Solution Structure of the PilZ Domain Protein PA4608 Complex with Cyclic di-GMP Identifies Charge Clustering as Molecular Readout*♦

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 13C and 15N 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 Arg8, Arg9, Arg10, and Arg13 of the PilZ motif, as well as β-barrel residues Asp35 and Trp77. 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.

GMP molecule induces a conformational change in the loop connecting the C-terminal PilZ domain to the N-terminal YcgR-N domain. This brings the two domains into close proximity, thereby forming a new allosteric interaction surface with c-di-GMP at their mutual interface. It has been shown recently that YcgR, a homolog of VCA0042 in Escherichia coli, is able to bind to the flagellar motor in its c-di-GMP-bound form (21). Although VCA0042 appears to bind only one molecule of c-di-GMP and does not alter its quaternary structure upon ligand binding, PP4397 from Pseudomonas putida binding of two molecules of c-di-GMP in the junction of its YcgR-N and PilZ domains results in a dimer-monomer transition (22). This suggested that different PilZ domain proteins exhibit distinct binding stoichiometries and mechanistic principles.
All structures of c-di-GMP protein complexes solved until today involve multidomain proteins (for review, see Ref. 15). In these structures, the binding of c-di-GMP within a hinge-like region induces an allosteric rearrangement of the neighboring domains via fixation to the ligand. It was proposed that this c-di-GMP-induced domain immobilization represents a general mechanism for signal transduction. In contrast to these multidomain proteins, the PilZ domain PA4608 from P. aeruginosa is a single domain c-di-GMP-binding protein. The apo solution structure of PA4608 was solved by Ramelot et al. (23), who also confirmed binding of c-di-GMP by chemical shift analysis. NMR chemical shift mapping was then used to localize the ligand binding site of PA4608 to one side of the protein surface and to demonstrate that c-di-GMP binds to PA4608 as a dimer (16). Furthermore, these analyses demonstrated that PA4608 remains monomeric after ligand binding (16).
Here we report the structure of the complex of PA4608 with c-di-GMP solved by advanced solution NMR techniques. Uniform 13 C, 15 N isotope labeling of both c-di-GMP and PA4608 allowed the observation of a large number of intermolecular and intraligand NOE contacts, 1 D HN and 1 D CH ligand and protein residual dipolar couplings (RDCs), and intermolecular hydrogen bond scalar couplings. Both termini of the protein undergo large structural changes upon ligand binding while retaining a certain amount of flexibility. In particular, the C-terminal helix is displaced by the ligand, thereby opening one side of the ␤-barrel as a binding site, and the N terminus containing the RXXXR PilZ domain motif wraps around the ligand and in turn ties the C-terminal helix in a loose conformation induced by a number of hydrophobic contacts. The rearranged termini expose a highly negatively charged surface on one side of the complex to a possible effector protein. This induced folding of the flexible N terminus containing the PilZ signature RXXXR and the resulting clustering of surface charges may present a general mechanism for c-di-GMP readout by PilZ domains.

EXPERIMENTAL PROCEDURES
Protein Expression and Purification-Overexpression, 13 C, 15 N labeling, and purification of PA4608 were achieved as described previously (16). In the protein construct used, the PA4608 sequence is preceded by a hexahistidine tag with the sequence MGSSHHHHHHSSGLVPRGSH. c-di-GMP was produced enzymatically from GTP by using a non-feedback-inhibited mutant of the diguanylate cyclase DgcA named DgcA0646, in which the inhibition site RESD is replaced by GRDC (24,25). Uniformly 13 C-and 15 N-labeled c-di-GMP was produced in the same manner but starting with 13 C-, 15 N-labeled GTP (Spectra Stable Isotopes) as a precursor.
