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 β-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 β-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.
Highlights
E-mail: stephan.grzesiek@ unibas.ch. 5 The abbreviations used are: c-di-GMP, cyclic diguanosine monophosphate; residual dipolar couplings (RDCs), residual dipolar coupling; r.m.s., root mean square; r.m.s.d., root mean bacteria to change from a motile single-cell state to an adhesive surface-attached multicellular state called biofilm [1,2,3]
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
It is synthesized from two molecules of guanosine triphosphate (GTP) by diguanylate cyclases (GGDEF-containing domains) and hydrolyzed into the linear dinucleotide 5Ј-phosphoguanylyl-(3Ј-5Ј)guanosine by the activity of c-di-GMP-specific phosphodiesterases (EAL-containing domains) [15]
Summary
Protein Expression and Purification—Overexpression, 13C, 15N labeling, and purification of PA4608 were achieved as described previously [16]. 13C- and 15N-labeled c-di-GMP was produced in the same manner but starting with 13C-, 15N-labeled GTP (Spectra Stable Isotopes) as a precursor. For complex formation, c-di-GMP was titrated to the apo PA4608 samples from a 20 mM stock solution, and the fraction of apo- and ligandbound protein was monitored by 1H-15N heteronuclear single quantum correlation spectra. Structure information, and assessment of backbone dynamics by 15N relaxation, standard two-dimensional and three-dimensional NMR spectra were acquired similar to the ones described [26]. Calculations started from an extended strand with c-di-GMP bound to the protein via the measured H-bond between H⑀1 of Trp and N7 of Gua, using intraprotein restraints only; the second step included all restraints. The 20-residue His tag and linker were not included in structure calculations.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
