No abstract

Cyclic diguanosine monophosphate (c-di-GMP) is a ubiquitous bacterial second messenger that controls the switch from a single-cell lifestyle to surface-attached, multicellular communities called biofilms. PilZ domain proteins are a family of bacterial c-di-GMP receptors, which control various cellular processes. We have solved the solution structure of the Pseudomonas aeruginosa single-domain PilZ protein PA4608 in complex with c-di-GMP by NMR spectroscopy. Isotope labeling by 13 C and 15 N of both the ligand and the protein made it possible to define the structure of c-di-GMP in the complex at high precision by a large number of intermolecular and intraligand NOEs and by two intermolecular hydrogen bond scalar couplings. Complex formation induces significant rearrangements of the C-and N-terminal parts of PA4608. c-di-GMP binds as an intercalated, symmetric dimer to one side of the ␤-barrel, thereby displacing the C-terminal helix of the apo state. The N-terminal RXXXR PilZ domain motif, which is flexible in the apo state, wraps around the ligand and in turn ties the displaced C terminus in a loose manner by a number of hydrophobic contacts. The recognition of the dimeric ligand is achieved by numerous H-bonds and stacking interactions involving residues Arg 8 , Arg 9 , Arg 10 , and Arg 13 of the PilZ motif, as well as ␤-barrel residues Asp 35 and Trp 77 . As a result of the rearrangement of the N and C termini, a highly negative surface is created on one side of the protein complex. We propose that the movement of the termini and the resulting negative surface form the basis for downstream signaling.The second messenger cyclic diguanosine monophosphate (c-di-GMP) 5 is widespread in and unique to the bacterial kingdom. Elevated intracellular levels of c-di-GMP generally cause bacteria to change from a motile single-cell state to an adhesive surface-attached multicellular state called biofilm (1-3). Moreover, c-di-GMP controls the virulence of animal and plant pathogens (4 -8), progression through the cell cycle (9), antibiotic production (10), and other cellular functions. In several pathogenic bacteria, c-di-GMP has been associated with the regulation of virulence factors and the development of chronic infections. During long term cystic fibrosis lung infections, increased persistence of Pseudomonas aeruginosa correlates with the generation of adaptive colony morphotypes. This includes mucoid colonies and small colony variants, auto-aggregative, hyper-adherent cells whose appearance correlates with poor lung function and persistence of infection. Both morphotypes are strongly linked to elevated levels of c-di-GMP, implicating a central role for this second messenger in chronic P. aeruginosa infections (8,(11)(12)(13)(14). These studies emphasize the clinical significance of c-di-GMP and stress the importance for understanding c-di-GMP signaling in the pseudomonads.c-di-GMP comprises two guanosine monophosphate molecules that are symmetrically connected by 5Ј-3Ј phosphodiester bonds, forming a 12-membered ring ...

The flagellar motor is a sophisticated rotary machine facilitating locomotion and signal transduction. Owing to its important role in bacterial behavior, its assembly and activity are tightly regulated. For example, chemotaxis relies on a sensory pathway coupling chemical information to rotational bias of the motor through phosphorylation of the motor switch protein CheY. Using a chemical proteomics approach, we identified a novel family of CheY-like (Cle) proteins in Caulobacter crescentus, which tune flagellar activity in response to binding of the second messenger c-di-GMP to a C-terminal extension. In their c-di-GMP bound conformation Cle proteins interact with the flagellar switch to control motor activity. We show that individual Cle proteins have adopted discrete cellular functions by interfering with chemotaxis and by promoting rapid surface attachment of motile cells. This study broadens the regulatory versatility of bacterial motors and unfolds mechanisms that tie motor activity to mechanical cues and bacterial surface adaptation.

Peptidyl prolyl cis-trans isomerases can enzymatically assist protein folding, but these enzymes exclusively target the peptide bond preceding proline residues. Here we report the identification of the Hsp70 chaperone DnaK as the first member of a novel enzyme class of secondary amide peptide bond cis-trans isomerases (APIases). APIases selectively accelerate the cis-trans isomerization of nonprolyl peptide bonds. Results from independent experiments support the APIase activity of DnaK: (i) exchange crosspeaks between the cis-trans conformers appear in 2D (1)H NMR exchange spectra of oligopeptides (ii) the rate constants for the cis-trans isomerization of various dipeptides increase and (iii) refolding of the RNase T1 P39A variant is catalyzed. The APIase activity shows both regio and stereo selectivity and is stimulated two-fold in the presence of the complete DnaK/GrpE/DnaJ/ATP refolding system. Moreover, known DnaK-binding oligopeptides simultaneously affect the APIase activity of DnaK and the refolding yield of denatured firefly luciferase in the presence of DnaK/GrpE/DnaJ/ATP. These results suggest a new role for the chaperone as a regioselective catalyst for bond rotation in polypeptides.

The fast reaction of the actin-based cytoskeleton in motile cells after stimulation with a chemoattractant requires a signal-transduction chain that creates a very specific environment at distinct regions beneath the plasma membrane. Dictyostelium hisactophilin, a unique actin-binding protein, is a submembranous pH sensor that signals slight changes of the H+ concentration to actin by inducing actin polymerization and binding to microfilaments only at pH values below seven. It has a relative molecular mass of 13.5K and its most unusual feature is the presence of 31 histidine residues among its total of 118 amino acids. The transduction of an external signal from the plasma membrane to the cytoskeleton is poorly understood. Here we report the protein's structure in solution determined by nuclear magnetic resonance spectroscopy. The nuclear Overhauser effect intensities of the three-dimensional nuclear Overhauser spectra were used directly in the calculations. The overall folding of histactophilin is similar to that of interleukin-1 beta and fibroblast growth factor, but the primary amino-acid sequence of hisactophilin is unrelated to these two proteins.