NMR Samples-Uniformly 13 C, 15 N-and 15 N-labeled samples of apo PA4608 (0.8 -1.2 mM) were prepared in 250 mM NaCl, 10 mM Tris-HCl at pH 6.7, 0.01% NaN 3 (w/v), and 5% (v/v) D 2 O as sample volumes of 200 -400 l. 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 1 H-15 N heteronuclear single quantum correlation spectra. The titration was stopped at two equivalents of c-di-GMP, i.e. when the protein was saturated. For preparation of D 2 O samples, the H 2 O samples were lyophilized and redissolved in D 2 O twice. Non-isotropic samples of complexed (apo) protein were prepared by adding 18 mg/ml (26 mg/ml) filamentous phage Pf1 (Asla Biotech).
NMR Spectroscopy and Resonance Assignments-NMR spectra were recorded at 293 K on Bruker DRX 600 and DRX 800 NMR spectrometers equipped with TXI and TCI probe heads, respectively. For protein assignment, structure information, and assessment of backbone dynamics by 15 N relaxation, standard two-dimensional and three-dimensional NMR spectra were acquired similar to the ones described (26). c-di-GMP ribose resonances were connected by three-dimensional HCCH-COSY and -TOCSY spectra recorded on 13 C/ 15 N-labeled c-di-GMP bound to 13 C/ 15 N-labeled PA4608. Ribose moieties and guanine H1/N1 and H8/C8 resonances were assigned via their NOE contacts. Details of the acquired spectra are indicated in the supplemental Table S1. NMR data were processed using the NMRPipe suite of programs (27). Spectra were displayed and analyzed with the programs SPARKY (28) and PIPP (29).
Structure Calculations-Structure calculations were performed with the program XPLOR-NIH (30) using two consecutive simulated annealing protocols (31). In a first step, calculations started from an extended strand with c-di-GMP bound to the protein via the measured H-bond between H⑀1 of Trp 77 and N7 of Gua1, using intraprotein restraints only; the second step included all restraints. c-di-GMP molecules were defined as two-residue, circular strands of RNA. The 20-residue His tag and linker were not included in structure calculations. A total of 200 structures were calculated, and the 20 lowest energy structures were selected for deposition (Protein Data Bank (PDB) code 2L74). The structural statistics are given in Table 1.

RESULTS AND DISCUSSION
NMR Spectroscopy, Resonance Assignments, and Structure Determination-Standard triple-resonance NMR spectra recorded on PA4608 saturated with c-di-GMP were used to assign backbone and side chain resonances of PA4608. Resonance assignments were obtained for about 92% of the protein backbone in the PA4608⅐c-di-GMP complex. Missing backbone assignments comprise parts of the N-terminal His tag sequence as well as residues His 12 and Asp 108 -Arg 114 . In almost all of these cases, resonances were not visible in the 1 H-15 N heteronuclear single quantum correlation spectra, presumably due to chemical exchange broadening resulting from flexibility on the microsecond to millisecond time scale.
The chemical shifts of c-di-GMP bound to PA4608 are very distinct from free c-di-GMP. In particular, four different imino H1 proton resonances are observed, indicative of four guanosines in an asymmetric environment. This is consistent with the binding of one c-di-GMP dimer to one protein monomer. Of these 4 guanosine residues, all one-bond 1 H1/ 15 N1 and 1 H/ 13 C resonance pairs could be assigned with the exception of Gua1-H5Љ and Gua4-H5Љ by using information from three-dimensional HCCH-TOCSY/-COSY and 15 N-or 13 C-edited NOESY experiments. For both Gua2 and Gua3, one of the two N2-bound protons could also be clearly observed. In free guanosines, these proton resonances are usually strongly broadened from the rotation around the C2-N2 bond. The observation of the resonances together with the measured NOEs indicates H-bond formation between the intercalated dimers in the following way ( Fig. 1B): Gua1-O1P⅐⅐⅐H1-N1-Gua3, Gua1-O1P⅐⅐⅐H21-N2-Gua3, Gua4-O1P⅐⅐⅐H1-N1-Gua2, and Gua4-O1P⅐⅐⅐H21-N2-Gua2. Two additional H-bonds between c-di-GMP and the protein, Gua1-N7⅐⅐⅐H⑀1-N⑀1-Trp 77 and Gua2-N7⅐⅐⅐H⑀-N⑀-Arg 9 , were detectable by the HNN-COSY experiment (32) (Fig. 1C) and are indicated in Fig. 1B as red dashed lines. The position of the c-di-GMP dimer on the protein is well defined by 103 intermolecular NOEs. A small section of the two-dimensional NOESY spectrum (Fig.  1C) shows some of these NOEs from the four imino protons of the c-di-GMP dimer and the Trp 77 H⑀1 proton, indicating contacts between the ligand and protein residues Arg 8 , Arg 9 , Phe 11 , Arg 13 , Arg 12 , Ile 41 , Leu 42 , Ala 76 , and Trp 77 . The c-di-GMP dimer itself is well defined by 59 intramolecular NOEs and a further 15 1 D CH RDCs. Structural restraints of the protein comprise 1734 intramolecular NOEs and 152 1 D HN and 1 D CH RDCs.
