystallized in spacegroup P2₁2₁2₁ (dark blue and light blue) on the homodimer of FimX EAL in complex with 5′-pGpG crystallized in spacegroup P4₃2₁2 (red and green).</p