Based on these restraints, a well defined structure of the PA4608⅐c-di-GMP complex was calculated using conventional simulated annealing protocols. Its quality is reflected in a heavy atom coordinate r.m.s.d. of 0.20 Å for the backbone in the ordered region of the protein and of 0.38 Å for the c-di-GMP dimer. Further structural statistics are given in Table 1.
Ligand-induced Changes in the Backbone Structure of PA4608-As compared with apo PA4608, the complexed protein shows strong chemical shift changes in the N-terminal region around residue Arg 9 , in the C-terminal region around residue Val 120 , as well as in the central ␤-barrel around residue Val 36 (16). An analysis of the secondary structure (Fig. 2) and backbone dynamics (see below) shows that structural changes mainly occur in the N-and C-terminal regions, whereas the central ␤-barrel is largely unaffected. Ligand binding induces a transition of the N-terminal region from an unfolded state to an ordered structure and a strong destabilization of the C-terminal region in particular between residues Asp 108 and Arg 114 . For residues Glu 110 to Arg 114 , no resonances can be detected at all due to exchange broadening, indicating the disappearance of the apo 3 10 helix ␥ 1 (Glu 109 -Glu 113 ). Residues Glu 115 up to the C terminus are again observable but correspond to an ␣-helix (␣ 2 ) between residues Ala 117 and Ser 121 rather than to the apo 3 10 helix ␥ 2 (Leu 116 -Leu 118 ). Fig. 2 shows a sequence and secondary structure alignment of PA4608 with the other PilZ domains, for which structures complexed with c-di-GMP have been solved, i.e. P. putida PP4397 (22) and V. cholerae VCA0042 (20). In addition, Caulobacter crescentus DgrA and DgrB and Salmonella typhimurium YcgR have been included because they have similar nanomolar affinities for c-di-GMP as PA4608, and key residues for ligand binding are identified by point mutations (16). Despite a rather low sequence similarity, the ␤-barrel and helix ␣ 1 secondary structure elements of the solved structures are largely conserved. Most importantly, in all aligned proteins, the PilZ domain signature motifs RXXXR and DXSXXG are completely conserved apart from a single amino acid replacement (S/A) for DgrA. Residues that have van der Waals contacts to the ligand in the solved holo structures (yellow) are also largely conserved. These comprise the two RXXXR and DXSXXG motifs and residues around Trp 77 (PA4608) and Gly 84 (PA4608), which are located on adjacent ␤-strands. Together with the DVSLHG 40 region, the latter form the surface of the ␤-barrel that is in direct contact with the ligand. Not surprisingly, mutations of the conserved residues Arg 8 , Arg 9 , Asp 35 , or Trp 77 (PA4608 numbering) strongly reduce or abolish c-di-GMP binding in DgrA (16).
Description of the PA4608⅐c-Di-GMP Complex Structure-The core structure of PA4608 in complex with c-di-GMP consists of a six-stranded antiparallel ␤-barrel with an elongated ␣-helix across one barrel opening (Fig. 3, A and B). The antiparallel ␤-strands are in the order 1-2-3-6-5-4 with 1 and 4 closing the barrel in an antiparallel manner. The intercalated c-di-GMP dimer is located at one side of the barrel. It makes contacts with residues of the barrel strands ␤5, ␤6, ␤3, and ␤2 as well as with side chains from the N-terminal part of the sequence, which is unstructured in the apo state. This N-terminal end wraps around the external side of the intercalated c-di-GMP dimer. The face of the N-terminal region, which points away from the ligand, makes contacts to the C-terminal region, thereby also anchoring helix ␣2 toward the ligand. Fig. 3, C and D, show bundle representations of the 20 lowest energy structures in a best fit superposition of the barrel backbone atoms and the ligand atoms, respectively. The high coordinate precision of the protein core, c-di-GMP, and the N terminus is evident. In contrast, the C-terminal region is less well defined, but clearly not random.
Details of the recognition of c-di-GMP dimer by PA4608 are shown in Fig. 4. The main residues involved are Trp 77 and Arg 8 through Arg 13 . Trp 77 recognizes the side of the Gua1 base by the h2 J NN -detected H-bond from its indole N⑀1 donor to the Gua1 N7 acceptor (Fig. 1). This locates the Gua1 base in the same plane as the aromatic ring of Trp 77 . A further H-bond may be possible from the Gua1 H1 hydrogen to the side chain of Asp 35 but is not clearly detectable due to low definition of the Asp 35 side chain (not shown). The Gua1 base also packs against the side chain of Arg 13 . The pyrimidine ring of Gua3 stacks below the pyrrole ring of Trp 77 . The N7 and O6 atoms of Gua3 are recognized by two further H-bonds from the Arg 13 N1 and N2 atoms. Besides the structural proximity, evidence for these H-bonds comes from the observation of resonances and NOE contacts from the N1 and N2 atoms of Arg 13 . Usually these proton resonances are not detectable in free arginine side chains due to chemical exchange broadening from the rotation around the C-N1 and C-N2 bonds. The imino N1 and the amino N2 groups of Gua3 are in H-bonding distance to the Gua1 phosphate group of the first c-di-GMP monomer.
The pyrimidine ring of Gua2 stacks below the pyrimidine ring of Gua3. Its imino N1 and the amino N2 groups are in H-bonding distance to the Gua4 phosphate group of the second c-di-GMP monomer. The N7 and O6 atoms of Gua2 are recog-FIGURE 1. Detection of the dimeric c-di-GMP structure and coordination in the complex with PA4608 by heteronuclear NMR methods. A, the chemical structure c-di-GMP consisting of two GMP moieties linked by O3Ј-P phosphodiester bonds. B, the intercalated dimeric c-di-GMP as it is bound to PA4608. Because c-di-GMP comprises two guanosine monophosphate molecules that are symmetrically connected by 5Ј-3Ј phosphodiester bonds to a single molecule, we kept the nomenclature of the atoms but numbered the guanosine moieties as guanosines 1 and 2 for the first monomer and guanosines 3 and 4 for the second monomer. For space reasons, guanosines are abbreviated as Gu in the figures. Hydrogen bonds, for which there is experimental evidence, are shown as dashed lines. Two of these result from direct detection via h2 J NN couplings (see C) and are shown by thick red dashes. C, extracted regions of two HNN-COSY spectra for direct detection of N-H⅐⅐⅐N H-bonds (32). The red (negative) signals are cross-peaks resulting from H-bond J-coupling transfer from the H-bond donor nucleus Trp 77 -15 N⑀1 to the acceptor nucleus Gua1-15 N7 ( h2 J NN ϭ 4.7 Ϯ 0.3 Hz) magnetization and from Arg 9 -15 N⑀ to Gua2-15 N7 ( h2 J NN ϭ 4.8 Ϯ 0.4 Hz), respectively. D, extracted region from the 1 H twodimensional NOESY spectrum showing the NOE contacts from the four imino protons H1 of guanines 1-4 and of H⑀1 of Trp 77 . A large number of intermolecular c-di-GMP-to-protein as well as c-di-GMP intradimer contacts are observed that define the c-di-GMP coordination in the complex with PA4608.
nized by H-bonds from the N⑀ and the N2 atoms of Arg 9 , respectively. The Arg 9 -N⑀-H⑀⅐⅐⅐N7-Gua2 H-bond is directly detected by h2 J NN couplings (Fig. 1), whereas again the close proximity and the detection of both Arg 9 H21 and H22 pro-tons indicate the presence of the Arg 9 -N2-H21⅐⅐⅐O6-Gua2 H-bond. Finally, the base of the last guanosine Gua4 is packing against the side chain of Arg 9 and is turned away from the stacked Trp 77 /Gua3/Gua2 ring system. Nevertheless, all four guanine bases and the indole ring of Trp 77 are nearly coplanar.
The observed H-bond interactions of the N7 and O6 acceptors of Gua3 and Gua2 with the N and N⑀ donors of Arg 13 and Arg 9 , respectively, are a very common mode of recognition of guanosines by arginines in protein-DNA complexes (33). A further common recognition motif is identified in the favorable cation-interactions (34,35) that can be assumed for the packing of Gua1 against Arg 13 and Gua4 against Arg 9 . The high degree of conservation of Arg 9 and Arg 13 (Fig. 2) is fully consistent with this observed key role in c-di-GMP dimer recognition. The side chains of Arg 8 and Arg 10 are not as well defined in the structure, and hence do not allow the identification of nonambiguous H-bonds. However, the proximity of Arg 8 to the guanine ring of Gua4 and of Arg 10 to the negatively charged phosphate of Gua2 suggests that Arg 8 and Arg 10 are also involved in c-di-GMP recognition.
Residues Phe 11 and His 12 are in the center of the RXXXR 13 c-di-GMP recognition motif. Their backbone 1 H-15 N resonances are very weak (Phe 11 ) or not observed at all (His 12 ), indicating flexibility on the microsecond to millisecond time scale. Nevertheless, the side chains of Phe 11 and His 12 could be assigned, and 15 unambiguous NOEs are detected from these residues to the side chains of Leu 118 and Leu 119 in the C-terminal helix ␣2 (Fig. 4). Despite the apparently high structural flexibility in this region, these hydrophobic contacts clearly dock the C-terminal helix ␣2 onto the top of the N-terminal c-di-GMP recognition site. Thus, the RXXXR signature motif couples ligand binding directly to the reorientation of the C-terminal part of the protein.
Comparison of the PA4608 c-Di-GMP Binding Mode to Other Known Structures-Besides PA4608, PilZ structures have been solved in both apo and holo forms for two additional proteins, PP4397 (22) and VCA0042 (20). PP4397 consists of a C-terminal PilZ and an N-terminal YcgR domain. It dimerizes in its apo form via interactions between the RXXXR signature motif of one protomer and the PilZ domain of the other protomer. Binding of an intercalated dimer of c-di-GMP to the RXXXR motif in the linker region between the two PP4397 domains induces the separation of the protein dimer into monomers. Fig. 5, A and B, show a comparison of the c-di-GMP dimer recognition in PA4608 and PP4397. The similarity of the c-di-GMP coordination is very strong but may not be surprising based on the high sequence conservation of the recognition motifs. Thus, (i) Arg 123 and Arg 127 in PP4397 recognize the bases of Gua2 and Gua3 by the same H-bonds as Arg 9 and Arg 13 in PA4608; (ii) the side chain of His 201 in PP4397 replaces the Trp 77 side chain in PA4608 to recognize Gua1 by H-bonds; and (iii) Arg 122 in PP4397 has well defined H-bonds to Gua4 that correspond to H-bonds from Arg 8 in PA4608 but are not well detected in the NMR structure due to the low definition of the side chain. Further similarities are (iv) the H-bond recognition of the Gua1 imino group by the side chain of Asp 157 in PP4397 and (v) the H-bond rec- .5Å. f These comprise 147 RDCs from N-HN and C ␣ -H ␣ , and 5 RDCs from aromatic rings. g 11 RDCs are from the ribose moieties and four of the guanine rings. h Backbone torsion angle restraints were derived from 13 C ␣ and 13 C ␤ chemical shifts using the program TALOSϩ (43). ognition of Gua2 phosphate by Asn 124 in PP4397, which are replaced by Asp 35 and Arg 10 in PA4608, respectively. VCA0042 also consists of a C-terminal PilZ and an N-terminal YcgR domain, dimerizes via the YcgR domain, and binds c-di-GMP in the linker region between the two domains. However, in contrast to PP4397, the bound c-di-GMP is in monomeric form. It was recently proposed that the replacement of the arginine, which precedes the RXXXR motif, by a leucine (Fig. 2) is responsible for the recognition of a c-di-GMP monomer instead of a dimer (20,22). It is interesting to note that the respective residues in PA4608 (Arg 8 ) and PP4397 (Arg 122 ) are both arginines and recognize the base of Gua4 in the second c-di-GMP monomer by hydrogen bonds, consistent with the binding of dimeric c-di-GMP to both PA4608 and PP4397.
PleD, a multidomain diguanylate cyclase from C. crescentus, is a member of an additional class of c-di-GMP-binding proteins (36,37). In this protein family, the c-di-GMP binding site constitutes an allosteric site responsible for tight feedback inhibition of the enzymatic activity (24). Similar to PA4608 and PP4397, PleD binds a dimer of c-di-GMP. Fig. 5C shows the dimer coordination in the same orientation as for PA4608 in Fig. 5A. Again, the specific recognition is mainly achieved by arginines. Thus, Arg 390 , Arg 359 , and Arg 178 assume the role of the base-recognizing Arg 8 , Arg 9 , and Arg 13 in PA4608. However, these side chains originate obviously from very different regions of the primary sequence and even from different domains of the protein. In addition, no equivalent of the recognition of Gua1 by the aromatic residue Trp 77 (His 201 ) in PA4608 (PP4397) is present in the PleD structure. Thus, although the principle of base recognition by arginine fingers is similar, the overall architecture of the c-di-GMP binding sites is distinct for the two protein families.
Comparison of PA4608 Apo and Holo Forms-Pronounced changes in the chemical shifts between the apo and holo form of PA4608 indicate significant structural rearrangements in the N-and C-terminal regions upon ligand binding. Fig. 6 shows a comparison between the two states. In the apo form, the c-di-GMP binding side of the ␤-barrel comprising strands ␤2, ␤3, ␤5, and ␤6 is partially covered by the C-terminal 3 10 helices ␥ 1 and ␥ 2 . Binding of c-di-GMP to this site pushes the C-terminal region away from the ␤-barrel and causes the unfolding of helix ␥ 1 . In turn, the N-terminal region, which is flexible in the apo state, buries the outer side of c-di-GMP with the residues RRRFHR 13 of the recognition motif. The end of the C-terminal region comprising the ␣-helix ␣2 covers this part of the N terminus by hydrophobic contacts in a lid-like fashion.
During this process, the ␤-barrel remains basically unchanged. This is reflected by an r.m.s.d. of 0.9 Å between the heavy backbone atom coordinates of the barrel in the apo and the holo structures. However, a certain rearrangement of helix ␣1 connecting the barrel to the C-terminal part is evident in Fig. 6. It corresponds to a rotation of 11°of the helix axis away from the barrel. The helix is structurally well defined with all its residues recognized as ␣-helical by PROCHECK-NMR (38) for all 20 lowest energy structures. Its change in orientation was confirmed experimentally from RDCs of the complex and the apo state, which were additionally acquired (supplemental Fig. S1 and Table S2). In the region of helix ␣1, the RDCs of the apo and holo form agree well with the apo and holo structures, respectively, but deviate for an interchange of structures. The difference in helix orientation is apparently caused by the hydrophobic interactions between helix residues Leu 98 , Leu 101 , and Val 102 and the N-terminal residues Ile 14 and Phe 16 in the apo state and rearrangement of the N-and C-terminal regions, which pull the helix away from the barrel in the complexed state.
The changes in the protein backbone dynamics between the apo and holo form were also characterized by 15 N relaxation experiments carried out at 293 K (Fig. 7). For most residues, the 15 N T 1 and T 2 relaxation times as well as the heteronuclear { 1 H}-15 N NOEs are very uniform and correspond to a well folded structure. Moreover, they are also almost identical between the apo and holo forms. An analysis by the program TENSOR (16,39) yields isotropic rotational correlation times ( c ) of 11.3 ns (12.3 ns) for the apo (holo) form, which are in reasonable agreement with values expected for monomeric PA4608 in its apo form (16.7 kDa) and bound to a dimer of c-di-GMP (18.1 kDa). Also, in agreement with the monomeric state of the complex, no NOEs were detected that would indicate intermolecular dimer  contacts. Thus, PA4608 is monomeric in the apo and holo form.
The relaxation data also give evidence for changes in dynamics upon complex formation for certain parts of the sequence. Thus, the N-terminal residues around Arg 8 have low { 1 H}-15 N NOE and large T 2 values corresponding to large amplitude motions on the nanosecond time scale for the apo state, whereas these values become comparable with folded residues in the rest of the sequence in the holo state. This is consistent with folding upon ligand binding of the N terminus. In contrast, the C-terminal end around residue Ala 122 has the opposite behavior, indicating that the very C-terminal end becomes flexible upon ligand binding. Nevertheless, the adjacent helix ␣2 of the complexed state appears relatively stable because residues Ala 117 and Leu 118 of this helix have 15 N relaxation parameters that correspond to a folded structure. Consistent with this stability of helix ␣2, all its 5 residues (Ala 117 to Ser 121 ) are found in ␣-helical conformations in 90% of the lowest energy structures. In the apo state, the region of residues Asp 108 to Leu 116 , which contains the 3 10 helix ␥1, has relaxation parameters of a well folded protein. In contrast, resonances from these residues are strongly broadened or not detectable at all in the holo state, which must be caused by micro-to millisecond dynamics of the protein backbone. Thus, this region undergoes an unfolding transition upon ligand binding.
Clustering of PA4608 Surface Charges upon c-di-GMP Binding-The conformational changes in PA4608 induced by c-di-GMP binding lead to a severe rearrangement of surface charges (Fig. 8). Besides the four positive arginines (Arg 8 , Arg 9 , Arg 10 , Arg 13 ), the N-terminal region contains 3 negative residues (Asp 3 , Asp 6 , Glu 7 ). The coordination of the ligand by the four arginines causes their side chains to point inward. The concomitant transition to a folded structure of this region reorients the side chains of Asp 3 , Asp 6 , and Glu 7 toward the  outside and creates a negative, surface-exposed cluster (Fig. 8).
Likewise, the C terminus contains 7 negative residues centered on the 3 10 helix ␥1 (Asp 108 , Glu 109 , Glu 110 , Glu 113 , Glu 115 ) and at the very C terminus (Asp 124 , Asp 125 ). The ligand-induced rearrangement of this region positions these negative charges on top of the negative cluster around residue Glu 7 . Together with further negative residues of the ␤-barrel (Asp 17 , Asp 19 , Glu 21 , Glu 30 , Asp 35 , Asp 48 , Asp 80 ) and helix ␣1 (Glu 103 ), which do not move upon ligand binding, the combined structural changes create one face of the protein, which is strongly negatively charged and devoid of any positive charges. It is attractive to speculate that this newly generated molecular surface constitutes the readout of this small signaling protein by providing a highly charged interaction surface for high affinity regulatory interactions with downstream target proteins.
Although the PilZ domain associates with a plethora of known signaling, catalytic, or transport domains (40), standalone or single domain PilZ proteins represent the largest subclass of this protein family (see NCBI/CDART (Conserved Domain Architecture Retrieval Tool)). It is interesting to note that some of the surface-exposed charged side chains that were found to cluster on one side of the c-di-GMP-bound form of  PA4608 show conservation within the subclass of stand-alone PilZ domain proteins. In particular, positions corresponding to Glu 7 and to a lesser extent Glu 115 have conserved negative charges within this family but not within the YcgRN-PilZ family represented by the VCA0042 and PP4397 proteins (supplemental Fig. S2). Consistent with this view, the C terminus of PilZ domains varies substantially between these two PilZ protein families (supplemental Fig. S2). Although the PilZ domains of YcgR homologs have rather conserved sequences of defined length, the C termini of single domain PilZ proteins share little sequence homology, vary substantially in length, and are usually strongly charged. This indicates a high degree of modularity within the C termini of single domain PilZ proteins and suggests that the clustering of key C-terminal residues engaged in potential electrostatic interactions might be a conserved principle for many of these c-di-GMP-binding proteins.
The motional freedom of the C-terminal region of the complexed form of PA4608 together with the abundant negative surface charges may indicate that more than one downstream effector protein with a positively charged surface site exists. Patterns of spatially clustered surface charges that have co-evolved with their respective interaction partners may provide binding specificity for a large variety of proteinprotein interactions. This emphasizes the potential of this large family of proteins for functional diversification as signal transducer. Given the diverse cellular functions controlled by c-di-GMP in bacteria, rapid adaptation of this sensing module to different signaling pathways could explain the wide distribution and evolutionary success of stand-alone PilZ domain proteins.
Despite the similar binding of c-di-GMP by the PilZ domains in PA4608, VCA0042, and PP4397 (Fig. 5), the three proteins show fundamental differences in how the ligand binding translates into a molecular readout. Although PA4608 is a monomer in its apo-and ligand-bound form, both VCA0042 and PP4397 form dimers. Ligand binding to YcgR homologs induces large conformational changes within the dimer (VCA0042 (20)) or causes a dimer-to-monomer transition (PP4397 (22)). In both structures, the RXXXR binding motif, also called the c-di-GMP switch (20), forms a hinge-like connector between the N-terminal YcgR and the C-terminal PilZ domain. In VCA0042, the switch is flexible and disordered in the apo structure but undergoes conformational changes upon ligand binding that cause a rotation of the entire PilZ domain and make it switch back toward the N-terminal YcgR domain (20). In contrast, in apo PP4397, the switch forms a short helix with residues within and next to the switch participating in dimerization. Upon c-di-GMP binding, this helix unwinds and wraps around the ligand, thereby contributing to the dimer-to-monomer transition. Thus, in all three proteins, the c-di-GMP switch region is critically involved in signal transduction by making extensive contacts both to c-di-GMP and to the flanking domains (VCA0042 and PP4397) or the C terminus of the same PilZ protomer (PA4608). Despite the different structural context, the central role of the c-di-GMP switch in all three PilZ proteins seems to be to "decode" ligand binding and to transfer this information to flanking regions within the same domain or interacting domains. Although this translates into repositioning of the two domains in the YcgR homologs, it amounts to a striking clustering of charged residues on one side of the PilZ domain protein PA4608. This stresses the versatility and evolutionary flexibility of the PilZ c-di-GMP binding module, which allows for rapid functional expansion in cellular signaling pathways.
During the preparation of this manuscript, Shin et al. (41) published a structural model of PA4608 bound to c-di-GMP based on solution NMR data. The overall structural arrangement is similar. However, in contrast to our work, the position and coordination of the ligand are not well defined because the ligand and the intermolecular interactions were not assigned. Furthermore, NOEs indicating the coordination of the C terminus to the N terminus were not assigned or detected.